vendredi 24 avril 2009
par   G. Grégori

Abboudi M, Surget SM, Rontani JF, Sempere R, Joux F (2008) Physiological alteration of the marine bacterium Vibrio angustum S14 exposed to simulated sunlight during growth. Curr Microbiol 57 :412-417


Growth experiments on the marine bacterium Vibrio angustum S14 were conducted under four light conditions using a solar simulator : visible light (V), V + ultraviolet A (UV-A), V + UV-A + UV-B radiation, and dark. Growth was inhibited mainly by UV-B and slightly by UV-A. UV-B radiation induced filaments containing multiple genome copies with low cyclobutane pyrimidine dimers. These cells did not show modifications in cellular fatty acid composition in comparison with dark control cultures and decreased in size by division after subsequent incubation in the dark. A large portion of the bacterial population grown under visible light showed an alteration in cellular DNA fluorescence as measured by flow cytometry after SYBR-Green I staining. This alteration was not aggravated by UV-A and was certainly due to a change in DNA topology rather than DNA deterioration because all the cells remained viable and their growth was not impaired. Ecological consequences of these observations are discussed.

Adkins I, Koberle M, Grobner S, Autenrieth SE, Bohn E, Borgmann S, Autenrieth IB (2008) Y. enterocolitica inhibits antigen degradation in dendritic cells. Microbes Infect 10 :798-806


Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.

Allegra S, Berger F, Berthelot P, Grattard F, Pozzetto B, Riffard S (2008) Use of flow cytometry to monitor Legionella viability. Appl Environ Microbiol 74 :7813-7816


Legionella viability was monitored during heat shock treatment at 70 degrees C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.

Amalfitano S, Fazi S (2008) Recovery and quantification of bacterial cells associated with streambed sediments. J Microbiol Methods 75 :237-243


Efficient detachment and purification of bacterial cells associated with streambed sediments are required in order to quantify cell abundance and to assess community composition through the application of epifluorescence microscopy techniques. We applied chemical (i.e., sodium pyrophosphate and polysorbate) and physical treatments (i.e., shaking and sonication), followed by Nycodenz density gradient centrifugation to efficiently recover benthic bacteria. This procedure resulted in a highly purified cell suspension allowing for a precise cell quantification through the application of fluorescent dyes. About 93% of total cells were recovered from the original sediment, with higher recovery from the finer grain-size class (90%) in comparison to the coarse fraction (69%). The potential damaging effects of the applied procedures on cell integrity were assessed on planktonic bacteria in a pre-filtered water control. As a consequence of the high purity of the extracted bacteria, flow cytometry was successfully applied as counting method for sediment cell suspension. However, a significant decrease of protein synthesis in purified samples was measured by estimating the (3)H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification.

Arnot DE, Cavanagh DR, Remarque EJ, Creasey AM, Sowa MP, Morgan WD, Holder AA, Longacre S, Thomas AW (2008) Comparative testing of six antigen-based malaria vaccine candidates directed toward merozoite-stage Plasmodium falciparum. Clin Vaccine Immunol 15 :1345-1355


Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-1(19) and five Pichia pastoris candidates, including two versions of MSP-1(19), AMA-1 (domains I and II), AMA-1+MSP-1(19), and fused AMA-1/MSP-1(19)). Animals were immunized with equimolar amounts of each antigen, formulated in Montanide ISA720. The specificities and titers of antibodies were compared using immunofluorescence assays and enzyme-linked immunosorbent assay (ELISA). The antiparasite activity of immunoglobulin G (IgG) in in vitro cultures was determined by growth inhibition assay, flow cytometry, lactate dehydrogenase assay, and microscopy. Baculovirus MSP-1(19) immunizations produced the highest parasite-specific antibody titers in immunofluorescence assays. In ELISAs, baculovirus-produced MSP-1(19) induced more antibodies than any other single MSP-1(19) immunogen and three times more MSP-1(19) specific antibodies than the AMA-1/MSP-1(19) fusion. Antibodies induced by baculovirus MSP-1(19) gave the highest levels of growth inhibition in HB3 and 3D7 parasite cultures, followed by AMA-1+MSP-1(19) and the AMA-1/MSP-1(19) fusion. With the FCR3 isolate (homologous to the AMA-1 construct), antibodies to the three AMA-1-containing candidates gave the highest levels of growth inhibition at high IgG concentrations, but antibodies to baculovirus MSP-1(19) inhibited as well or better at lower IgG concentrations. The two P. pastoris-produced MSP-1(19)-induced IgGs conferred the lowest growth inhibition. Comparative analysis of immunogenicity of vaccine antigens can be used to prioritize candidates before moving to expensive GMP production and clinical testing. The assays used have given discriminating readouts but it is not known whether any of them accurately reflect clinical protection.

Atapattu DN, Albrecht RM, McClenahan DJ, Czuprynski CJ (2008) Dynamin-2-dependent targeting of mannheimia haemolytica leukotoxin to mitochondrial cyclophilin D in bovine lymphoblastoid cells. Infect Immun 76 :5357-5365


Exotoxins which belong to the family containing the RTX toxins (repeats in toxin) contribute to a variety of important human and animal diseases. One example of such a toxin is the potent leukotoxin (LKT) produced by the bovine respiratory pathogen Mannheimia haemolytica. LKT binds to CD18, resulting in the death of bovine leukocytes. In this study, we showed that internalized LKT binds to the outer mitochondrial membrane, which results in the release of cytochrome c and collapse of the mitochondrial membrane potential (psi(m)). Incubation of bovine lymphoblastoid cells (BL-3 cells) with the mitochondrial membrane-stabilizing agent cyclosporine (CSA) reduced LKT-mediated cytotoxicity, cytochrome c release, and collapse of the psi(m). Coimmunoprecipitation and intracellular binding studies suggested that LKT binds to the mitochondrial matrix protein cyclophilin D. We also demonstrated that LKT mobilizes the vesicle scission protein dynamin-2 from mitochondria to the cell membrane. Incubation with CSA depleted mitochondrial dynamin-2 in BL-3 cells, making it unavailable for vesicle scission and LKT internalization. The results of this study show that LKT trafficking and LKT-mediated cell death involve dynamin-2 and cyclophilin D, in a process that can be prevented by the mitochondrial membrane-protecting function of CSA.

Bakdash N, Williams MS (2008) Spatially distinct production of reactive oxygen species regulates platelet activation. Free Radic Biol Med 45 :158-166


Platelets play a key role in hemostasis and changes in redox balance are known to alter platelet activation and aggregation. Interestingly, activation of platelets leads to production of reactive oxygen species (ROS), but the role(s) of these ROS remain unclear. Using flow cytometry and chemiluminescence, agonist-induced ROS generation was found to be spatially distinct with stimulation through the major collagen receptor GPVI inducing only intraplatelet ROS while thrombin induced production of extracellular ROS. Platelet activation by either the GPVI-selective agonist convulxin or thrombin was differentially regulated by ROS generation. Thus, surface expression of CD62P, CD40L, or activated integrin alphaIIbbeta3 was abrogated by pharmacologic antioxidants but externalization of phosphatidylserine was not inhibited. Furthermore, extracellular antioxidants SOD/catalase markedly inhibited thrombin-, but not convulxin-, induced CD62P expression and alphaIIbbeta3 activation. The data suggest that ROS selectively regulate biochemical steps in platelet activation and that distinct source(s) of ROS and discrete redox-sensitive pathway(s) may control platelet activation in response to GPVI or thrombin stimulation. Thus, targeting ROS with site-specific antioxidants may differentially regulate platelet activation via thrombin or collagen.

Ballesta S, Velasco C, Borobio MV, Arguelles F, Perea EJ (2008) [Fresh versus pasteurized yogurt : comparative study of the effects on microbiological and immunological parameters, and gastrointestinal comfort]. Enferm Infecc Microbiol Clin 26 :552-557


OBJECTIVE : To determine whether the beneficial effects of yogurt are dependent on the viability of lactic bacteria and exclusive to fresh yogurt, by comparison with the effects of yogurt that is pasteurized after fermentation. MATERIAL AND METHOD : Using a double-blind design in a healthy adult population over 75 days, we compared the effects of fresh and pasteurized yogurt on microbiological (presence of viable bacteria in yogurt and DNA detection in feces) and immunological (nephelometry, hematometry, and flow cytometry) parameters. A questionnaire was used to assess gastrointestinal comfort. Differences in lactose absorption after ingestion of fresh or pasteurized yogurt were determined by breath hydrogen analysis. RESULTS : There were no significant differences in the results obtained for microbiological or immunological parameters, gastrointestinal comfort, or lactose test between the two types of yogurt ingested. Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) was isolated in 0.7% of the fecal samples analyzed. Streptococcus thermophilus was not found in any sample. DNA from lactic bacteria was detected in only 12.5% of the samples analyzed. CONCLUSION : Transit through the gastrointestinal tract affects survival of L. bulgaricus and S. thermophilus. No differences were found in the immunological parameters, gastrointestinal comfort, or lactose overload after intake of fresh or pasteurized yogurt.

Bansal S, Lucia MS, Wiseman A (2008) A case of polyomavirus-associated nephropathy presenting late after transplantation. Nat Clin Pract Nephrol 4 :283-287


BACKGROUND : A 36-year-old white female, who had received a deceased-donor kidney transplant for end-stage renal disease secondary to reflux nephropathy 8 years previously, was referred to a transplant clinic for evaluation following an increase in her serum creatinine level from 123.8 micromol/l to 185.6 micromol/l (1.4 mg/dl to 2.1 mg/dl) over the preceding 9 months. Her immunosuppression regimen included mycophenolate mofetil, ciclosporin and prednisone, with ciclosporin trough levels ranging from 100 ng/ml to 150 ng/ml, as detected by fluorescence polarization immunoassay, over the preceding year. The following possible causes of subacute renal failure were ruled out : post-obstructive nephropathy, altered hemodynamics (hypotension and renal artery stenosis), and toxicity from medications other than calcineurin inhibitors. Potential etiologies such as acute T-cell-mediated rejection, acute and chronic antibody-mediated rejection, polyomavirus-associated nephropathy, and calcineurin inhibitor nephrotoxicity were considered. INVESTIGATIONS : Physical examination, urine and blood analysis, analysis of human leukocyte antigen antibodies by flow cytometry (Luminex, Luminex Corporation, Austin, TX), ultrasound of the transplanted kidney, polymerase chain reaction assay for the detection of BK virus in the serum, and biopsy of the transplanted kidney with staining for simian virus 40 antigen. DIAGNOSIS : Polyomavirus-associated nephropathy with advanced nephrosclerosis and moderate to marked hyaline arteriolosclerosis. MANAGEMENT : Reduction of immunosuppression by discontinuation of mycophenolate mofetil, dose reduction of ciclosporin, and initiation of leflunomide.

Barbier M, Oliver A, Rao J, Hanna SL, Goldberg JB, Alberti S (2008) Novel phosphorylcholine-containing protein of Pseudomonas aeruginosa chronic infection isolates interacts with airway epithelial cells. J Infect Dis 197 :465-473


Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP) epitope, a structure crucial for the virulence of several respiratory pathogens. In this study, ChoP expression analysis comparing organisms from acute and chronic infections revealed that expression of ChoP at 37 degrees C was higher among strains from chronic infections. Coimmunoprecipitation experiments and mass spectrometry analysis demonstrated that ChoP was on the protein elongation factor Tu. The presence of ChoP at the surface was confirmed by immunofluorescence and flow cytometry analysis of intact bacteria. Pretreatment of bronchial epithelial cells or mice with a platelet-activating factor receptor (PAFR) antagonist reduced adhesion and invasion of the ChoP-positive P. aeruginosa isolates. Results of this study suggest that ChoP expression may represent a novel phenotype expressed by the chronic infection isolates that could mediate P. aeruginosa colonization of the epithelial airway by means of the interaction with the PAFR.

Barbosa J, Costa-de-Oliveira S, Rodrigues AG, Pina-Vaz C (2008) Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia. Travel Med Infect Dis 6 :234-239


Giardia lamblia is responsible for causing diarrhoeal diseases in humans. Infection occurs by fecal-oral route and is considered an important water pathogen, since many water surfaces are infected by cysts. Most studies involve cyst concentration procedures, followed by conventional microscopy, but are often tedious and influenced by fatigue. Our main objective was to optimize a specific flow cytometric (FC) protocol for detection of G. lamblia, to establish its sensibility limit and also the cyst viability. G. lamblia cysts (Waterborne, Inc., USA) were used for protocol optimization. FC analysis was performed using cyst suspensions stained with serial concentrations of a fluorescein-labelled mouse monoclonal antibody (Giardia-a-Glo, Waterborne). Serial concentrations (2 x 10(5), 1 x 10(5), 2 x 10(4), 1 x 10(4), 2 x 10(3), 1 x 10(3), 2 x 10(2) and 1 x 10(2)cysts/ml) were stained with the optimized antibody concentration and analysed by FC. Specificity and sensibility limit of the method were established using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Dead cysts were stained with 5.0 microg/ml of propidium iodide (PI, Sigma), with and without the specific fluorescent antibody. As the antibody concentration decreased, a decline of peak intensity was registered ; 1.5 microg/ml was considered as the optimal antibody concentration, yielding a histogram clearly separated. We established a threshold of detection of 2 x 10(2)cysts/ml. Below threshold limit fluorescence was not enough to allow the discrimination of cysts. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. When using both antibody and PI, we could distinguish the viable cyst. With the use of specific antibodies, a distinct cellular population corresponding to cysts could be represented in the FC histogram.

Barbosa JM, Costa-de-Oliveira S, Rodrigues AG, Hanscheid T, Shapiro H, Pina-Vaz C (2008) A flow cytometric protocol for detection of Cryptosporidium spp. Cytometry A 73 :44-47


Cryptosporidium parvum is transmitted through water and can cause severe diarrhea. The diagnosis is usually based upon observer-dependent microscopic detection of oocysts, with rather low sensitivity and specificity. Our objective was to optimize a flow cytometric (FC) protocol for the detection of C. parvum. A specific monoclonal antibody conjugated with R-phycoerythrin was incubated with dead oocysts to determine the optimal antibody concentration. Serial concentrations of oocysts were stained with the optimized concentration and analyzed by FC. The lower detection limit was determined, and the possibility of cross-reaction was investigated using prokaryotic and eukaryotic microorganisms. A FC protocol was optimized to detect oocysts in spiked human stools. The optimal antibody concentration was found to be 3.0 mug/ml. The lowest number detectable was 2 x 10(3) oocysts/ml. Staining procedure was specific, as no cross-reactions were observed. This reliable and easy FC protocol allow the specific detection of Cryptosporidium oocysts, even at very low concentrations, which is important for public health and further studies of treatment efficacy.

Barnes RJ, Leung KT, Schraft H, Ulanova M (2008) Chromosomal gfp labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon : application for studies of bacteria-host interactions. Can J Microbiol 54 :48-57


Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.

Basu A, Jain P, Gangodkar SV, Shetty S, Ghosh K (2008) Dengue 2 virus inhibits in vitro megakaryocytic colony formation and induces apoptosis in thrombopoietin-inducible megakaryocytic differentiation from cord blood CD34+ cells. FEMS Immunol Med Microbiol 53 :46-51


Thrombocytopenia is frequently associated with dengue virus infection in humans. Although antiplatelet immunopathogenic processes have been implicated in the origin of dengue-associated thrombocytopenia, the effect of dengue viruses on megakaryocyte differentiation remains incompletely understood. In this study, we examined the effect of human dengue 2 virus isolates on the in vitro growth and differentiation of thrombopoietin-induced megakaryopoiesis of cord blood CD34+ cells. Dengue 2 viruses, but not Japanese encephalitis virus, showed a dose-dependent inhibition of CFU-Mk. Viral antigens could be detected by an immunohistochemical technique in 3-5% of the early megakaryocytic progenitors by the 5th postexposure day in liquid cultures with cell loss, increased annexin V binding and active caspase-3 expression. In summary, dengue 2 viruses can inhibit in vitro megakaryopoiesis, as well as infect and induce apoptotic cell death in a subpopulation of early megakaryocytic progenitors. These events might contribute towards the origin of thrombocytopenia in dengue disease.

Berney M, Vital M, Hulshoff I, Weilenmann HU, Egli T, Hammes F (2008) Rapid, cultivation-independent assessment of microbial viability in drinking water. Water Res 42 :4010-4018


Fast and accurate monitoring of chemical and microbiological parameters in drinking water is essential to safeguard the consumer and to improve the understanding of treatment and distribution systems. However, most water utilities and drinking water guidelines still rely solely on time-requiring heterotrophic plate counts (HPC) and plating for faecal indicator bacteria as regular microbiological control parameters. The recent development of relative simple bench-top flow cytometers has made rapid and quantitative analysis of cultivation-independent microbial parameters more feasible than ever before. Here we present a study using a combination of cultivation-independent methods including fluorescence staining (for membrane integrity, membrane potential and esterase activity) combined with flow cytometry and total adenosine tri-phosphate (ATP) measurements, to assess microbial viability in drinking water. We have applied the methods to different drinking water samples including non-chlorinated household tap water, untreated natural spring water, and commercially available bottled water. We conclude that the esterase-positive cell fraction, the total ATP values and the high nucleic acid (HNA) bacterial fraction (from SYBR Green I staining) were most representative of the active/viable population in all of the water samples. These rapid methods present an alternative way to assess the general microbial quality of drinking water as well as specific events that can occur during treatment and distribution, with equal application possibilities in research and routine analysis.

Bhide M, Yilmaz Z, Golcu E, Torun S, Mikula I (2008) Seroprevalence of anti-Borrelia burgdorferi antibodies in dogs and horses in Turkey. Ann Agric Environ Med 15 :85-90


The aim of the study was to determine the seroprevalence of anti-Borrelia burgdorferi antibodies in a population of Turkish dogs and horses, as well as to compare the sensitivity of novel flow-cytometry-based borreliacidal antibody test (BAT) with ELISA assay. Serum samples collected from 400 dogs and 300 horses were tested with enzyme-linked protein A/G assay (ELPAGA), using Borrelia whole cell antigens. ELPAGA test showed 93 dogs (23.2%) and 18 horses (6%) serologically positive for anti-Borrelia antibodies. In parallel testing of sera with BAT, we found 27.75% positive dogs and 6.33% positive horses. When the results of these serological testes were compared with the health status of the animals, the most common clinical signs noticed in dogs were skin manifestations, urinary tract disorder and anemia ; however, no clinical symptoms were observed in horses positive for the anti-Borrelia antibodies. This is a first time that seroprevalence of Lyme disease in dogs and horses has been reported from Turkey, as well as the use of novel BAT in animals.

Brennan LJ, Keddie BA, Braig HR, Harris HL (2008) The endosymbiont Wolbachia pipientis induces the expression of host antioxidant proteins in an Aedes albopictus cell line. PLoS ONE 3 :e2083


Wolbachia are obligate intracellular bacteria which commonly infect arthropods. They are maternally inherited and capable of altering host development, sex determination, and reproduction. Reproductive manipulations include feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility. The mechanism by which Wolbachia avoid destruction by the host immune response is unknown. Generation of antimicrobial peptides (AMPs) and reactive oxygen species (ROS) by the host are among the first lines of traditional antimicrobial defense. Previous work shows no link between a Wolbachia infection and the induction of AMPs. Here we compare the expression of protein in a cell line naturally infected with Wolbachia and an identical cell line cured of the infection through the use of antibiotics. Protein extracts of each cell line were analyzed by two dimensional gel electrophoresis and LC/MS/MS. Our results show the upregulation of host antioxidant proteins, which are active against ROS generated by aerobic cell metabolism and during an immune response. Furthermore, flow cytometric and microscopic analysis demonstrates that ROS production is significantly greater in Wolbachia-infected mosquito cells and is associated with endosymbiont-containing vacuoles located in the host cell cytoplasm. This is the first empirical data supporting an association between Wolbachia and the insect antioxidant system.

Brouwer N, Dolman KM, van Houdt M, Sta M, Roos D, Kuijpers TW (2008) Mannose-binding lectin (MBL) facilitates opsonophagocytosis of yeasts but not of bacteria despite MBL binding. J Immunol 180 :4124-4132


Mannose-binding lectin (MBL) is a serum protein of the innate immune system. After binding to a microorganism, MBL in complex with MBL-associated serine proteases activates the complement system, resulting in cleavage of complement factor C3. Cleaved C3 on the surface of the microorganism mediates opsonization for clearance, but the impact of MBL on subsequent phagocytosis has not been widely studied. We investigated the role of MBL in complement activation and phagocytosis of various bacteria and yeast species by flow cytometry. We measured both the C3 deposition during serum opsonization of fluorescent-labeled microorganisms as well as subsequent uptake of these microorganisms by human neutrophils. In MBL-deficient sera, a consistently decreased C3 deposition on both zymosan and Candida albicans was found and a reduced phagocytosis by neutrophils that was restored by exogenous MBL. This indicates that the lectin pathway of complement activation is important for the opsonophagocytosis of yeasts. In contrast, the C1q-dependent classical pathway dominated in the opsonization and phagocytosis of Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, whereas no effect of MBL was found. Both the lectin and the classical pathway of complement activation were highly amplified by the alternative route for opsonophagocytosis by neutrophils of yeast as well as microbial species. In summary, our data demonstrate that yeast species are preferentially opsonized and subsequently phagocytosed via activation of the lectin pathway of complement, whereas the uptake of bacterial strains was found to be largely MBL independent.

Cai H, Jiao N (2008) Diversity and abundance of nitrate assimilation genes in the northern South china sea. Microb Ecol 56 :751-764


Marine heterotrophic microorganisms that assimilate nitrate play an important role in nitrogen and carbon cycling in the water column. The nasA gene, encoding the nitrate assimilation enzyme, was selected as a functional marker to examine the nitrate assimilation community in the South China Sea (SCS). PCR amplification, restriction fragment length polymorphism (RFLP) screening, and phylogenetic analysis of nasA gene sequences were performed to characterize in situ nitrate assimilatory bacteria. Furthermore, the effects of nutrients and other environmental factors on the genetic heterogeneity of nasA fragments from the SCS were evaluated at the surface in three stations, and at two other depths in one of these stations. The diversity indices and rarefaction curves indicated that the nasA gene was more diverse in offshore waters than in the Pearl River estuary. The phylotype rank abundance curve showed an abundant and unique RFLP pattern in all five libraries, indicating that a high diversity but low abundance of nasA existed in the study areas. Phylogenetic analysis of environmental nasA gene sequences further revealed that the nasA gene fragments came from several common aquatic microbial groups, including the Proteobacteria, Cytophaga-Flavobacteria (CF), and Cyanobacteria. In addition to the direct PCR/sequence analysis of environmental samples, we also cultured a number of nitrate assimilatory bacteria isolated from the field. Comparison of nasA genes from these isolates and from the field samples indicated the existence of horizontal nasA gene transfer. Application of real-time quantitative PCR to these nasA genes revealed a great variation in their abundance at different investigation sites and water depths.

Cai K, Wang H, Bao S, Shi J, Hou X, Gao X, Liu H, Yin J (2008) Novel human 3-domain disulfide-stabilized antibody fragment against glycoprotein of rabies virus. Microbes Infect 10 :548-555


Mutated disulfide bond sites VH (Cys44) and VL (Cys100) were constructed in variable domains (Fvs) of the human anti-glycoprotein antigen of the rabies virus (anti-GPRV), and the light chain variable (VL) and heavy chain variable (VH) fragments were linked using the heavy chain constant region 1 (CH1) of the human immunoglobulin (Ig) to successfully construct a 3-domain disulfide-stabilized fragment of variables (3d-dsFv). 3d-dsFv was mainly expressed as an inclusion body. After refolding by the conventional dilution method, 3d-dsFv was purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. Enyzme-linked immunosorbent assay (ELISA) was used to determine the binding activity of 3d-dsFv to GPRV. Flow cytometry studies and rapid fluorescent focus inhibition test were used to evaluate the function of 3d-dsFv. The results showed that the stability of 3d-dsFv was improved notably in some aspects such as thermal kinetics, ability to withstand urea denaturation, etc. 3d-dsFv could bind specially to infective cells and the GPRV. The titration of 3d-dsFv to RV-CVS is 83.3 IU/mg, and it can easily reach 2.5IU/mL, which is the value suggested by the WHO as effective for neutralization titration of the rabies virus.

Cao J, Chen T, Li D, Wong CK, Chen D, Xu W, Zhang X, Lam CW, Yin Y (2008) Mucosal immunization with purified ClpP could elicit protective efficacy against pneumococcal pneumonia and sepsis in mice. Microbes Infect 10 :1536-1542


Caseinolytic protease (ClpP) has been found to be highly conserved among different strains of Streptococcus pneumoniae and intraperitoneal immunization with ClpP could elicit protection against invasive pneumococcal infections. In this study, mucosal immunization with ClpP antigen induced both systemic and mucosal antibodies, and in this way reduced lung colonization in an invasive pneumococcal pneumonia model and also protected mice against death in an intraperitoneal-sepsis model. Surface localization of ClpP was confirmed using flow cytometry analysis. Furthermore, characterization of human sera for anti-ClpP IgG antibody levels demonstrated that ClpP protein was immunogenic in healthy children and was expressed during disease based on the elevated antibody levels in infected individuals. Finally, we describe that in vitro functional anti-ClpP antibody could kill streptococcus pneumoniae by polymorphonuclear leukocytes in a complement-dependent assay. To our knowledge, this is the first study about the protective efficacy of mucosal immunization with ClpP as a promising pneumococcal protein antigen.

Chang HC, Peng YT, Chang HL, Chaung HC, Chung WB (2008) Phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus. Vet Microbiol 129 :281-293


It is well documented that there is a delay in the development of effective immunity to porcine reproductive and respiratory syndrome virus (PRRSV) in infected and vaccinated pigs. This suggests that PRRSV might possess some inherent properties to evade host defense mechanisms during the early stage of infection. Dendritic cells (DCs) play a crucial role in the activation and control of T-cells in response to viral antigens. In this study, we investigated the phenotypic and functional property changes of bone marrow-derived immature DCs (BM-imDCs) that take place after infection by PRRSV. Results showed that BM-imDCs were permissive to PRRSV infection, as productive replication took place in these cells. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 is observed at 48 h following infection. Also at 48h following PRRSV infection, a significant increase of IL-10 secretion by BM-imDCs was noticed. Results suggest that the inhibited expression of MHC I and the enhanced secretion of IL-10 by BM-imDCs after PRRSV infection might be among the strategies used by the virus to evade the host immune defenses.

Chapman-McQuiston E, Wu XL (2008) Stochastic receptor expression allows sensitive bacteria to evade phage attack. Part I : experiments. Biophys J 94 :4525-4536


It has long been suspected that population heterogeneity, either at a genetic level or at a protein level, can improve the fitness of an organism under a variety of environmental stresses. However, quantitative measurements to substantiate such a hypothesis turn out to be rather difficult and have rarely been performed. Herein, we examine the effect of expression heterogeneity of lambda-phage receptors on the response of an Escherichia coli population to attack by a high concentration of lambda-phage. The distribution of the phage receptors in the population was characterized by flow cytometry, and the same bacterial population was then subjected to different phage pressures. We show that a minority population of bacteria that produces the receptor slowly and at low levels determines the long-term survivability of the bacterial population and that phage-resistant mutants can be efficiently isolated only when the persistent phage pressure >10(10) viruses/cm(3) is present. Below this phage pressure, persistors instead of mutants are dominant in the population.

Chen ZY, Liu H, Li ZQ, Zhang QY (2008) Development and characterization of monoclonal antibodies to spring viraemia of carp virus. Vet Immunol Immunopathol 123 :266-276


Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Western blotting analysis revealed that three mAbs (1H5, 1E10, and 1H7) recognized specifically to a single protein of 47kDa (N), the mAb 3G4 reacted with two SVCV0504 proteins of 69kDa (G) and 47kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69kDa (G), 50kDa (P), and 47kDa (N). By indirect ELISA, two mAbs (1H5 and 1H7) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID(50) (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28h after inoculation with the virus (0.01PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus.

Cheng TP, French AR, Plougastel BF, Pingel JT, Orihuela MM, Buller ML, Yokoyama WM (2008) Ly49h is necessary for genetic resistance to murine cytomegalovirus. Immunogenetics 60 :565-573


Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.

Chessa D, Dorsey CW, Winter M, Baumler AJ (2008) Binding specificity of Salmonella plasmid-encoded fimbriae assessed by glycomics. J Biol Chem 283 :8118-8124


The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome encodes 12 intestinal colonization factors of the chaperone/usher fimbrial assembly class ; however, the binding specificity is known for only one of these adhesins, known as type 1 fimbriae. Here we explored the utility of glycomics to determine the carbohydrate binding specificity of plasmid-encoded fimbriae from S. Typhimurium. A cosmid carrying the pef operon was introduced into Escherichia coli and expression of fimbrial filaments composed of PefA confirmed by flow cytometry and immune-electron microscopy. Plasmid-encoded fimbriae were purified from the surface of E. coli, and the resulting preparation was shown to contain PefA as the sole major protein component. The binding of purified plasmid-encoded fimbriae to a glycanarray suggested that this adhesin specifically binds the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc, also known as the Lewis X (Le(x)) blood group antigen. Results from the glycanarray were validated by enzyme-linked immunosorbent assay (ELISA) in which plasmid-encoded fimbriae bound Le(x)-coated wells in a concentration-dependent manner. The binding of plasmid-encoded fimbriae to Le(x)-coated wells could be inhibited by co-incubation with soluble Le(x) antigen. Our results establish glycomic analysis as a promising new approach for determining the carbohydrate binding specificity of bacterial adhesins.

Collado MC, Isolauri E, Laitinen K, Salminen S (2008) Distinct composition of gut microbiota during pregnancy in overweight and normal-weight women. Am J Clin Nutr 88 :894-899


BACKGROUND : Results of experimental studies suggest that deviations in gut microbiota composition predispose to excessive energy storage and obesity. The mother influences the original inoculum and the development of infant microbiota, which in turn is associated with later weight gain. OBJECTIVE : We characterized the gut microbiota in women according to their body mass index (BMI) and the effect of weight gain over pregnancy on the composition of microbiota before delivery. DESIGN : Overweight women (n = 18) were selected according to their prepregnancy BMI from a prospective follow-up study. Normal-weight women (n = 36) were selected as controls in consecutive order of recruitment. Excessive weight gain during pregnancy was defined as >16.0 kg for normal-weight and >11.5 kg for overweight states according to Institute of Medicine recommendations. The composition of gut microbiota was analyzed by fluorescent in situ hybridization coupled with flow cytometry (FCM-FISH) and by quantitative real-time polymerase chain reaction (qPCR). RESULTS : Bacteroides and Staphylococcus were significantly higher in the overweight state than in normal-weight women as assessed by FCM-FISH and qPCR. Mother’s weight and BMI before pregnancy correlated with higher concentrations of Bacteroides, Clostridium, and Staphylococcus. Microbial counts increased from the first to third trimester of pregnancy. High Bacteroides concentrations were associated with excessive weight gain over pregnancy (P = 0.014). CONCLUSIONS : Gut microbiota composition and weight are linked, and mother’s weight gain is affected by microbiota. Microbiota modification before and during pregnancy may offer new directions for preventive and therapeutic applications in reducing the risk of overweight and obesity.

Costa-de-Oliveira S, Araujo R, Silva-Dias A, Pina-Vaz C, Rodrigues AG (2008) Propofol lipidic infusion promotes resistance to antifungals by reducing drug input into the fungal cell. BMC Microbiol 8 :9


BACKGROUND : The administration of non-antifungal drugs during patient hospitalization might be responsible for discrepancies between in vitro and in vivo susceptibility to antifungals. Propofol is often administered to intensive care units as a sedative.The purpose of this study was to evaluate the effect of propofol lipidic infusion upon the growth and susceptibility profile of pathogenic fungi.Candida and Aspergillus were studied regarding the ability to grow and its susceptibility profile to antifungals in the presence of propofol infusion (Fresenius(R)) (1.25, 2.5 and 5 mg.ml-1) and its lipidic vehicle. The intensity of fluorescence after staining with FUN1, in the presence and absence of propofol infusion, was determined by flow cytometry. Radioactivity assays were also performed in order to quantify the input of [3H]- itraconazole into the fungal cell in the presence of propofol. Assays were repeated after addition of sodium azide, in order to block efflux pumps. RESULTS : Propofol infusion promoted budding of Candida and the germination of Aspergillus, latter forming a lipid layer around the hypha. An increase of minimal fungicidal concentrations regarding both Candida and Aspergillus strains was found for all antifungals when incubated simultaneously with propofol infusion. A decrease of the intensity of fluorescence of Candida cells was systematically observed, as well as a significant reduced intracellular uptake of [3H] itraconazole in cells treated with propofol infusion, even after the blockade of efflux pumps. The results obtained when testing with the lipid vehicle were similar. CONCLUSION : Propofol infusion, due to its lipidic vehicle, increased the fungal germination and promoted resistance to antifungals. This effect seems to be related to the reduced access and/or permeabilization to fungal cells by antifungals.

Cronin UP, Wilkinson MG (2008) Bacillus cereus endospores exhibit a heterogeneous response to heat treatment and low-temperature storage. Food Microbiol 25 :235-243


Bacillus cereus endospores were challenged by heat treatments simulating typical domestic/industrial cooking regimes and the resulting effects on germination, viability and sub-lethal heat damage determined using differential plate counting on a rich versus selective medium, flow cytometry (FCM), beta-D-glucuronidase (GUD) activity and OD(600) measurement. Additionally, these techniques were used to investigate the effect on endospores of storage in a non-nutrient medium at 4 degrees C for 1 month. Plate counting revealed that heating generated sub-populations of sub-lethally damaged endospores, with the more severe heat treatments generating larger proportions of sub-lethally damaged endospores. These findings were also reflected in FCM analyses, which detected large amounts of heterogeneity among the populations of heat-treated endospores and uncovered differences in the proportions of membrane-damaged endospores and those displaying esterase activity pre- and post-treatment. Plate count data suggested that both the control and heat-treated endospores lost viability during storage, with FCM data indicating that the proportion of membrane-damaged endospores increased and those displaying the esterase activity decreased. The FCM, GUD and OD(600) data suggested that germination rates decreased with the increasing severity of heat treatment. This study demonstrates that a combination of plate counting and FCM can be used to detect heterogeneity in the response of endospores to insults.

Cronin UP, Wilkinson MG (2008) Physiological response of Bacillus cereus vegetative cells to simulated food processing treatments. J Food Prot 71 :2168-2176


Vegetative cells of the spore-former Bacillus cereus were exposed to a number of treatments commonly used in commercial food preparation or during equipment cleaning and decontamination. Treated suspensions were then analyzed for reductions (CFU per milliliter) by plate counting and changes in levels of ATP and ADP released from cells with a bioluminescence-based assay. With the use of flow cytometry (FCM), the physiological status of individual cells before and after exposure to treatments was determined by staining of control and treated cells with three pairs of physiological dyes (SYTO 9/propidium iodide, carboxyfluorescein diacetate/Hoechst 33342, and C12-resazurin/SYTOX Green). Good agreement was found between plate counting and FCM. In general, treatments giving rise to the highest count reductions also had the greatest effects on cell membrane permeability (measured with the use of propidium iodide or SYTOX Green), esterase activity (measured with carboxyfluorescein diacetate), or redox activity (C12-resazurin). FCM data demonstrated the extent of heterogeneity of vegetative cell responses to treatments in, for example, the treatment with 5% H2O2, which caused a 6-log reduction in which approximately 95% of the population was composed of membrane-damaged cells (as reflected by their permeability to SYTOX Green), whereas in treatment with 0.09% (wt/vol) potassium sorbate, which caused only a 1-log reduction, not more than 40% of cells were membrane damaged. The approaches described in this work can be applied to gain a greater understanding of bacterial responses to food control measures, generate more accurate inactivation models, or screen novel prospective food control measures.

Czechowska K, Johnson DR, van der Meer JR (2008) Use of flow cytometric methods for single-cell analysis in environmental microbiology. Curr Opin Microbiol 11 :205-212


Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays.

Das S, Basu A (2008) Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation. J Neurochem 106 :1624-1636


Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.

Dassanayake RP, Liu W, Davis WC, Foreyt WJ, Srikumaran S (2008) Bighorn sheep beta2-integrin LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin. J Wildl Dis 44 :743-747


Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS ; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.

de Werra P, Baehler E, Huser A, Keel C, Maurhofer M (2008) Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry. Appl Environ Microbiol 74 :1339-1349


The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Delaporte M, McKenna P, Siah A, Berthe FC (2008) Immunophenotyping of Mya arenaria neoplastic hemocytes using propidium iodide and a specific monoclonal antibody by flow cytometry. J Invertebr Pathol 99 :120-122


Disseminated neoplasia (DN) is a disorder referred to as hemic neoplasia (HN) in the soft-shell clam Mya arenaria. Traditionally, diagnosis is performed by hematocytology or histology. The intensity of the disease is generally given as the percentage of transformed neoplastic cells out of total number of hemocytes. Flow cytometry techniques have found a field of application in diagnosis of HN with analysis of ploidy. Hemocytes of the soft-shell clams with HN display tetraploid DNA content, as shown by propidium iodide staining. This feature makes difficult HN diagnosis in the soft-shell clam, especially for early stages of the condition, since the percentage of normal circulating cells undergoing mitosis, which also are tetraploid, remains unknown in molluscs. Use of specific monoclonal antibodies in a flow cytometry assay was foreseen as a way to overcome the difficulty. The purpose of this study was to develop a double staining protocol using propidium iodide for hemocyte cycle analysis and the MAb 1E10 for staining of HN cells. Our results showed a correlation between tetraploid and MAb 1E10-stained hemocytes in a single clam with moderate HN. This protocol offers some potential for further investigation of this cell disorder. However, a validation step will be necessary to confirm our preliminary results.

Delaporte M, Synard S, Pariseau J, McKenna P, Tremblay R, Davidson J, Berthe FC (2008) Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry. J Invertebr Pathol 98 :190-197


Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase ; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.

Derrien M, Collado MC, Ben-Amor K, Salminen S, de Vos WM (2008) The Mucin degrader Akkermansia muciniphila is an abundant resident of the human intestinal tract. Appl Environ Microbiol 74 :1646-1648


A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract.

Deshpande A, Wheeler CM, Hunt WC, Peyton CL, White PS, Valdez YE, Nolan JP (2008) Variation in HLA class I antigen-processing genes and susceptibility to human papillomavirus type 16-associated cervical cancer. J Infect Dis 197 :371-381


BACKGROUND : Persistent infection with human papillomavirus type 16 (HPV16) is a primary etiological factor for the development of cervical cancer. Genes involved in antigen processing influence both the repertoire of antigens presented by HPV16-infected cells and the nature of HPV16-specific immune responses. Genetic variation in these genes may affect protein structure and function and, consequently, the ability of an individual to clear HPV infection. METHODS : Thirty-five single-nucleotide polymorphisms (SNPs) in 5 genes (LMP2, TAP1, LMP7, TAP2, and Tapasin) were investigated for association with susceptibility to HPV16-associated cervical cancer. Sequencing of these genes resulted in the discovery of 15 previously unreported SNPs. Microsphere-array flow cytometry-based genotyping was conducted on 787 samples from Hispanic and non-Hispanic white women (241 randomly selected control subjects, 205 HPV16-positive control subjects, and 341 HPV16-positive case subjects with cervical cancer). RESULTS : For 9 SNPs, 8 of which had not previously been reported in the context of cervical cancer, there were statistically significant differences between the genotype distribution in case subjects and that in control subjects. Haplotype analysis of 3 haplotype blocks revealed 3 haplotypes with significant differences in frequency in case-control comparisons. Both HPV16-specific and non-type-specific differences in genotype distribution were seen. CONCLUSIONS : Genes involved in antigen processing for HLA class I presentation may contribute to susceptibility to cervical cancer.

Dixon BR, Parrington LJ, Parenteau M, Leclair D, Santin M, Fayer R (2008) Giardia duodenalis and Cryptosporidium spp. in the intestinal contents of ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) in Nunavik, Quebec, Canada. J Parasitol 94 :1161-1163


The prevalence of Giardia duodenalis and Cryptosporidium spp. was determined for ringed and bearded seals harvested for food in the Nunavik region in northern Quebec, Canada. Flow cytometric results demonstrated that G. duodenalis was present in the intestinal contents of 80% of the ringed seals and 75% of the bearded seals tested, while Cryptosporidium spp. were present in 9% of the ringed seals and none of the bearded seals. Prevalence of both parasites was highest in animals less than 1 yr of age. Giardia sp. isolates from ringed seals were identified as G. duodenalis Assemblage B, which is commonly identified in human infections. The high prevalence of G. duodenalis in ringed seals, and the presence of Assemblage B in these animals, highlights the potential for zoonotic transmission to the Inuit people, who consume dried seal intestines and uncooked seal meat.

Dong C, Weng S, Shi X, Xu X, Shi N, He J (2008) Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV). Virus Res 135 :273-281


Infectious spleen and kidney necrosis virus (ISKNV) is a typical species of the genus Megalocytivirus in the family Iridoviridae. However, until recently, no suitable piscine cell line is stably susceptible to ISKNV. Here, a continuous cell culture derived from the mandarin fish fry (MFF-1) was developed and has been subcultured over 60 passages during a period of 18 months. MFF-1 consists predominantly of epithelial-like cells and grows well in DMEM supplemented with 10% fetal bovine serum. MFF-1 could produce high titers of ISKNV by continuous viral passages which were further confirmed by indirect immunofluorescence assay and RT-PCR analysis. Flow cytometry analyse showed that approximately 80.3% cells could be infected by ISKNV at 3 days post-infection. Abundant ISKNV particles were observed in the cytoplasm of the ISKNV infected MFF-1 cells by transmission electron microscopy. Mandarin fish injected with the filtrate from the infected cell suspension developed clinical signs and died, which is in accordance with the infectious spleen and kidney necrosis virus disease (ISKNVD). In addition, apoptosis was observed in the MFF-1 cells upon ISKNV infection by FITC-annexin V staining. ISKNV was purified and the viral protein profiles were also determined in this research. To our knowledge, MFF-1 is the first cell line originated from mandarin fish and it can be an efficient tool for the study of ISKNV.

Duhamel S, Gregori G, Van Wambeke F, Mauriac R, Nedoma J (2008) A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry. J Microbiol Methods 75 :269-278


It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g ; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4’,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.

Enevold A, Lusingu JP, Mmbando B, Alifrangis M, Lemnge MM, Bygbjerg IC, Theander TG, Vestergaard LS (2008) Reduced risk of uncomplicated malaria episodes in children with alpha+-thalassemia in northeastern Tanzania. Am J Trop Med Hyg 78 :714-720


The prevalence of human red blood cell (RBC) polymorphisms is high in areas of intense Plasmodium falciparum transmission, and individuals carrying these genetic traits are believed to be partially protected against severe malaria. However, it remains uncertain how RBC polymorphisms affect the susceptibility to uncomplicated malaria. We compared the risk of suffering from febrile, uncomplicated malaria between individuals carrying three common RBC polymorphisms (sickle cell trait, alpha(+)-thalassemia, and glucose-6-phosphate-dehydrogenase deficiency) and controls. The study was performed in an area of intense malaria transmission where 202 individuals 0-19 years of age were monitored clinically for a period of 6 months. RBC polymorphisms were assessed with molecular methods, and plasma antibodies to P. falciparum variant surface antigens (anti-VSA IgG) and glutamate-rich protein (anti-GLURP IgG) were measured with flow cytometry and ELISA assays, respectively. Regression analyses showed that alpha(+)-thalassemia was associated with a reduced risk of uncomplicated malaria episodes and that this advantageous effect seemed to be more predominant in children older than 5 years of age, but was independent of levels of antibodies to VSA and GLURP.

Falcioni T, Papa S, Gasol JM (2008) Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl Environ Microbiol 74 :1767-1779


Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Gregori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability : active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 microg ml(-1) of propidium iodide, simultaneous to a 10x concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.

Fidan I, Bozdayi G, Rota S, Biri A, Gurelik FC, Yuksel S, Imir T (2008) The relationship between cervical human papillomavirus infection and apoptosis. Clin Invest Med 31 :E168-175


PURPOSE : Cervical carcinoma is the second most common cancer among women worldwide. Viral infections, especially human papillomavirus (HPV) infections, are important factors in its etiology. Changes in apoptotic regulation are considered to have an important role in the carcinogenesis development. In this study, the relationship between apoptosis and HPV infection was investigated. METHODS : HPV DNA and HPV DNA type 16 positivity were detected in 110 cervical smear samples with Real Time PCR and sequencing was performed for HPV DNA type 18. The presence of apoptosis was investigated using TUNEL and Annexin V staining methods and analyzed by fluorescence microscope and flow cytometry. RESULTS : HPV DNA type 16 was detected in 9 samples (8.1%), HPV DNA type 18 positive in 6 samples (5.4%) and HPV types other than HPV type 16 and HPV type 18 in 9 samples (8.1%). A decrease apoptosis was found in HPV DNA positive samples compared with controls (P < 0.05). CONCLUSION : The decrease of apoptosis during HPV infection might cause cellular immortality and then malignant transformation.

Flori L, Rogel-Gaillard C, Cochet M, Lemonnier G, Hugot K, Chardon P, Robin S, Lefevre F (2008) Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection. BMC Genomics 9 :123


BACKGROUND : Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry. RESULTS : An increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection. CONCLUSION : Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

Folkesson A, Haagensen JA, Zampaloni C, Sternberg C, Molin S (2008) Biofilm induced tolerance towards antimicrobial peptides. PLoS ONE 3 :e1891


Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.

Foresta C, De Toni L, Di Mambro A, Ferlin A, Perilli L, Bertuzzi I, Galan A, Zuccarello D (2008) Role of estrogen receptors in menstrual cycle-related neoangiogenesis and their influence on endothelial progenitor cell physiology. Fertil Steril

OBJECTIVE : To study whether estrogen receptors (ERs) are expressed in vitro and in vivo by female circulating endothelial progenitor cells (EPCs) ; and the role of ERs in the periodic vascular damage and repair that occurs during the menstrual cycle. DESIGN : Quantification of circulating progenitor cells, EPCs, and relative CXCR4+ fraction by flow cytometry. Quantification of plasma 17beta-E(2) by electrochemiluminescent immunoassay. Expression of ERs by immunofluorescence and immunohistochemistry. Estrogen receptor, CXCR4, and matrix metalloproteinase 9 gene expression by reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction. SETTING : University clinic and academic research laboratory. PATIENT(S) : Twelve young fertile women (aged 22-27 years) observed for 6 months, 10 postmenopausal women (aged 52-63 years), and 50 male control subjects (aged 24-61 years). INTERVENTION(S) : Blood (35 mL) was collected at each observation point. MAIN OUTCOME MEASURE(S) : Correlation between 17beta-E(2) exposure and neoangiogenesis markers. RESULT(S) : Estrogen receptors are expressed both in cultured EPCs after prolonged estrogen stimulation and in circulating EPCs, such as in CD34+ cells in bone marrow. The number of ER-beta+ and CXCR4+ EPCs increased during the ovulatory phase, and this increase is probably mediated by ER-beta and matrix metalloproteinase 9. CONCLUSION(S) : Estrogens play a key role in neoangiogenesis processes, such as endometrium recovery, and this mechanism involves both a central action (on bone marrow) and a cytokine-mediated peripheral one (on endothelium).

Frigimelica E, Bartolini E, Galli G, Grandi G, Grifantini R (2008) Identification of 2 hypothetical genes involved in Neisseria meningitidis cathelicidin resistance. J Infect Dis 197 :1124-1132


Cathelicidins play a pivotal role in innate immunity, providing a first barrier against bacterial infections at both mucosal and systemic sites. In this work, we have investigated the mechanisms by which Neisseria meningitidis serogroup B (MenB) survives at the physiological concentrations of human and mouse cathelicidin LL37 and CRAMP, respectively. By analyzing the global transcription profile of MenB in the presence or absence of CRAMP, 21 genes were found to be differentially expressed. Among these genes, the hypothetical genes NMB0741 and NMB1828 were up-regulated. When either of the 2 genes was deleted, MenB resistance to cathelicidins was impaired in vitro. Furthermore, the deletion of either of the 2 genes substantially reduced MenB virulence, as measured by the number of bacteria found in the blood of infected animals. Altogether, these results indicate that NMB0741 and NMB1828 are novel genes that have never been described before and that are involved in MenB cathelicidin resistance.

Fukiya S, Yokota A (2008) [FISH-flow cytometry, a new tool for the analysis of intestinal microbiota]. Seikagaku 80 :421-425


Gerard P, Brezillon C, Quere F, Salmon A, Rabot S (2008) Characterization of cecal microbiota and response to an orally administered lactobacillus probiotic strain in the broiler chicken. J Mol Microbiol Biotechnol 14 :115-122


A probiotic Lactobacillus strain was given in drinking water to young broiler chickens from 1 to 19 days of age. Cecal contents were collected from 4- and 19-day-old chickens in treated and control groups. Enumeration of bacteria by culture on selective media showed a decrease in Clostridium perfringens carriage in the 4-day-old treated chickens, whereas coliforms and Lactobacillus populations were not significantly affected by the treatment. Fluorescent in situ hybridization analysis with 7 phylogenetic probes targeting the major groups of intestinal bacteria revealed that the Clostridium coccoides group accounted for more than 50% of the total bacteria in the cecum of 4-day-old chickens, whereas the bacterial community of 19-day-old chickens evolved towards a more diverse microbiota with Faecalibacterium prausnitzii (36%) and C. coccoides (22%) groups representing the predominant bacteria. No effect of the Lactobacillus strain supplementation was observed in the composition of the cecal microbiota assessed by fluorescent in situ hybridization with the 7 probes. Nevertheless, profiling of the cecal microbiota using temporal temperature gradient gel electrophoresis in combination with principal component analysis demonstrated an impact of the probiotic treatment on the overall bacterial community as well as on the Lactobacillus population.

Gonen E, Nedvetzki S, Naor D, Shpigel NY (2008) CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium. Vet Res 39 :29


Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals.Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.

Grimberg BT, Erickson JJ, Sramkoski RM, Jacobberger JW, Zimmerman PA (2008) Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy. Cytometry A 73 :546-554


The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite’s life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy.

Grisafi D, Piccoli M, Pozzobon M, Ditadi A, Zaramella P, Chiandetti L, Zanon GF, Atala A, Zacchello F, Scarpa M, De Coppi P, Tomanin R (2008) High transduction efficiency of human amniotic fluid stem cells mediated by adenovirus vectors. Stem Cells Dev 17 :953-962


In the last few years some studies have shown the possibility of deriving progenitors with various potential from the amniotic fluid. Amniocentesis is a widely accepted method for prenatal diagnosis ; it is associated with low risk both for the mother and the fetus and overcomes the ethical problems commonly associated to other sources. Recently we have described that amniotic fluid stem (AFS) cells, for their ability to differentiate to various lineages, could represent a good candidate for therapeutic applications. For gene therapy purposes human AFS (hAFS) cells should be genetically modified with a therapeutic gene and delivered systematically or injected directly into the tissue of interest. The aim of this study was to investigate the feasibility of transducing hAFS cells with adenoviral vectors and to determine whether transduced stem cells retain the ability to differentiate into different lineages. Herein, we showed that hAFS cells could be efficiently infected by first generation adenovirus vectors. In addition, we demonstrated that infection and expression of two different marker genes, LacZ and EGFP, have no effect on cells phenotype and differentiation potential. In particular, on undifferentiated status, hAFS cells continued to express both the transgenes and stemness cell markers OCT4 and SSEA4. When cultured under mesenchymal conditions, infected cells could still differentiate into osteocytes and adipocytes expressing lineage specific genes. These preliminary findings suggest that adenovirus may be useful to engineer populations of pluripotent stem cells, which may be used in a wide range of gene therapy treatments.

Gunther S, Hubschmann T, Rudolf M, Eschenhagen M, Roske I, Harms H, Muller S (2008) Fixation procedures for flow cytometric analysis of environmental bacteria. J Microbiol Methods 75 :127-134


Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.

Hahn MA, Keng PC, Krauss TD (2008) Flow cytometric analysis to detect pathogens in bacterial cell mixtures using semiconductor quantum dots. Anal Chem 80 :864-872


Compared to a common green organic dye, semiconductor quantum dots (QDs) composed of CdSe/ZnS core/shell bioconjugates display brighter fluorescence intensities, lower detection thresholds, and better accuracy in analyzing bacterial cell mixtures composed of pathogenic E. coli O157:H7 and harmless E. coli DH5alpha using flow cytometry. For the same given bacterial mixture, QDs display fluorescence intensity levels that are approximately 1 order of magnitude brighter compared to the analogous experiments that utilize the standard dye fluorescein isothiocyanate. Detection limits are lowest when QDs are used as the fluorophore label for the pathogenic E. coli O157:H7 serotype : limits of 1% O157:H7 in 99% DH5alpha result, corresponding to 106 cells/mL, which is comparable to other developing fluorescence-based techniques for pathogen detection. Finally, utilizing QDs to label E. coli O157:H7 in cell mixtures results in greater accuracy and more closely approaches the ideal fluorophore for pathogen detection using flow cytometry. With their broader absorption spectra and narrower emission spectra than organic dyes, QDs can make vast improvements in the field of flow cytometry, where single-source excitation and simultaneous detection of multicolor species without complicating experimental setups or data analysis is quite advantageous for analyzing heterogeneous cell mixtures, both for prokaryotic pathogen detection and for studies on eukaryotic cell characteristics.

Hallen U, Bjorkner AE, Hallberg EC (2008) Binding of the periodontitis associated bacterium Porphyromonas gingivalis to glycoproteins from human epithelial cells. Oral Microbiol Immunol 23 :367-371


INTRODUCTION : In the present study we examined the ability of the periodontal pathogen Porphyromonas gingivalis to adhere to glycoconjugates on intact cells and to protein preparations of epithelial cells (KB cells). METHODS : The KB cell protein preparation was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by Western blotting. The membranes were used in overlay assays with labeled P. gingivalis. Flow cytometry was used to analyze attachment of bacteria to intact KB cells. RESULTS : Glycoconjugate expression on the KB cells and in the protein preparation was confirmed. Binding was detected to several bands on the Western blots. Flow cytometry showed a distinct increase in fluorescence for strain FDC 381. Preincubation of the bacteria with mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine inhibited the binding to KB cells by approximately 30% whereas preincubation with N-acetylneuraminic acid reduced the binding by 60%. CONCLUSION : These results indicate that carbohydrate structures are involved in the binding process of P. gingivalis to oral epithelial cells and that neuraminic acid plays a significant role in the adhesion process.

Hammes F, Berney M, Wang Y, Vital M, Koster O, Egli T (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Res 42 :269-277


There are significantly more microbial cells in drinking water than what can be cultured on synthetic growth media. Nonetheless, cultivation-based heterotrophic plate counts (HPCs) are used worldwide as a general microbial quality parameter in drinking water treatment and distribution. Total bacterial cell concentrations are normally not considered during drinking water treatment as a design, operative or legislative parameters. This is mainly because easy and rapid methods for quantification of total bacterial cell concentrations have, up to now, not been available. As a consequence, the existing lack of data does not allow demonstrating the practical value of this parameter. In this study, we have used fluorescence staining of microbial cells with the nucleic acid stain SYBR((R)) Green I together with quantitative flow cytometry (FCM) to analyse total cell concentrations in water samples from a drinking water pilot plant. The plant treats surface water (Lake Zurich) through sequential ozonation, granular active carbon (GAC) filtration and membrane ultrafiltration (UF). The data were compared with adenosine tri-phosphate (ATP) measurements and conventional HPCs performed on the same water samples. We demonstrated that the impact of all three major treatment steps on the microbiology in the system could accurately be described with total cell counting : (1) ozonation caused chemical destruction of the bacterial cells ; (2) GAC filtration facilitated significant regrowth of the microbial community ; and (3) membrane UF physically removed the bacterial cells from the water. FCM typically detected 1-2 log units more than HPC, while ATP measurements were prone to interference from extracellular ATP released during the ozonation step in the treatment train. We have shown that total cell concentration measured with FCM is a rapid, easy, sensitive and importantly, a descriptive parameter of several widely applied drinking water treatment processes.

Hanley BP (2008) Variance in multiplex suspension array assays : a distribution generation machine for multiplex counts. Theor Biol Med Model 5 :3


BACKGROUND : This study attempted to replicate Luminex experimental results for large numbers of beads per classifier using multiplexed assays and routine instrument use conditions. CONCLUSION : Using larger numbers of microspheres per classifier highlights a fundamental stochastic distribution of bead counts issue complicated by other factors. The more classifiers and the higher the count required per classifier there are, the more apparent the distribution of counts per classifier will be, and the more microspheres are required. Additional problems have been identified. Alternate methods of improving precision and reliability are recommended such as intraplexing and multi-well sample replicates to improve precision and confidence.

Heinecke L, Proud D, Sanders S, Schleimer RP, Kim J (2008) Induction of B7-H1 and B7-DC expression on airway epithelial cells by the Toll-like receptor 3 agonist double-stranded RNA and human rhinovirus infection : In vivo and in vitro studies. J Allergy Clin Immunol 121 :1155-1160


BACKGROUND : T-cell infiltration of the epithelium is a key feature of chronic rhinosinusitis and asthma. Viral infections are an important cause of disease exacerbations. We have found virus-induced expression of T cell-interacting ligands, B7 homolog costimulatory molecules, on airway epithelium. OBJECTIVE : We tested the ability of human rhinovirus (HRV) 16 and double-stranded RNA (dsRNA) to alter the expression of B7 homologs on human airway epithelial cells. METHODS : BEAS2B and primary human airway epithelial cells were exposed in vitro to dsRNA (25 microg/mL) or HRV-16, and then expression of cell-surface protein and mRNA for B7 homologs was assessed by means of flow cytometry and real-time PCR, respectively. Additionally, human subjects were infected with HRV-16 in vivo, and mRNA for B7 homologs was assessed by means of real-time PCR in fresh nasal epithelial cell scrapings obtained before and daily up to 4 days after infection. RESULTS : dsRNA exposure of BEAS2B and human primary bronchial epithelial cells resulted in increased levels of cell-surface and mRNA expression of B7-H1 and B7-DC but not B7-H2 or B7-H3. Exposure of primary cells to HRV-16 resulted in induction of cell-surface expression of B7-H1 and B7-DC. Pretreatment with fluticasone propionate failed to suppress the induction of B7-H1 and B7-DC. Nasal scrapings taken at the time of peak symptom scores (3 days) after infection of 6 human subjects with HRV-16 displayed selective induction of levels of mRNA for B7-H1 and B7-DC. CONCLUSION : These data show that HRV-16 infection or exposure to dsRNA induces epithelial B7-H1 and B7-DC.

Hinton TM, Doran TJ (2008) Inhibition of chicken anaemia virus replication using multiple short-hairpin RNAs. Antiviral Res 80 :143-149


RNA interference is becoming a powerful tool in gene-specific silencing. New generation vaccines against many pathogens will attempt to incorporate these molecules. Here we report the efficient silencing of chicken anaemia virus (CAV) genes in vitro using short-hairpin RNAs (shRNAs) targeting the region of the CAV transcript encoding either viral protein (VP) 1, or overlapping sections of VP2/3 and VP1/2. The shRNAs were first validated against a EGFP-CAV fusion transcript reporter system and then against CAV grown in MDCC-MSB1 cells. The decrease in CAV replication was shown with a flow cytometry assay specific for VP3. Overall the results showed efficient silencing of CAV replication in tissue culture using shRNAs. It was also shown that the combination of three shRNAs being expressed from a single plasmid is less effective at silencing CAV replication than the most active shRNA alone.

Hoopman TC, Wang W, Brautigam CA, Sedillo JL, Reilly TJ, Hansen EJ (2008) Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase. J Bacteriol 190 :1459-1472


Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

Hopfe M, Henrich B (2008) OppA, the ecto-ATPase of Mycoplasma hominis induces ATP release and cell death in HeLa cells. BMC Microbiol 8 :55


BACKGROUND : In the facultative human pathogen Mycoplasma hominis, which belongs to the cell wall-less Mollicutes, the surface-localised substrate-binding domain OppA of the oligopeptide permease was characterised as the main ecto-ATPase. RESULTS : With the idea that extra-cellular ATP could only be provided by the infected host cells we analysed the ATP release of HeLa cells after incubation with different preparations of Mycoplasma hominis : intact bacterial cells, the membrane fraction with or without OppA, recombinant OppA as well as an ATPase-deficient OppA mutant. Release of ATP into the supernatant of the HeLa cells was primarily determined in all samples lacking ecto-ATPase activity of OppA. In the presence of the ATPase inhibitor DIDS the amount of ATP in the OppA-containing samples increased. This increase was maximal after incubation with fractions containing OppA protein indicating that OppA is involved in ATP release and subsequent hydrolysis. Real-time PCR analyses revealed that the proliferation of HeLa cells is reduced after infection with M. hominis and flow cytometry experiments established that OppA induces greater apoptosis than necrosis of HeLa cells whereas the preservation of ecto-ATPase activity of OppA induces apoptosis. CONCLUSION : The OppA induced ATP-release and -hydrolysis induced cell death of M. hominis infected HeLa cells was predominantly due to apoptosis rather than necrosis. Future work will elucidate whether the induction of apoptosis is indispensable for survival of these non-invasive pathogen.

Hsieh SC, Liu IJ, King CC, Chang GJ, Wang WK (2008) A strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles. Virology 374 :338-350


Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem-anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170-180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem-anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem-anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.

Hubner MP, Thomas Stocker J, Mitre E (2008) Inhibition of type 1 diabetes in filaria-infected non-obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3(+) regulatory T cells. Immunology

Summary We sought to determine whether Litomosoides sigmodontis, a filarial infection of rodents, protects against type 1 diabetes in non-obese diabetic (NOD) mice. Six-week-old NOD mice were sham-infected or infected with either L3 larvae, adult male worms, or adult female worms. Whereas 82% of uninfected NOD mice developed diabetes by 25 weeks of age, no L. sigmodontis-infected mice developed disease. Although all mice had evidence of ongoing islet cell inflammation by histology, L. sigmodontis-infected mice had greater numbers of total islets and non-infiltrated islets than control mice. Protection against diabetes was associated with a T helper type 2 (Th2) shift, as interleukin-4 (IL-4) and IL-5 release from alpha-CD3/alpha-CD28-stimulated splenocytes was greater in L. sigmodontis-infected mice than in uninfected mice. Increased circulating levels of insulin-specific immunoglobulin G1, showed that this Th2 shift occurs in response to one of the main autoantigens in diabetes. Multicolour flow cytometry studies demonstrated that protection against diabetes in L. sigmodontis-infected NOD mice was associated with significantly increased numbers of splenic CD4(+) CD25(+) FoxP3(+) regulatory T cells. Interestingly, injection of crude worm antigen into NOD mice also resulted in protection against type 1 diabetes, though to a lesser degree than infection with live L. sigmodontis worms. In conclusion, these studies demonstrate that filarial worms can protect against the onset of type 1 diabetes in NOD mice. This protection is associated with a Th2 shift, as demonstrated by cytokine and antibody production, and with an increase in CD4(+) CD25(+) FoxP3(+) regulatory T cells.

Itoh S, Takeshita K, Susuki C, Shige-Eda K, Tsuji T (2008) Redistribution of P-selectin ligands on neutrophil cell membranes and the formation of platelet-neutrophil complex induced by hemodialysis membranes. Biomaterials 29 :3084-3090


The formation of platelet-neutrophil microaggregates and successive activation of neutrophils are closely related to hemodialysis-associated complications. The microaggregate is mediated primarily by the interaction between P-selectin (CD62P) expressed on activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1, CD162) expressed on neutrophils. We previously reported that the clustered distribution of PSGL-1 on the cell membranes of chemokine-treated neutrophils caused upregulation of the microaggregate formation. In this study, we found that neutrophils treated with human plasma that had been incubated with hemodialysis membranes greatly enhanced the microaggregate formation. The membrane-treated plasma also induced PSGL-1 to form a cap-like cluster on the neutrophil surface. Analysis of several hemodialysis membranes with different materials indicated that the inducibility for the cap-like cluster formation of PSGL-1 parallels their ability to activate the complement system. Both the enhancement of microaggregate formation and the redistribution of PSGL-1 induced by the hemodialysis membrane-treated plasma were almost completely abrogated in the presence of a specific antagonist for the complement component C5a receptor, W-54011. These results strongly suggest that the generation of anaphylatoxin C5a through complement activation induced by hemodialysis membranes is responsible for the clustered redistribution of PSGL-1 in neutrophils leading to the increase in the platelet-neutrophil microaggregate formation. The present study indicates the importance of synergistic exacerbation of complement activation and platelet-neutrophil microaggregate formation in developing hemodialysis-associated complications.

Jafri M, Donnelly B, Allen S, Bondoc A, McNeal M, Rennert PD, Weinreb PH, Ward R, Tiao G (2008) Cholangiocyte expression of alpha2beta1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia. Am J Physiol Gastrointest Liver Physiol 295 :G16-G26


Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as alpha2beta1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express alpha2beta1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether alpha2beta1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the alpha2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the alpha2beta1-integrin. Newborn mice were pretreated with a monoclonal antibody against the alpha2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed alpha2beta1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-alpha2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the alpha2beta1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

Jakiela B, Brockman-Schneider R, Amineva S, Lee WM, Gern JE (2008) Basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection. Am J Respir Cell Mol Biol 38 :517-523


We used an in vitro model of differentiated tracheobronchial epithelium to analyze the susceptibility of different cell types to infection with rhinoviruses (RVs). Primary cells from control subjects were cultured in an air-liquid interface to form differentiated epithelia. Suprabasal and basal fractions were separated after trypsin digestion, and cell suspensions were infected with serotypes RV16 and RV1A. These cell fractions were analyzed for expression of viral capsid protein VP2 (flow cytometry), viral replication (real-time PCR), cytokeratin-14, and intercellular adhesion molecule-1 (ICAM-1). Compared with suprabasal fraction, basal cells had increased percentages of cells staining positive for VP2 (RV1A : 37.8% versus 9.1%, P < 0.01 ; RV16 : 12.0 versus 3.0%, P < 0.05). The average number of viral RNA copies per cell was also higher in basal cells (2.2- and 2.4-fold increase in RV1A- and RV16-infected cells, respectively) compared with suprabasal cells. Furthermore, ICAM-1 was expressed by 33.3% of basal cells, compared with 8.1% of suprabasal cells (P < 0.05). Finally, in culture models of epithelial injury (detached suprabasal cells or scratched surface), there was significantly greater replication of RV1A compared with intact cell layer. These findings demonstrate that basal cells are more susceptible to RV infection than suprabasal cells. For major group RV, this may be in part due to increased expression of ICAM-1 ; however, minor group RV also replicated more effectively in basal cells. These results suggest the possibility that epithelial cell differentiation is associated with the maturation of antiviral defense mechanisms.

Jarzembowski T, Wisniewska K, Jozwik A, Bryl E, Witkowski J (2008) Flow cytometry as a rapid test for detection of penicillin resistance directly in bacterial cells in Enterococcus faecalis and Staphylococcus aureus. Curr Microbiol 57 :167-169


We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.

Johnson LR, Starkey CR, Palmer J, Taylor J, Stout S, Holt S, Hendren R, Bock B, Waibel E, Tyree G, Miller GC (2008) A comparison of two methods to determine the presence of high-risk HPV cervical infections. Am J Clin Pathol 130 :401-408


Clinical tests for human papillomavirus (HPV) DNA require clinical validation before being offered for use by laboratories. To determine the clinical viability of a laboratory-developed test using the Invader HPV reagents (Third Wave Technologies, Madison, WI), a retrospective study was designed using 213 patient cervical cytologic samples. The results of the Invader assay were directly compared with the results obtained using the Hybrid Capture 2 High-Risk HPV assay (Digene, Gaithersburg, MD). The results of both assays were also compared with cytologic evaluation. In addition, clinical performance was evaluated using a standard-of-care approach in which colposcopically guided biopsies were done in cases where standard of care dictated, and the histologic features of the biopsy specimens were noted. The Invader-based test demonstrated a clinical sensitivity in atypical squamous cells of undetermined significance cases of 98% for cervical intraepithelial neoplasia (CIN) 2 or worse and 100% for CIN 3 or worse and a negative predictive value of 96.9% (confidence interval, 89.3%-99.6%) using data generated mostly from the use of an earlier version of reagents. These findings support the clinical and laboratory benefits of the Invader method.

Jonjic S, Krmpotic A, Arapovic J, Koszinowski UH (2008) Dissection of the antiviral NK cell response by MCMV mutants. Methods Mol Biol 415 :127-149


Our understanding of virus control by natural killer (NK) cells relies mainly on in vitro observations. The significance of these findings for virus control in vivo is not yet fully understood. Complexity is added by the fact that many viruses, particularly herpesviruses, are equipped with sets of genes that, dependent on the genetic background of the host, modify the NK cell response. The advent of recombinant DNA technology and mutagenesis procedures for BAC-cloned viral genomes has made it possible not only to screen for viral proteins with such functions but also to assess their biological relevance. Mutant viruses with gene defects reveal the efficacy and complexity of NK cell control. Here, we describe procedures to assess the NK cell response to mouse cytomegalovirus (MCMV), a prominent virus model for studying NK cell functions in vivo.

Kalliomaki M, Collado MC, Salminen S, Isolauri E (2008) Early differences in fecal microbiota composition in children may predict overweight. Am J Clin Nutr 87 :534-538


BACKGROUND : Experimental studies suggest that gut microbiota deviations predispose toward energy storage and obesity. OBJECTIVE : We wanted to establish whether early gut microbiota composition can guide weight development throughout early childhood. DESIGN : Overweight and obese children (n = 25) were selected from a prospective follow-up study at the age of 7 y and identified according to the International Obesity Task Force criteria. Normal-weight children (n = 24) were selected from the same cohort and matched for gestational age and body mass index at birth, mode of delivery, probiotic supplementation, duration of breastfeeding, use of antibiotics during infancy, and frequencies of atopic diseases and atopic sensitization. Early fecal microbiota composition was analyzed by fluorescent in situ hybridization (FISH) with microscopic and flow cytometry detection and by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS : The bifidobacterial numbers in fecal samples during infancy, as assessed by the FISH with flow cytometry, were higher in children remaining normal weight, [median : 2.19 x 10(9) cells/g (interquartile range : 1.10-5.28 x 10(9) cells/g)] than in children becoming overweight [1.20 x 10(9) cells/g (0.48-1.59x 10(9) cells/g) ; P = 0.02]. A similar tendency was found by FISH with microscopic detection and qRT-PCR. The microbiota aberrancy during infancy in children becoming overweight was also associated with a greater number of Staphylococcus aureus [0.64 x 10(6) cells/g (0.33-1.00 x 10(6) cells/g)] than in children remaining normal weight [0.27 x 10(6) cells/g (0.17-0.50 x 10(6) cells/g) ; P = 0.013]. CONCLUSION : Aberrant compositional development of the gut microbiota precedes overweight, offering new possibilities for preventive and therapeutic applications in weight management.

Kalyuzhnaya MG, Lidstrom ME, Chistoserdova L (2008) Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington. Isme J 2 :696-706


Redox sensor green (RSG), a novel fluorescent dye from Invitrogen was employed as a tool for real-time detection of microbes metabolically active in situ, in combination with flow cytometry and cell sorting. Lake Washington sediment, an environment known for high rates of methane oxidation, was used as a model, and methylotrophs were targeted as a functional group. We first tested and optimized the performance of the dye with pure methylotroph cultures. Most cells in actively growing cultures were positive for staining, whereas in starved cultures, few cells fluoresced. However, starved cells could be activated by addition of substrate. High numbers of fluorescing cells were observed in a Lake Washington sediment sample, and activation of subpopulations of cells was demonstrated in response to methane, methanol, methylamine and formaldehyde. The fraction of the population activated by methane was investigated in more detail, by phylogenetic profiling. This approach showed that the major responding species were the Methylomonas species, previously isolated from the site, and Methylobacter species that have not yet been cultivated from Lake Washington. In addition, from the methane-stimulated fraction, uncultivated bacterial sequences were obtained that belonged to unclassified Deltaproteobacteria, unclassified Verrucomicrobiles and unclassified Acidobacteria, suggesting that these microbes may also be involved in methane metabolism. The approach was further tested for its utility in facilitating enrichment for functional types that possess specific metabolic activities but resist cultivation. It was demonstrated that in enrichment cultures inoculated with cells that were sorted after stimulation with methane, Methylobacter sequences could be detected, whereas in enrichment cultures inoculated by randomly sorted cells, Methylomonas species quickly outcompeted all other types.

Karo O, Wahl A, Nicol SB, Brachert J, Lambrecht B, Spengler HP, Nauwelaers F, Schmidt M, Schneider CK, Muller TH, Montag T (2008) Bacteria detection by flow cytometry. Clin Chem Lab Med 46 :947-953


Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37 degrees C, and measuring the contaminating bacteria in a flow cytometer.

Khandavilli S, Homer KA, Yuste J, Basavanna S, Mitchell T, Brown JS (2008) Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence. Mol Microbiol 67 :541-557


Cell surface lipoproteins are important for the full virulence of several bacterial pathogens, including Streptococcus pneumoniae. Processing of prolipoproteins seems to be conserved among different bacterial species, and requires type II signal peptidase (Lsp) mediated cleavage of the N-terminal signal peptide to form the mature lipoprotein. Lsp has been suggested as a target for new antibiotic therapies, but at present there are only limited data on the function of Lsp for Gram-positive bacterial pathogens. We have investigated the function and role during disease pathogenesis of the S. pneumoniae Lsp, which, blast searches suggest, is encoded by the gene Sp0928. Expression of Sp0928 protected Escherichia coli against the Lsp antagonist globomycin, and proteomics and immunoblot analysis demonstrated that deletion of Sp0928 prevented processing of S. pneumoniae prolipoproteins to mature lipoproteins. These data strongly suggest that Sp0928 encodes the S. pneumoniae Lsp. However, immunoblots of membrane-associated proteins, immunoelectron microscopy and flow cytometry assays all confirmed that in the absence of Lsp, immature lipoproteins were still attached to the cell surface. Despite preservation of lipoprotein attachment to the cell membrane, loss of S. pneumoniae Lsp resulted in several phenotypes associated with impaired lipoprotein function and reduced S. pneumoniae replication in animal models of infection.

Klouche M, Schroder U (2008) Rapid methods for diagnosis of bloodstream infections. Clin Chem Lab Med 46 :888-908


Direct detection technologies for pathogenic microorganisms are emerging to be applied in the diagnosis of serious bloodstream infections and infections at sterile body sites, as well as for quality control measures prior to the release of sterile blood products and to ascertain microbial safety of food. Standard blood cultures as the current gold standard for detection of bacteraemia/sepsis and other culture-based microbiological identification procedures are comparatively slow and have limited sensitivity for fastidious or slow-growing microorganisms. Rapid nucleic acid-based technologies with PCR amplification or hybridisation probes for specific pathogens, broad-range bacterial or fungal assays, flow cytometry, as well as protein-based characterisation by mass spectrometry, aim at identification of pathogenic microorganisms within minutes to hours. Interpretation of direct detection of panbacterial or panfungal nucleic acids instead of living microorganisms in blood is complex, given the risk of contamination, the ubiquitous presence of bacterial and fungal DNA, and the lack of a gold standard. Since many of the infections at sterile sites, particularly sepsis, are medical emergencies requiring immediate therapeutic responses, rapid technologies could contribute to reduction of morbidity, mortality, and of the economic burden. This review summarises the currently available data on rapid non-culture-based technologies and outlines the potential clinical usefulness in infectious disease diagnosis.

Kristian SA, Birkenstock TA, Sauder U, Mack D, Gotz F, Landmann R (2008) Biofilm formation induces C3a release and protects Staphylococcus epidermidis from IgG and complement deposition and from neutrophil-dependent killing. J Infect Dis 197 :1028-1035


BACKGROUND : Biofilm formation is considered to be an important virulence factor of the opportunistic pathogen Staphylococcus epidermidis. We hypothesized that biofilm formation could interfere with the deposition of immunoglobulins and complement on the bacterial surface, leading to diminished activation of the complement system and protection from killing by human phagocytes. METHODS : The killing of biofilm-encased and planktonically grown wild-type (wt) S. epidermidis and the killing of an isogenic biofilm-negative ica mutant (ica(-)) by human polymorphonuclear neutrophils (PMNs) were compared. C3a induction and deposition of C3b and immunoglobulin G (IgG) on the bacteria after opsonization with human serum were assessed by enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. The virulence of the bacterial strains was compared in a mouse model of catheter-associated infection. RESULTS : Biofilm-embedded wt S. epidermidis was killed less well by human PMNs and induced more C3a than planktonically grown wt and ica(-) S. epidermidis. However, the deposition of C3b and IgG on the bacterial surface was diminished in biofilm-encased staphylococci. wt S. epidermidis was more virulent in implant-associated infections and was killed more slowly than ica(-) in ex vivo assays of killing by PMNs. CONCLUSIONS : The results indicate that prevention of C3b and IgG deposition on the bacterial surface contributes to the biofilm-mediated protection of S. epidermidis from killing by PMNs.

Krutzik PO, Crane JM (2008) High-content single-cell drug screening with phosphospecific flow cytometry. Orthopedics 31

Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose- response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.

Lam HS, Ng PC (2008) Biochemical markers of neonatal sepsis. Pathology 40 :141-148


The use of biochemical markers in neonatal infection has remained an important area of research in the past decades. Many infection markers are components of the inflammatory cascade and reflect the host’s immunological status and response to infection. Cytokines and chemokines such as interleukin (IL)-6 and IL-8 have been demonstrated to have good diagnostic utilities as early phase markers, while acute phase reactants such as C-reactive protein and procalcitonin have superior diagnostic properties during the later phases. Other markers, including inter-alpha-inhibitor proteins, IL-10 and regulated upon activation normal T cells expressed and secreted (RANTES) have been demonstrated to yield important prognostic information and may help the clinician identify infants who will develop fulminant infection from the outset of presentation. The advent of flow cytometry and molecular techniques have made crucial contributions to the field and promise to further improve the diagnostic accuracy and clinical management of infected infants.

Landolfo S, Politi H, Angelozzi D, Mannazzu I (2008) ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium. Biochim Biophys Acta 1780 :892-898


To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ’fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.

Ledru S, Canonne JP (2008) [Comparison of IRIS IQ ELITE and microscopy for urinalysis and evaluation of performance in predicting outcome of urine cultures]. Ann Biol Clin (Paris) 66 :555-559


Since apprearance of reagent strip chemical determinations, different flow cytometers for urine sediments analysis appears (Sysmex UF-100, UF-50 and UF 1000). Recently has been introduced a new analyser (Iris IQ Elite) that analyses urine specimen by microscopy. Five hundred microscopic fields are photographed and analysed. Each particle of the microscopic field is separated and analysed with the software for recognition : Auto Particle Recognition (APR). In most of cases, automatic classification leads to correct results. For abnormal particles, the possibilities of visualisation by the operator permit us to avoid confusion between, for example : red cells and yeasts, spermatozoa and bacillus ; that are identified as artefacts in flow cytometer. L’Iris IQ Elite presents a sensitivity of 68%, a specificity of 80%, a predictive positive value of 60% and a negative predictive value of 86% versus urine specimen culture. By combination of differents parameters given by the Iris machine, we can forget 43% of microscopic direct examination and predict the negativity of 32% of culture with a sensitivity and a negative predictive value of 100%.

Lemaire R, Marcelino M, Yuan Z (2008) Achieving the nitrite pathway using aeration phase length control and step-feed in an SBR removing nutrients from abattoir wastewater. Biotechnol Bioeng 100 :1228-1236


Aeration phase length control and step-feed of wastewater are used to achieve nitrogen removal from wastewater via nitrite in sequencing batch reactors (SBR). Aeration is switched off as soon as ammonia oxidation is completed, which is followed by the addition of a fraction of the wastewater that the SBR receives over a cycle to facilitate denitrification. The end-point of ammonia oxidation is detected from the on-line measured pH and oxygen uptake rate (OUR). The method was implemented in an SBR achieving biological nitrogen and phosphorus removal from anaerobically pre-treated abattoir wastewater. The degree of nitrite accumulation during the aeration period was monitored along with the variation in the nitrite oxidizing bacteria (NOB) population using fluorescence in situ hybridization (FISH) techniques. It is demonstrated that the nitrite pathway could be repeatedly and reliably achieved, which significantly reduced the carbon requirement for nutrient removal. Model-based studies show that the establishment of the nitrite pathway was primarily the result of a gradual reduction of the amount of nitrite that is available to provide energy for the growth of NOB, eventually leading to the elimination of NOB from the system.

Lemonnier M, Levin BR, Romeo T, Garner K, Baquero MR, Mercante J, Lemichez E, Baquero F, Blazquez J (2008) The evolution of contact-dependent inhibition in non-growing populations of Escherichia coli. Proc Biol Sci 275 :3-10


In the course of liquid culture, serial passage experiments with Escherichia coli K-12 bearing a mutator gene deletion (DeltamutS) we observed the evolution of strains that appeared to kill or inhibit the growth of the bacteria from where they were derived, their ancestors. We demonstrate that this inhibition occurs after the cells stop growing and requires physical contact between the evolved and ancestral bacteria. Thereby, it is referred to as stationary phase contact-dependent inhibition (SCDI). The evolution of this antagonistic relationship is not anticipated from existing theory and experiments of competition in mass (liquid) culture. Nevertheless, it occurred in the same way (parallel evolution) in the eight independent serial transfer cultures, through different single base substitutions in a gene in the glycogen synthesis pathway, glgC. We demonstrate that the observed mutations in glgC, which codes for ADP-glucose pyrophosphorylase, are responsible for both the ability of the evolved bacteria to inhibit or kill their ancestors and their immunity to that inhibition or killing. We present evidence that without additional evolution, mutator genes, or known mutations in glgC, other strains of E. coli K-12 are also capable of SCDI or sensitive to this inhibition. We interpret this, in part, as support for the generality of SCDI and also as suggesting that the glgC mutations responsible for the SCDI, which evolved in our experiments, may suppress the action of one or more genes responsible for the sensitivity of E. coli to SCDI. Using numerical solutions to a mathematical model and in vitro experiments, we explore the population dynamics of SCDI and postulate the conditions responsible for its evolution in mass culture. We conclude with a brief discussion of the potential ecological significance of SCDI and its possible utility for the development of antimicrobial agents, which unlike existing antibiotics, can kill or inhibit the growth of bacteria that are not growing.

Lesch HP, Turpeinen S, Niskanen EA, Mahonen AJ, Airenne KJ, Yla-Herttuala S (2008) Generation of lentivirus vectors using recombinant baculoviruses. Gene Ther 15 :1280-1286


In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses : the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 x 10(6) TU ml(-1), which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.

Leser TD, Knarreborg A, Worm J (2008) Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs. J Appl Microbiol 104 :1025-1033


AIMS : To determine if orally ingested Bacillus spores used as probiotics or direct-fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract. METHODS AND RESULTS : Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore-feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4-6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid-colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2.14-fold compared to time zero. CONCLUSIONS : The experiments showed that 70-90% of dietary-supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract. SIGNIFICANCE AND IMPACT OF THE STUDY : A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.

Liu J, Liu B, Cao Z, Inoue S, Morita K, Tian K, Zhu Q, Gao GF (2008) Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein. J Virol Methods 154 :20-26


Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA ; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID(50)/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.

Liu T, Konig R, Sha J, Agar SL, Tseng CT, Klimpel GR, Chopra AK (2008) Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice : potential use as live-attenuated vaccines. Microb Pathog 44 :224-237


We generated and characterized Salmonella enterica serovar Typhimurium mutants that were deleted for the genes encoding Braun lipoprotein (lpp) alone or in conjunction with the msbB gene, which codes for an enzyme required for the acylation of the lipid A moiety of lipopolysaccharide. Two copies of the lpp gene, designated as lppA and lppB, exist on the chromosome of S. Typhimurium. These mutants were highly attenuated in a mouse infection model and induced minimal histopathological changes in mouse organs compared to those seen in infection with wild-type (WT) S. Typhimurium. The lppB/msbB and the lppAB/msbB mutants were maximally attenuated, and hence further examined in this study for their ability to induce humoral and cellular immune responses. Importantly, infection of out-bred Swiss-Webster mice with the mutant S. Typhimurium generated superior T helper cell type 2 (Th2) responses compared to WT S. Typhimurium, as determined by measuring IgG subclasses and cytokines. WT S. Typhimurium induced higher levels of IgG2a in sera of infected mice, while the lppB/msbB and lppAB/msbB mutants mounted higher levels of IgG1 as determined by an enzyme-linked immunosorbent assay. Mice immunized with lppB/msbB and lppAB/msbB mutants rapidly cleared WT S. Typhimurium upon subsequent rechallenge, and naive mice passively immunized with sera from animals infected with S. Typhimurium mutants were protected against subsequent challenge with WT S. Typhimurium. Splenic T cells produced higher levels of interferon-gamma following ex vivo exposure to WT S. Typhimurium, while splenic T cells infected with the above-mentioned two mutants evoked higher levels of interleukin-6. Further, mice infected with lppB/msbB and lppAB/msbB mutants showed much higher levels of splenic T cell activation as measured by CD44(+) expression on CD4(+) T cells by flow cytometry and by incorporation of (3)H-thymidine compared to mice that were infected with WT S. Typhimurium. We expect the lppB/msbB and lppAB/msbB mutants to be excellent live-attenuated vaccine candidates, because they induced minimal inflammatory responses and evoked stronger and specific antibody and cellular immune responses.

Loehfelm TW, Luke NR, Campagnari AA (2008) Identification and characterization of an Acinetobacter baumannii biofilm-associated protein. J Bacteriol 190 :1036-1044


We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302 ::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm.

Ma CL, Hu XC, Hu FY, Zhou HJ, Xue L, Yu XB (2008) [The effects of Lysophospholipase from Clonorchis sinensis on the hepatic stellate cells and oval cells of rat.]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 24 :692-695


AIM : To clarify the effects of the recombinant protein of Lysophospholipase from Clonorchis sinensis (CsLysoPLA) on the hepatic stellate cells (HSC) and oval cells of rat. METHODS : Binding of the recombinant CslysoPLA protein to the membrane of HSC and oval cells was indentified by immunofluorescent staining. The HSC and oval cells were cultured and treated with the recombinant protein at different doses, and proliferation was quantified by MTT method. Cell cycle analysis was performed by flow cytometry. RESULTS : The recombinant CslysoPLA protein could bind to the membrane of HSC and oval cells. Compared to control, 2 mg/L and 20 mg/L the recombinant protein could promote HSC and oval cells growth (P<0.05), whereas 200 mg/L the recombinant protein could induce the cells necrosis, which associated with overt plasma membrane disruption. Oval cell number in G(2) phase of the recombinant protein 20 mg/L treated group was higher than that of control group. CONCLUSION : In vitro, the recombinant protein could induce HSC and oval cells proliferation at low concentrations (2 mg/L and 20 mg/L), whereas it also could induce the cells necrosis at high concentration (200 mg/L). These results suggested that CslysoPLA might play a role in the pathogenicity of C. sinensis.

Magistrado PA, Staalsoe T, Theander TG, Hviid L, Jensen AT (2008) CD36 selection of 3D7 Plasmodium falciparum associated with severe childhood malaria results in reduced VAR4 expression. Malar J 7 :204


BACKGROUND : A subset of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1(SM)) is involved in the cytoadherence of P. falciparum-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1(SM) are encoded by group A var genes that are composed of a more constrained range of amino acid sequences than groups B and C var genes encoding PfEMP1(UM) associated with uncomplicated malaria. Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding—a major cytoadhesion phenotype of P. falciparum isolates. METHODS : A 3D7 PfEMP1(SM) sub-line (3D7(SM)) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7(SM) parasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 var genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using var-specific primers. RESULTS : A selection-induced increased adhesion of 3D7(SM) iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression. CONCLUSION : VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

Malone KE, Stohlman SA, Ramakrishna C, Macklin W, Bergmann CC (2008) Induction of class I antigen processing components in oligodendroglia and microglia during viral encephalomyelitis. Glia 56 :426-435


Glia exhibit differential susceptibility to CD8 T cell mediated effector mechanisms during neurotropic coronavirus infection. In contrast to microglia, oligodendroglia are resistant to CD8 T cell perforin-mediated viral control in the absence of IFN gamma. Kinetic induction of MHC Class I expression by microglia and oligodendroglia in vivo was thus analyzed to assess responses to distinct inflammatory signals. Flow cytometry demonstrated delayed Class I surface expression by oligodendroglia compared with microglia. Distinct kinetics of Class I protein upregulation correlated with cell type specific transcription patterns of genes encoding Class I heavy chains and antigen processing components. Microglia isolated from naive mice expressed high levels of these mRNAs, whereas they were near detection limits in oligodendroglia ; nevertheless, Class I protein was undetectable on both cell types. Infection induced modest mRNA increases in microglia, but dramatic transcriptional upregulation in oligodendroglia coincident with IFN alpha or IFN gamma mRNA increases in infected tissue. Ultimately mRNAs reached similar levels in both cell types at their respective time points of maximal Class I expression. Expression of Class I on microglia, but not oligodendroglia, in infected IFN gamma deficient mice supported distinct IFN requirements for Class I presentation. These data suggest an innate immune preparedness of microglia to present antigen and engage CD8 T cells early following infection. The delayed, yet robust, IFN gamma dependent capacity of oligodendroglia to express Class I suggests strict control of immune interactions to avoid CD8 T cell recognition and potential presentation of autoantigen to preserve myelin maintenance.

Mandal SM, Pati BR, Das AK, Ghosh AK (2008) Characterization of a symbiotically effective Rhizobium resistant to arsenic : Isolated from the root nodules of Vigna mungo (L.) Hepper grown in an arsenic-contaminated field. J Gen Appl Microbiol 54 :93-99


Bacteria were isolated from the root nodules of Vigna mungo (L.) Hepper, grown in an arsenic-contaminated field and the strain was selected by its nodulation ability as well as better arsenic tolerant capacity compared to others. The selected strain was identified as Rhizobium by 16S rDNA sequencing and designated as VMA301. Phylogenetic analysis of the gene sequences showed its close relatedness with Sinorhizobium fredii. LC(50) value of arsenate for the bacteria as determined by flow cytometry was found to be 2.8 mM and arsenic uptake was measured by atomic absorption spectrometry as 0.048 mg g(-1) biomass. The high amount of arsenic was toxic to the cell, which changed the morphology of the bacteria to an elongated shape. Presence of a transcriptional regulatory gene (ArsR) of the ars genetic system was confirmed by amplification and sequencing. The symbiotic property of the isolate was also confirmed by amplification and sequencing of the NodC gene. These results indicate that the isolated Rhizobium bacteria may exert dual roles in the environment, arsenic bioremediation from the soil as well as increase of soil fertility through nitrogen fixation.

Mannazzu I, Angelozzi D, Belviso S, Budroni M, Farris GA, Goffrini P, Lodi T, Marzona M, Bardi L (2008) Behaviour of Saccharomyces cerevisiae wine strains during adaptation to unfavourable conditions of fermentation on synthetic medium : cell lipid composition, membrane integrity, viability and fermentative activity. Int J Food Microbiol 121 :84-91


During must fermentation wine strains are exposed to a variety of biotic and abiotic stresses which, when prevailing over the cellular defence systems, can affect cell viability with negative consequences on the progression of the fermentative process. To investigate the ability of wine strains to survive and adapt to unfavourable conditions of fermentation, the lipid composition, membrane integrity, cell viability and fermentative activity of three strains of Saccharomyces cerevisiae were analysed during hypoxic growth in a sugar-rich medium lacking lipid nutrients. These are stressful conditions, not unusual during must fermentation, which, by affecting lipid biosynthesis may exert a negative effect on yeast viability. The results obtained showed that the three strains were able to modulate cell lipid composition during fermentation. However, only two of them, which showed highest viability and membrane integrity at the end of the fermentation process, reached a fatty acid composition which seemed to be optimal for a successful adaptation. In particular, C16/TFA and UFA/TFA ratios, more than total lipid and ergosterol contents, seem to be involved in yeast adaptation.

Manti A, Boi P, Falcioni T, Canonico B, Ventura A, Sisti D, Pianetti A, Balsamo M, Papa S (2008) Bacterial cell monitoring in wastewater treatment plants by flow cytometry. Water Environ Res 80 :346-354


The activated sludge process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment, in which bacteria constitute the majority and represent the main microorganisms responsible for the degradation process in a plant. In this work, we monitored bacterial charge in different wastewater treatment plants by flow cytometry, also evaluating chlorination effects on bacterial viability, both by flow cytometry and traditional plate counts. Maximum values of bacterial charge were registered in the aeration tank of all plants monitored. Cell viability did not show significant differences (p > 0.05) in samples collected in "before chlorination" and "wastewater effluent" treatment steps ; this suggests that the chlorination was not able to decrease total viable bacterial charge. In this work, we discuss the need to improve microbiological analyses, both in terms of measuring other potential pathogens and of using new methodological approaches in the traditional evaluation of the microbiological quality of effluents.

Marques CP, Cheeran MC, Palmquist JM, Hu S, Lokensgard JR (2008) Microglia are the major cellular source of inducible nitric oxide synthase during experimental herpes encephalitis. J Neurovirol 14 :229-238


Although production of reactive nitrogen and reactive oxygen species (RNS and ROS) is a component of innate defense against viral infection, their overproduction in the brain may also lead to deleterious consequences. To investigate potential immunopathologic roles of oxidative stress during herpes encephalitis, the authors examined the expression kinetics of inducible nitric oxide synthase (iNOS) as well as heme oxygenase-1 (HO-1), a marker of oxidative stress, and evaluated infection-induced oxidative brain damage. Results from these studies showed that both iNOS and HO-1 gene expression were highly elevated in the brain within 7 days post infection (d.p.i.) and remained elevated through 21 d.p.i. Real-time bioluminescence imaging of HO-1 promoter-luciferase transgenic mice confirmed HO-1 promoter activity in the brains of HSV-1-infected animals within 3 d.p.i., which peaked between 5 and 7 d.p.i. Immunohistochemical staining for both 3-nitrotyrosine and 8-hydroxydeoxyguanosine (8-OH-dG), as well as quantitative assessment of 8-isoprostane levels, demonstrated the presence of viral infection-induced oxidative brain damage. In addition, when brain leukocytes obtained from animals with experimental herpes encephalitis were sorted using fluorescence-activated cell sorting (FACS) and the individual cell populations analyzed, CD45(int)/CD11b(+) resident microglia were found to be the major cellular source of iNOS expression.

Mary I, Tarran GA, Warwick PE, Terry MJ, Scanlan DJ, Burkill PH, Zubkov MV (2008) Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean. FEMS Microbiol Ecol 63 :36-45


(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.

McCann AK, Schwartz KJ, Bangs JD (2008) A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes. Mol Biochem Parasitol 159 :146-149


The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH(4)Cl) then allows precise titration of steady state lysosomal pH (4.84+/-0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa.

McIlroy S, Hoefel D, Schroeder S, Ahn J, Tillett D, Saint C, Seviour RJ (2008) FACS enrichment and identification of floc-associated alphaproteobacterial tetrad-forming organisms in an activated sludge community. FEMS Microbiol Lett 285 :130-135


A precise phylogenetic identity of the Defluviicoccus-related glycogen-accumulating organisms (GAO) observed after FISH probing in a novel activated sludge process removing phosphorus was sought with the aim of exploring the phylogenetic diversity of this important group. These organisms, whose sequences were not revealed in previously generated community wide 16S rRNA gene clone libraries, were identified using flow cytometry cell sorting of FISH-positive cells. Sequencing of a 16S rRNA gene clone library created from this sorted population identified the Defluviicoccus-related GAO as being highly related to previous identified GAO from enhanced biological phosphorus removal systems, despite a marked environmental difference between the two systems.

Mesnage S, Chau F, Dubost L, Arthur M (2008) Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism. J Biol Chem 283 :19845-19853


Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously described N-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[14C]acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.

Moretta R, Ruybal P, Mesplet M, Petrigh R, Nunez P, Gil G, Wilkowsky S, Garbossa G, Farber M (2008) Flow cytometry to evaluate Anaplasma marginale parasitemia using a fluorescent nucleic acid stain. Ann N Y Acad Sci 1149 :111-113


In this work we describe a flow cytometry-based method using SYTO16 (a DNA intercalating agent) to quantify Anaplasma marginale-infected erythrocytes in blood from bovine animals. The linearity and reproducibility of the results obtained with SYTO16 labeling followed by flow cytometry analysis make it a suitable approach for measurement of parasitemia in A. marginale infections.

Morris MA, Dawson CW, Wei W, O’Neil JD, Stewart SE, Jia J, Bell AI, Young LS, Arrand JR (2008) Epstein-Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes. J Gen Virol 89 :2806-2820


SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell-cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1beta and IL-1alpha was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.

Muller TH, Mohr H, Montag T (2008) Methods for the detection of bacterial contamination in blood products. Clin Chem Lab Med 46 :933-946


Culture-based and molecular assays have been developed for the screening of platelet concentrates and other blood components for bacterial contaminations. In this review, the principles of the assays are outlined. The focus of this review is the assessment of the analytical qualities of the methods. Spiking studies by adding defined levels of a wide range of bacteria to the complex biological matrix provide the first basis to evaluate and compare the qualities of methods for bacterial detection. The sensitivity acceptable for reliable screening for bacteria critically depends on the timing of either early sampling (within a period of up to 24 h after preparation of the blood component) or late sampling (a few hours before issuing the blood component). Large screening studies are essential to confirm both adequate sensitivity and specificity of the testing. In the ideal setting, these studies are prospectively planned and include systematic surveillance of adverse events in response to the administration of the screened products. The findings from sterility testing (predominantly with automated systems for detection of bacteria based on CO(2) generation) of more than 550,000 platelet concentrates in 13 studies are summarised. The limitations of the early sampling and the "negative-to-date" strategy to issue platelet concentrates are addressed. A few reported cases of probable transmission of bacteria by platelet transfusion despite negative screening tests emphasise the need to further develop optimised methods for testing of bacteria blood components.

Munukka E, Lepparanta O, Korkeamaki M, Vaahtio M, Peltola T, Zhang D, Hupa L, Ylanen H, Salonen JI, Viljanen MK, Eerola E (2008) Bactericidal effects of bioactive glasses on clinically important aerobic bacteria. J Mater Sci Mater Med 19 :27-32


Bioactive glasses (BAGs) have been studied for decades for clinical use, and they have found many dental and orthopedic applications. BAGs have also been shown to have an antibacterial effect e.g., on some oral microorganisms. In this extensive work we show that six powdered BAGs and two sol-gel derived materials have a clear antibacterial effect on 29 clinically important bacterial species. We also incorporated a rapid and accurate flow cytometric (FCM) method to calculate and standardize the numbers of viable bacteria inoculated in the suspensions used in the tests for antibacterial activity. In all materials tested growth inhibition could be demonstrated, although the concentration and time needed for the effect varied depending on the BAG. The most effective glass was S53P4, which had a clear growth-inhibitory effect on all pathogens tested. The sol-gel derived materials CaPSiO and CaPSiO II also showed a strong antibacterial effect. In summary, BAGs were found to clearly inhibit the growth of a wide selection of bacterial species causing e.g., infections on the surfaces of prostheses in the body after implantation.

Neugebauer H, Hartmann P, Krenn S, Gluck T, Scholmerich J, Straub R, Wiest R (2008) Bacterial translocation increases phagocytic activity of polymorphonuclear leucocytes in portal hypertension : priming independent of liver cirrhosis. Liver Int 28 :1149-1157


AIM : Bacterial translocation (BT) to mesenteric lymph nodes (MLN) in cirrhosis has been linked to impaired host defence. Phagocytosis by polymorphonuclear leucocytes (PMNLs) is the primary event in the killing of bacteria but has not been investigated in relation to the presence of BT. METHODS : Mesenteric lymph nodes were harvested sterile and assessed for BT by culture techniques. Study groups included ascitic cirrhotic rats (LC), healthy controls (Con) as well as portal-vein-ligated (PVL) rats 2 days (acute PVL with and without norfloxacin) or 3 weeks after surgery (chronic PVL). PMNLs were isolated from systemic blood and the capacity to phagocytose opsonized Escherichia coli was evaluated by FACS analysis. RESULTS : No BT was observed in Con and chronic PVL animals but 11/20 LC (55%) and six out of six acute PVL (100%) presented with BT. In the presence of BT, PMNL from PVL as well as LC rats showed significantly increased phagocytic activity as compared with controls. In contrast, PMNL from animals without BT, whether PVL or LC, exhibited phagocytic activity similar to those from control rats. The number of PMNLs involved in the phagocytic process was significantly increased only in portal-hypertensive rats with but not without BT as compared with controls. Norfloxacin did prevent BT in acute PVL animals, thereby correcting the increase in phagocytic capacity in PMNL. CONCLUSIONS : Cirrhosis per se is not associated with alterations of the phagocytic capacity of PMNL. The occurrence of BT, however, increases the phagocytic capacity of PMNL, being observed likewise in prehepatic portal hypertension, indicating an in vivo’priming’ of PMNL by BT independent of cirrhosis.

Noack S, Kloden W, Bley T (2008) Modeling synchronous growth of bacterial populations in phased cultivation. Bioprocess Biosyst Eng 31 :435-443


The phasing technique is a method for synchronizing cell populations in a bioreactor. Periodic changes of substrate supply and depletion can provoke a cell cycle phasing of originally stochastic scattered proliferation patterns. Synchronized cell populations characterized by changes in DNA content distribution can be monitored by flow cytometry. Thus, studies of the dynamics of single cells in specific cell cycle phases are facilitated. Here we present an age structured model framework investigating synchronized populations using delay differential equations. Applying the framework not only cell populations synchronously increasing under balanced growth conditions, but also synchronized cultures growing in continuous phasing experiments can be described. A process model developed for describing phased cultures was fitted to growth data obtained from a synchronous cultivation of Cupriavidus necator. Its potential utility is demonstrated by a quantitative process description and by its ability to identify ways in which the grade of synchrony could be improved.

Parisi MG, Li H, Jouvet LB, Dyrynda EA, Parrinello N, Cammarata M, Roch P (2008) Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria. Fish Shellfish Immunol 25 :834-840


Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.

Pathirana RD, O’Brien-Simpson NM, Visvanathan K, Hamilton JA, Reynolds EC (2008) The role of the RgpA-Kgp proteinase-adhesin complexes in the adherence of Porphyromonas gingivalis to fibroblasts. Microbiology 154 :2904-2911


Porphyromonas gingivalis strains W50 and ATCC 33277 were shown to bind to cultured human fibroblast (MRC-5) cells using flow cytometry. As the concentration of P. gingivalis strain W50 cells was increased relative to the concentration of MRC-5 cells, the number of W50 cells bound per MRC-5 cell increased, as did the percentage of MRC-5 cells with bacteria bound. However, this relationship was only seen for P. gingivalis strain ATCC 33277 at low cell concentrations : at high bacterial cell concentrations strain ATCC 33277 auto-aggregated and binding to the MRC-5 cells decreased. Strain W50 was therefore chosen to study the role of the surface proteinase-adhesin complexes (RgpA-Kgp complexes) in binding to MRC-5 cells. P. gingivalis W50 cells treated with an inhibitor of the RgpA-Kgp complexes exhibited reduced binding to MRC-5 cells. The purified active and proteinase-inactive RgpA-Kgp complexes competitively inhibited binding of W50 to MRC-5 cells, and isogenic mutants of W50 lacking RgpA/B and Kgp displayed reduced binding. P. gingivalis W50 mutant cells lacking Kgp exhibited the lowest binding to MRC-5 cells, suggesting an important role for this proteinase and its associated adhesins in binding to fibroblasts.

Pezzicoli A, Santi I, Lauer P, Rosini R, Rinaudo D, Grandi G, Telford JL, Soriani M (2008) Pilus backbone contributes to group B Streptococcus paracellular translocation through epithelial cells. J Infect Dis 198 :890-898


We have recently shown that group B Streptococcus (GBS) crosses the epithelial barrier by a paracellular route. Here, we show that, although deletion of the pilus backbone protein did not affect GBS adhesiveness, it reduced the pathogen’s capacity to transcytose through differentiated human epithelial cells. In addition, contrary to our expectation, a strain with a mutant pilus ancillary protein and reduced adhesiveness translocated through the epithelial monolayer in a fashion identical to that of the isogenic wild-type strain. To monitor the localization of pili during GBS paracytosis, we performed 3-dimensional confocal experiments. By this approach, we observed that pili located in the intercellular space ahead of translocating bacteria. These results were also confirmed by a novel in vitro model of GBS infection in which bacteria bind to epithelial surfaces against the action of gravitation. These findings suggest a dual role for pilus components during the critical steps leading to GBS dissemination in the host.

Pozner RG, Collado S, de Giusti CJ, Ure AE, Biedma ME, Romanowski V, Schattner M, Gomez RM (2008) Astrocyte response to Junin virus infection. Neurosci Lett 445 :31-35


In a previous study of experimental murine encephalitis induced by Junin virus (JV), an arenavirus, we showed increased expression of iNOS by unidentified cells, concomitant with the astrocyte reaction. The specific inhibition of iNOS was associated with greater mortality but lower astrocytosis, suggesting that the protective role of nitric oxide (NO) synthesized by iNOS was related to enhanced astrocyte activation, representing a beneficial cellular response to virus-induced central nervous system damage. In the present work, cultured astrocytes were used to study whether JV infection could trigger iNOS expression and assess its eventual relationship with viral replication, glial fibrilary acidic protein (GFAP) expression levels and the presence of apoptosis. We found that JV infection of astrocytes did not induce apoptosis but produced both increased iNOS synthesis, detected by immunocytochemistry and fluorescence activated cell sorting (FACS) analysis, and increased NO, which was indirectly measured by nitrite/nitrate levels. These changes occurred early relative to the increases in GFAP expression, as detected by immunocytochemistry, FACS analysis and RT-PCR. The fact that iNOS inhibition abolished enhanced GFAP expression in infected monolayers suggests that NO was directly involved. In addition, iNOS inhibition enhanced virus replication. Together with data from confocal microscopy, these results suggest that JV induces iNOS expression in infected astrocytes and that the resulting NO has an important role both in reducing viral replication and in enhancing subsequent astrocyte activation.

Prorot A, Eskicioglu C, Droste R, Dagot C, Leprat P (2008) Assessment of physiological state of microorganisms in activated sludge with flow cytometry : application for monitoring sludge production minimization. J Ind Microbiol Biotechnol 35 :1261-1268


Many sludge reduction processes have been studied for the minimization of sludge production in biological wastewater treatment. The investigations on most of these processes have monitored the increase of the soluble chemical oxygen demand, the sludge mass reduction, or the decrease of the floc size, but little information has been obtained on cell lysis and the change of the biological cell activity. However, employing any strategy for reducing sludge production may have an impact of microbial community in biological wastewater treatment process. This impact may influence the sludge characteristics and the quality of effluent. The objective of this study concerns the determination of the physiological state of activated sludge microorganisms during a sludge minimization process. A thermal treatment at 80 degrees C for 5, 20, 40 and 60 min was chosen in this study. Staining bacteria with CTC and SYTOX green was used to evaluate biological cell activity and viability of cell types contained in activated sludge, respectively. The monitoring of cell activity and viability was performed using flow cytometry (FCM) analysis before and after thermal treatment of activated sludge. Results indicated an increase in the number of permeabilized cells and a decrease in the number of active cells, subsequent to the thermal treatment. The study also confirms the potential of FCM to successfully evaluate the physiological heterogeneity of an activated sludge bacterial population. Moreover, the experimentally observed correlations between the FCM results and the organic matter solubilization in activated sludge samples during thermal treatment revealed that the increase in the soluble organic matter concentration was predominantly due to an intracellular material release. Identifying the increase in activated sludge hydrolysis requires a precise knowledge of the involved mechanisms, and this study indicated that the FCM, used in conjunction with specific probes, could be a useful tool.

Quan DN, Cooper MD, Potter JL, Roberts MH, Cheng H, Jarvis GA (2008) TREM-2 binds to lipooligosaccharides of Neisseria gonorrhoeae and is expressed on reproductive tract epithelial cells. Mucosal Immunol 1 :229-238


Triggering receptor expressed on myeloid cells-2 (TREM-2) is an innate immune receptor that initiates cellular activation upon ligation. In this study, we examined the interaction of TREM-2 with Neisseria gonorrhoeae using murine TREM-2A, as it has been reported to recognize bacterial ligands. Using a whole-bacteria enzyme-linked immunosorbent assay (ELISA), TREM-2A bound to all six strains in variable degrees. Far-western blots of gonococcal outer membranes revealed TREM-2A binding to lipooligosaccharide (LOS) and opacity (Opa) protein, with predominant binding to LOS. Binding of TREM-2A to LOS was confirmed by ELISA and surface plasmon resonance. O-deacylation of the lipid A significantly reduced binding. Flow cytometry and reporter cell assays showed that gonococci bound to TREM-2A-transfected cells and induced transmembrane signaling. In humans, TREM-2 was constitutively expressed by genitourinary and fallopian tube epithelial cells, both of which are primary targets of gonococcal invasion. Ligation of TREM-2 by LOS induced interleukin-6 production in HeLa cervical carcinoma cells. To our knowledge, this is the first report of the expression of human TREM-2 by cells deriving from a non-myeloid lineage. We conclude that gonococci can interact with TREM-2 receptors through binding to LOS and Opa protein and initiate cell signaling and cytokine production.

Ragnarsson EG, Schoultz I, Gullberg E, Carlsson AH, Tafazoli F, Lerm M, Magnusson KE, Soderholm JD, Artursson P (2008) Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis. Lab Invest 88 :1215-1226


Crohn’s disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv+ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P<0.01) and ileal mucosa (268+/-47% of control ; P<0.01), whereas inv bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size- and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of beta1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of beta1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.

Ratcliff WC, Kadam SV, Denison RF (2008) Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia. FEMS Microbiol Ecol 65 :391-399


The carbon that rhizobia in root nodules receive from their host powers both N(2) fixation, which mainly benefits the host, and rhizobium reproduction. Rhizobia also store energy in the lipid poly-3-hydroxybutyrate (PHB), which may enhance rhizobium survival when they are carbon limited, either in nodules or in the soil between hosts. There can be a conflict of interest between rhizobia and legumes over the rate of PHB accumulation, due to a metabolic tradeoff between N(2) fixation and PHB accumulation. To quantify the benefits of PHB to carbon-limited rhizobia, populations of genetically uniform rhizobia with high vs. low PHB (confirmed by flow cytometry) were generated by fractionating Sinorhizobium meliloti via density gradient centrifugation, and also by harvesting cells at early vs. late stationary phase. These rhizobia were starved for 165 days. PHB use during starvation was highly predictive of both initial reproduction and long-term population maintenance. Cultured S. meliloti accumulated enough PHB to triple their initial population size when starved, and to persist for c. 150 days before the population fell below its initial value. During the first 21 days of nodule growth, undifferentiated S. meliloti within alfalfa nodules accumulated enough PHB to support significant increases in reproduction and survival during starvation.

Rochet V, Rigottier-Gois L, Ledaire A, Andrieux C, Sutren M, Rabot S, Mogenet A, Bresson JL, Cools S, Picard C, Goupil-Feuillerat N, Dore J (2008) Survival of Bifidobacterium animalis DN-173 010 in the faecal microbiota after administration in lyophilised form or in fermented product - a randomised study in healthy adults. J Mol Microbiol Biotechnol 14 :128-136


The survival of Bifidobacterium animalis strain DN-173 010 was assessed after its ingestion in a fermented product or in a lyophilised form. Twelve healthy subjects were included in a randomised, open study with 2 parallel groups. The composition and activities of the faecal microbiota were monitored before (10-day baseline step), during (1-week product administration step) and after (10-day follow-up step) the ingestion of 1 of the 2 products. A colony immunoblotting method, fluorescent in situ hybridisation with group-specific DNA probes, and temporal temperature gradient gel electrophoresis using group-specific primers were carried out to compare survival of B. animalis strain DN-173 010 after ingestion of the 2 products, together with analyses of enzyme activities and faecal metabolites. At the end of the supplementation step, the mean number of B. animalis DN-173 010 quantified by immunodetection in the faeces of 5 of 6 subjects in each treatment group was >/=10(8) colony-forming units/g faeces. These numbers corresponded to an average survival of 22% for the lyophilised form and 20% for the fermented product. At the same step, the PCR temporal temperature gradient gel electrophoresis profiles showed a double band corresponding to the B. animalis DN-173 010 pattern for 11 subjects. No major modification was observed during the trial in either the dominant members of the faecal microbiota assessed by fluorescent in situ hybridisation or their activities. In conclusion, we show that the lyophilised form of B. animalis DN-173 010 survives transit and could represent a more convenient form to administer for long-term clinical trials.

Rodriguez SB, Thornton RJ (2008) Use of flow cytometry with fluorescent antibodies in real-time monitoring of simultaneously inoculated alcoholic-malolactic fermentation of Chardonnay. Lett Appl Microbiol 46 :38-42


AIMS : To monitor in real-time the changes in microbial populations and chemistry of grape juice simultaneously inoculated with Saccharomyces cerevisiae and Oenococcus oeni. METHODS AND RESULTS : Viable populations of S. cerevisiae and O. oeni in Chardonnay fermentations were identified and quantified using fluorescent dyes and fluorescently labelled antibodies in a flow cytometric assay. Fermentation chemistry was monitored using Fourier transform infrared (FTIR) spectroscopy, except for malic acid which was measured enzymatically. Malic acid utilization by O. oeni was greatest in the presence of the yeast Cepage. Growth of O. oeni was substantially slower in the presence of the yeast VL1. The three yeasts had similar fermentation rates in the presence and absence of O. oeni. CONCLUSIONS : Viable and nonviable yeast and bacterial populations can be rapidly discriminated in simultaneous malolactic-alcoholic wine fermentations using antibodies, fluorescent dyes and flow cytometry. SIGNIFICANCE AND IMPACT OF THE STUDY : This is the first study using fluorescently labelled antibodies to discriminate and monitor yeast and bacterial populations in wine fermentations and offers a new approach to investigating microbial interactions in wine fermentations.

Rodriguez-Porrata B, Novo M, Guillamon J, Rozes N, Mas A, Otero RC (2008) Vitality enhancement of the rehydrated active dry wine yeast. Int J Food Microbiol 126 :116-122


In winemaking, spontaneous grape must fermentations have been replaced by inoculation of commercial active dry wine yeast (ADWY). Yeast rehydration is the key to avoiding stuck and sluggish fermentations. Despite the importance of this step, not enough is known about what this process implies for winemaking as a whole or about what kind of practices could help to improve it. The main aim of this study is to determine the best yeast rehydration conditions for ensuring good cell viability and vitality before inoculation into the must. The experimental rehydration media in this study can be divided into four groups : carbon and nitrogen compounds, metallic ions, oxidant and antioxidant agents, and membrane fluidity agents. We studied the biochemical and biophysical behaviour of ADWY after rehydration in the various media under oenological conditions, i.e. incubation at 37 degrees C for 30 min. The viability of rehydrated yeast cells was evaluated by plating, and assessed by fluorescence microscopy and flow cytometry. The vitality of rehydrated cells was estimated by indirect impedance. The rehydrating solution complemented with magnesium provided the best vitality rate because the time taken to reach the activity threshold was cut by two thirds. This improvement was also illustrated by the less time needed to stop the leakage of intracellular compounds during the rehydration process.

Ronald LS, Yakovenko O, Yazvenko N, Chattopadhyay S, Aprikian P, Thomas WE, Sokurenko EV (2008) Adaptive mutations in the signal peptide of the type 1 fimbrial adhesin of uropathogenic Escherichia coli. Proc Natl Acad Sci U S A 105 :10937-10942


Signal peptides (SPs) are critical for protein transport across cellular membranes, have a highly conserved structure, and are cleaved from the mature protein upon translocation. Here, we report that naturally occurring mutations in the SP of the adhesive, tip-associated subunit of type 1 fimbriae (FimH) are positively selected in uropathogenic Escherichia coli. On the one hand, these mutations have a detrimental effect, with reduced FimH transport across the inner membrane, fewer FimH and fimbriae expressed on the bacterial surface, and decreased bacterial adhesion under flow conditions. On the other hand, the fimbriae expressed by the mutants are significantly longer on average, with many fimbriae able to stretch to >20 microm in length. More surprisingly, the SP mutant bacteria display an increased ability to resist detachment from the surface upon a switch from high to low flow. This functional effect of longer fimbriae highlights the importance of the nonadhesive fimbrial rod for adhesive function. Also, whereas bacterial adhesion to bladder epithelial cells was preserved in most mutants, binding to and killing by human neutrophils was decreased, providing an additional reason the SP mutations are relatively common among uropathogenic strains. Thus, this study demonstrates how mutations in an SP, while decreasing transport function and not affecting the final structure of the translocated protein, can lead to functional gains of the extracellular organelles that incorporate the protein and overall adaptive changes in the organism’s fitness.

Sahin-Yilmaz A, Baroody F, Markaryan A, Thompson K, Wall GM, Naclerio R (2008) Effect of topical ciprofloxacin/dexamethasone or dexamethasone alone on acute Streptococcus pneumoniae rhinosinusitis in mice. Otolaryngol Head Neck Surg 138 :340-346


OBJECTIVE : To test whether intranasal ciprofloxacin/dexamethasone or dexamethasone alone affects the course of acute bacterial rhinosinusitis in mice. STUDY DESIGN : We performed a randomized, double-blind, parallel, placebo-controlled study in mice. SUBJECTS AND METHODS : Three groups of 10 C57Bl/6 mice were infected with Streptococcus pneumoniae, and then 1 day later randomized to treatment with placebo, ciprofloxacin plus dexamethasone, or dexamethasone. The mice were killed 3 or 10 days after treatment was begun. RESULTS : The placebo-treated mice became infected and developed an inflammatory cell infiltration in their sinuses. None of the treatments significantly affected the course of the illness. CONCLUSION : The lack of topical, intranasal efficacy of ciprofloxacin and dexamethasone could be attributed to subpotent dosage, rapid nasal clearance, or inability of the drops to reach the site of infection. Treatment with dexamethasone neither improved nor worsened the bacterial infection.

Sato T, Fujieda M, Maeda A, Tanaka E, Miyamura M, Chikamoto H, Hisano M, Akioka Y, Ishiura Y, Dohno S, Hattori M, Wakiguchi H (2008) Monitoring of Epstein-Barr virus load and killer T cells in pediatric renal transplant recipients. Clin Nephrol 70 :393-403


AIM : The aim of this study is to establish a monitoring method to prevent Epstein-Barr virus (EBV)-associated symptoms including post-transplant lymphoproliferative disorder (PTLD) that occur after pediatric renal transplantation. SUBJECTS AND METHODS : Circulating EBV loads were quantified by real-time PCR every 1 - 3 months after grafting in 22 pediatric recipients (13 EBV-seronegative [R(-)] and 9 EBV-seropositive [R(+)] recipients before grafting). The peripheral blood cell populations of non-specific activated killer cells (CD8+HLA-DR+ phenotype) in 13 R(-) recipients and EBV-specific cytotoxic T cells (CTLs) reactive with a tetramer expressing HLA-A24-restricted EBV-specific antigens in 8 of 13 R(-) recipients were determined by flow cytometry. RESULTS : EBV-associated symptoms including PTLD (2 cases) were found in 4 R(-) and none of the R(+) recipients. The maximum of EBV load in the R(-) group was significantly higher that in the R(+) group. In R(-) recipients, 4 symptomatic cases had significantly more EBV genome than asymptomatic cases. EBV-specific CTLs were detected in 6 of the 8 R(-) recipients, but these CTLs could not be detected in 1 of the 2 cases at onset of PTLD. The percentage of CD8+HLA-DR+ cells was significantly higher in asymptomatic recipients than in recipients with EBV-associated symptoms whose EBV loads were over 400 copies/microg DNA. CONCLUSION : Monitoring of killer T cells and EBV loads may allow assessment of the risk of EBV-associated symptoms, and high EBV loads and low EBV-specific and/or non-specific CTL responses may be predictive for development of EBV-associated symptoms such as PTLD.

Schellenberg J, Blake Ball T, Lane M, Cheang M, Plummer F (2008) Flow cytometric quantification of bacteria in vaginal swab samples self-collected by adolescents attending a gynecology clinic. J Microbiol Methods 73 :216-226


Bacterial vaginosis (BV) is an important risk factor in reproductive health outcomes, such as pre-term birth and sexually transmitted infections including HIV. However, its etiology, diagnosis and treatment remain poorly defined. We evaluated flow cytometry as a tool to quantify total bacterial cells in vaginal specimens self-collected longitudinally by adolescents. BV was diagnosed by Gram-stain (criteria of Hay and Ison). Average flow cytometric counts of bacterial cell-units (BCU) was log(10) 8.04 per gram sample and was found to correlate with sample weight (p<0.0001). BV was frequently observed in this group, with 22 of 32 participants (69%) diagnosed with BV for at least one timepoint. Surprisingly, increased BCU was associated with normal Hay-Ison score (p=0.0003), even when adjusting for sample weight (p=0.02). Since presence and quantity of Lactobacillus defines normal vaginal microbiology (ie. absence of BV), this result indicates a possible bias towards dominance of Lactobacillus cells in measurements of "total" BCU. Increased BCU per gram was associated in multivariate analysis with longer self-reported time since last menstruation (p=0.004) and last sexual intercourse (p=0.007). Sperm was detected in 3 samples provided by those reporting sexual intercourse in the previous 24 h. Light-scattering profiles of bacteria and vaginal cells in samples collected over time from an individual were often identical and distinct from other individuals. To our knowledge, this is the first description of flow cytometry for analysis of commensal bacteria in vaginal specimens. Further development may help to illuminate the complex dynamics of vaginal microbial communities underlying BV.

Sellman BR, Timofeyeva Y, Nanra J, Scott A, Fulginiti JP, Matsuka YV, Baker SM (2008) Expression of Staphylococcus epidermidis SdrG increases following exposure to an in vivo environment. Infect Immun 76 :2950-2957


SdrG is a surface-associated fibrinogen binding protein present in most strains of Staphylococcus epidermidis. Surface expression of SdrG was not detected by flow cytometry or immunofluorescence microscopy on S. epidermidis 0-47 grown in nutrient broth or in the presence of human serum. sdrG transcript levels increased 1 hour following a shift from growth in nutrient broth to growth in the bloodstream of a mouse and resulted in a concomitant increase in protein levels as detected by immunofluorescence microscopy. The environmental signal(s) resulting in the increase in expression is elusive, as growth under conditions known to mimic in vivo conditions (elevated CO(2), iron limitation, human serum, and citrated human blood) did not affect expression of SdrG. Immunizing mice with either the N1N2N3 (amino acids 50 to 597) or N2N3 (amino acids 273 to 597) subdomain of the N-terminal A domain of recombinant SdrG (rSdrG) elicited a robust antibody response ; however, only mice vaccinated with rSdrG(N23) exhibited a significant reduction in 0-47 recovered after experimental infection. Since SdrG is expressed early during infection in response to specific host environmental cues present in the bloodstream and since antibodies to it are effective in reducing bacteremia, SdrG possesses attributes of a vaccine component effective against the pathogenic form of the ubiquitous human commensal S. epidermidis.

Senigl F, Plachy J, Hejnar J (2008) The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing. J Virol 82 :7818-7827


Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

Shah P, Romero DG, Swiatlo E (2008) Role of polyamine transport in Streptococcus pneumoniae response to physiological stress and murine septicemia. Microb Pathog 45 :167-172


Streptococcus pneumoniae has a potential ABC-type transporter (Pot) for extracellular polyamines. Polyamine transport protein D (PotD) is a membrane-associated, surface protein that putatively binds polyamines such as putrescine and spermidine. In this study we used quantitative PCR (qPCR) to analyze potD mRNA expression under physiologically relevant stress conditions in vitro, during in vivo infection, and in the presence of polyamines and choline. Expression of potD mRNA was elevated 2- and 4-fold when cells were grown at either 34 or 42 degrees C, respectively, in a choline restricted environment. Expression increased by 5- and 11-fold in response to oxidative stress in either low or high choline environments, respectively. Putrescine led to an increase in potD mRNA transcription, while choline and spermidine resulted in decreased gene expression. Transcription of potD in pneumococci harvested from blood of systemically infected mice was 43-fold higher compared to in vitro transcription levels. Flow cytometry analysis using PotD antiserum confirmed increased PotD expression on the pneumococcal surface. These results indicate that polyamines and polyamine transport systems potentially play an important role in Streptococcus pneumoniae pathogenesis, and may be important for bacterial response to temperature shock, oxidative stress, choline limitation and in vivo growth.

Shi L, Muller S, Harms H, Wick LY (2008) Effect of electrokinetic transport on the vulnerability of PAH-degrading bacteria in a model aquifer. Environ Geochem Health 30 :177-182


There has been increasing interest in employing electro-bioremediation, a hybrid technology of bioremediation and electrokinetics, to overcome the low bioavailability of hydrophobic organic contaminants (HOC) by homogenizing sorption-retarded HOC and immobilised microorganisms. Present electro-remediation approaches mainly aim at macroscale pollutant extraction and tend to neglect possible impacts of direct current (DC) on the physiology of microorganisms. The effect of weak electric fields (X = 1 V cm(-1)) on the fitness of electrokinetically dispersed fluorene-degrading Sphingomonas sp. LB126 in bench-scale model aquifers was investigated by flow cytometry using propidium iodide (PI) as an indicator that distinguishes between PI-permeable (cells with porous membranes, i.e. dead or vulnerable) and PI-impermeable bacteria. After 15.5 h of DC treatment 56% of all cells recovered were dispersed at the centimetre scale relative to 29% in the absence of DC. There was no overall negative effect of the 15.5-h DC treatment on cell vulnerability, as 7.0% of the DC-treated bacteria exhibited PI-staining compared to 6.5% of the control population. Minor differences were observed in the subpopulation that had been mobilised by electroosmosis with an approximately twofold increase in the percentage of PI-stained cells relative to the control. Enhanced PI staining did not correlate with reduced culturability of the cells on rich-medium agar plates. Relative to the control, DC-treated cells mobilised by electroosmosis were threefold more culturable, confirming earlier data that that PI-cell membrane permeability does not always indicate reduced viability of oligotrophic environmental bacteria. Our findings suggest that electrokinetics is a valuable mechanism to transport viable and culturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria in soil or sediments.

Shi L, Muller S, Harms H, Wick LY (2008) Factors influencing the electrokinetic dispersion of PAH-degrading bacteria in a laboratory model aquifer. Appl Microbiol Biotechnol 80 :507-515


Despite growing interest in the electro-bioremediation of contaminated soil it is still largely unknown to which degree weak electric fields influence the fate of contaminant-degrading microorganisms in the sub-surface. Here we evaluate the factors influencing the electrokinetic transport and deposition of fluorene-degrading Sphingomonas sp. LB126 in a laboratory model aquifer exposed to a direct current (DC) electric field (1 V cm(-1)) typically used in electro-bioremediation measures. The influence of cell size, cell membrane integrity, cell chromosome contents (all assessed by flow cytometry), cell surface charge and cell hydrophobicity on the spatial distribution of the suspended and matrix-bound cells after 15 h of DC-treatment was evaluated. In presence of DC the cells were predominantly mobilised by electroosmosis to the cathode with an apparent velocity of 0.6 cm h(-1), whereas a minor fraction only of the cells augmented was mobilised to the anode by electrophoresis. Different electrokinetic behaviour of individual cells could be solely attributed to intra-population heterogeneity of the cell surface charge. In the absence of DC by contrast, a Gaussian-type distribution of bacteria around the point of injection was found. DC had no influence on the deposition efficiency, as the glass beads in presence and absence of an electric field retained quasi-equal fractions of the cells. Propidium iodide staining and flow cytometry analysis of the cells indicated the absence of negative influences of DC on the cell wall integrity of electrokinetically mobilised cells and thus point at unchanged physiological fitness of electrokinetically mobilised bacteria.

Shin J, Ji S, Choi Y (2008) Ability of oral bacteria to induce tissue-destructive molecules from human neutrophils. Oral Dis 14 :327-334


AIM : The induction of tissue-destructive molecules from neutrophils by periodontopathic bacteria has been suggested as one of the mechanisms of periodontal destruction. The aim of this study was to determine whether the ability to stimulate neutrophils is an authentic characteristic of periodontopathic bacteria.METHODS : We evaluated, along with phagocytosis, the production of reactive oxygen species (ROS), matrix metalloproteinase-8 (MMP-8), and interleukin-1beta by neutrophils in response to non-periodontopathic Streptococcus sanguinis and periodontopathic bacteria Fusobacterium nucleatum and Treponema denticola, in the absence or presence of antibodies. Phagocytosis, the death of neutrophils, and intracellular ROS production were measured by flow cytometry and the concentrations of MMP-8 and interleukin-1beta secreted into medium were determined by enzyme-linked immunosorbent assay.RESULTS : S. sanguinis and F. nucleatum induced greater production of ROS, MMP-8, and interleukin-1beta than did T. denticola. The levels of tissue-destructive molecules produced by neutrophils had a positive correlation with phagocytosis. Opsonization of bacteria with antibodies significantly increased phagocytosis and ROS production and release, thus increasing both bacterial clearance and potential tissue damage.CONCLUSION : The ability of oral bacteria to induce tissue-destructive molecules from neutrophils is not an inherent characteristic of periodontopathic bacteria, which would provide a new insight into the role of neutrophils in periodontal destruction.

Sillanpaa J, Nallapareddy SR, Prakash VP, Qin X, Hook M, Weinstock GM, Murray BE (2008) Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium. Microbiology 154 :3199-3211


Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10 ; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene ; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a ’ladder’ pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Slobodskaya O, Laarman A, Spaan WJ (2008) Intracellular restriction of a productive noncytopathic coronavirus infection. J Virol 82 :451-460


Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10(7) to 10(8) PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.

Smelt JP, Bos AP, Kort R, Brul S (2008) Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells. Int J Food Microbiol 128 :34-40


Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell.

Sporri R, Joller N, Hilbi H, Oxenius A (2008) A novel role for neutrophils as critical activators of NK cells. J Immunol 181 :7121-7130


Neutrophils are essential players in innate immune responses to bacterial infection. Despite the striking resistance of Legionella pneumophila (Lpn) to bactericidal neutrophil function, neutrophil granulocytes are important effectors in the resolution of legionellosis. Indeed, mice depleted of neutrophils were unable to clear Lpn due to a lack of the critical cytokine IFN-gamma, which is produced by NK cells. We demonstrate that this can be ascribed to a previously unappreciated role of neutrophils as major NK cell activators. In response to Lpn infection, neutrophils activate caspase-1 and produce mature IL-18, which is indispensable for the activation of NK cells. Furthermore, we show that the IL-12p70 response in Lpn-infected neutropenic mice is also severely reduced and that the Lpn-induced IFN-gamma production by NK cells is strictly dependent on IL-12. However, since dendritic cells, and not neutrophils, are the source of Lpn-induced IL-12, its paucity is a consequence of the absence of IFN-gamma produced by NK cells rather than the absence of neutrophils per se. Therefore, neutrophil-derived IL-18, in combination with dendritic cell-produced IL-12, triggers IFN-gamma synthesis in NK cells in Lpn-infected mice. We propose a novel central role for neutrophils as essential IL-18 producers and hence NK cell "helpers" in bacterial infection.

Srivastava SY, de Silva AM (2008) Reciprocal expression of ospA and ospC in single cells of Borrelia burgdorferi. J Bacteriol 190 :3429-3433


Outer surface proteins (Osp) A and C of the Lyme disease spirochete (Borrelia burgdorferi) are selectively produced and of functional significance in the tick vector and mammalian host, respectively. Some studies indicate a simple, reciprocal relationship where the signals and pathways that turn on ospC also turn off ospA. Other studies indicate a more complex regulation where many spirochetes produce both proteins and others produce one of the proteins or neither protein. Here, we have used flow cytometry to characterize ospA and ospC transcript and protein levels in individual bacterial cells grown in culture. The results support a simple, reciprocal model where, at the level of single cells, the transcription of ospC is linked to the repression of ospA. We also demonstrate that under conditions conducive for OspC production, spirochetes display an "all or none" response, with some cells displaying high levels of ospC transcription and others demonstrating little or no transcription. Despite the reciprocal regulation of ospA and ospC at the single-cell level, we propose that spirochetes display an array of phenotypes due to stochasticity in the pathways that regulate osp expression and the slow turnover of outer surface proteins.

Stachowski-Haberkorn S, Becker B, Marie D, Haberkorn H, Coroller L, de la Broise D (2008) Impact of Roundup on the marine microbial community, as shown by an in situ microcosm experiment. Aquat Toxicol 89 :232-241


The effects of the herbicide Roundup (glyphosate) on natural marine microbial communities were assessed in a 7-day field experiment using microcosms. Bottles were maintained underwater at 6m depth, and 10% of their water content was changed every other day. The comparison of control microcosms and surrounding surface water showed that the microcosm system tested here can be considered as representative of the natural surrounding environment. A temporal temperature gradient gel electrophoresis (TTGE) was run on 16S and 18S rDNA-amplified extracts from the whole microbial community. Cluster analysis of the 16S gel showed differences between control and treatment fingerprints for Roundup at 1 microg L(-1) (ANOSIM, p=0.055 ; R=0.53), and 10 microg L(-1) (ANOSIM, p=0.086 ; R=0.40). Flow cytometry analysis revealed a significant increase in the prasinophyte-like population when Roundup concentration was increased to 10 microg L(-1). This study demonstrates that a disturbance was caused to the marine microbial community exposed to 1 microg L(-1) Roundup concentration, a value typical of those reported in coastal waters during a run-off event.

Stauffer BA, Schaffner RA, Wazniak C, Caron DA (2008) Immunofluorescence flow cytometry technique for enumeration of the brown-tide alga, Aureococcus anophagefferens. Appl Environ Microbiol 74 :6931-6940


A new immunologically based flow cytometry (IFCM) technique was developed to enumerate Aureococcus anophagefferens, a small pelagophyte alga that is the cause of "brown tides" in bays and estuaries of the mid-Atlantic states along the U.S. coast. The method utilizes a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) to label the surface of A. anophagefferens cells which are then detected and enumerated by using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations, were determined. The FITC-MAb method was tested for cross-reactivity with nontarget, similarly sized, photoautotrophic protists, and the method was compared to an enzyme-linked immunosorbent assay (ELISA) using the same MAb. Comparisons of the IFCM technique to traditional microscopy enumeration of cultures and spiked environmental samples showed consistent agreement over several orders of magnitude (r(2) > 0.99). Comparisons of the IFCM and ELISA techniques for enumerating cells from a predation experiment showed a substantial overestimation (up to 10 times higher) of the ELISA in the presence of consumers of A. anophagefferens, presumably due to egested cell fragments that retained antigenicity, using the ELISA method, but were not characterized as whole algal cells by the IFCM method. Application of the IFCM method to environmental "brown-tide" samples taken from the coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundance levels throughout the course of a bloom and over a large range of abundance values. IFCM counts of the brown-tide alga from natural samples were consistently lower than those obtained using the ELISA method and were equivalent to those of the polyclonal immunofluorescence microscopy technique, since both methods discriminate intact cells. Overall, the IFCM approach was an accurate and relatively simple technique for the rapid enumeration of A. anophagefferens in natural samples over a wide range of abundance values (10(3) to 10(6) cells ml(-1)).

Stout-Delgado HW, Yang X, Walker WE, Tesar BM, Goldstein DR (2008) Aging impairs IFN regulatory factor 7 up-regulation in plasmacytoid dendritic cells during TLR9 activation. J Immunol 181 :6747-6756


Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-alpha in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with HSV-2, a viral pathogen that activates TLR9. We found that impaired IFN-alpha production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective up-regulation of IFN-regulatory factor 7, a key adaptor in the type I IFN pathway, as compared with younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-alpha defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.

Sung WS, Lee DG (2008) In vitro candidacidal action of Korean red ginseng saponins against Candida albicans. Biol Pharm Bull 31 :139-142


Korean red ginseng saponins (ginsenosides) have been reported as having various biological properties, but the antifungal effects and the mode of action of ginsenosides remain mostly unknown. In this study, saponins were isolated from Korean red ginseng, and the antifungal effects of ginsenosides were investigated. Ginsenosides showed fungicidal effects toward pathogenic fungi tested. To elucidate the antifungal mode of action of ginsenosides, flow cytometry analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest that ginsenosides may exert antifungal activity by disrupting the structure of cell membrane. The present study indicates that ginsenosides have considerable antifungal activity, deserving further investigation for clinical applications.

Szczotko M, Krogulska B, Krogulski A (2008) [Elaboration of the method for assessment of susceptibility to microbial growth of materials contacting with drinking water]. Rocz Panstw Zakl Hig 59 :103-111


Initial methodological study was conducted to determine the susceptibility to microbial growth of different materials applied in contact with drinking water. The purpose of this assay was to elaborate the method for determining the ability of different materials contacting with drinking water to promote microbial growth and to determine possible correlation of results obtained by two analytic methods : inoculation of microorganisms swabbed from materials surface into a medium and ATP level examination. The assay was conducted during 16 weeks in dynamic conditions using continuous flow reactor. Every two weeks swabbes from examinated materials were collected and the total number of microorganisms was determined after incubation in 22 degrees C for 72 hours and in 37 degrees C for 48 hours respectively. Determination of microorganisms number and ATP level were both examinated in water inlet. Difference in susceptibility to microbial growth of different materials used in contact with drinking water was observed. This was confirmed by both analytic methods. Microbial growth on the surface of negative control materials (glass, poliethylene and stainless steel) was several time less intensive than on positive control material (floor finish not destined for contact with drinking water). Correlation between the quantity of microorganisms and ATP level on the surface of the same kind of materials was confirmed. Usefulness of continous flow reactor was confirmed.

Tadepalli S, Stewart GC, Nagaraja TG, Jang SS, Narayanan SK (2008) Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity. Vet Microbiol 127 :89-96


Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs ; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.

Tadepalli S, Stewart GC, Nagaraja TG, Narayanan SK (2008) Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity. J Med Microbiol 57 :225-231


Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies : subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

Takizawa H, Eto K, Yoshikawa A, Nakauchi H, Takatsu K, Takaki S (2008) Growth and maturation of megakaryocytes is regulated by Lnk/Sh2b3 adaptor protein through crosstalk between cytokine- and integrin-mediated signals. Exp Hematol 36 :897-906


OBJECTIVE : Various cytokines and growth factors control the differentiation and maturation of megakaryocytes (MKs). However, the mechanism regulating platelet release from MKs is not well understood. Here, we investigated a role of Lnk/Sh2b3, an intracellular adaptor protein, in megakaryopoiesis. MATERIALS AND METHODS : Number of MK progenitor in bone marrow (BM) of wild-type or Lnk(-/-) mice and their sensitivity to thrombopoietin (TPO) were determined in colony-forming unit assay. Using BM-derived wild-type or Lnk(-/-) MKs stimulated with TPO, activation of the signaling molecules was biochemically analyzed and effect of integrin stimulation on TPO signals was studied by addition of vascular cell adhesion molecule (VCAM-1). Platelet production from MKs in the presence of VCAM-1 was counted by flow cytometry and their morphological change was observed by time-lapse microscopy. RESULTS : Lnk(-/-) mice showed elevated platelets and mature MKs due to enhanced sensitivity of progenitors to TPO. Erk1/2 phosphorylation induced by TPO was augmented and prolonged in Lnk(-/-) MKs while activation of signal transducers and activators of transcription (Stat)3, Stat5, and Akt was normal. Wild-type MKs, but not in Lnk(-/-) MKs on VCAM-1 showed reduced Stat5 phosphorylation and mitogen-activated protein kinases activation upon stimulation with TPO. Additionally, the presence of VCAM in culture accelerated spontaneous platelet release from mature wild-type MKs, but not from Lnk(-/-) MKs. CONCLUSIONS : Results suggest that contact of MKs with adhesion molecules via integrins might contribute to platelet release, which is under Lnk-mediated regulation of Stat-5 activation and show that Lnk functions in responses controlled by cell adhesion and in crosstalk between integrin- and cytokine-mediated signaling.

Tanikawa T, Ishikawa T, Maekawa T, Kuronane K, Imai Y (2008) Characterization of monoclonal immunoglobulin a and g against shiga toxin binding subunits produced by intranasal immunization. Scand J Immunol 68 :414-422


Immunoglobulin A (IgA) is considered to play a major role in protection of the mucosal surface. However, its immunological and biological properties have not been extensively studied because the production of IgA class monoclonal antibodies (mAbs) is difficult. We compared the properties of IgA and IgG mAbs against Shiga toxin B subunits (Stx1B). These mAbs were secreted from hybridomas that had been produced from mice after intranasal immunization with recombinant Stx1B and cholera toxin. The dose response curves for the binding of the IgA (clone G2G7) and IgG (clone D11C6) mAbs to immobilized Stx1B were similar, as revealed on ELISA. The majority of the IgA mAb formed dimers while the IgG mAb was monomeric, as judged by immunoblot analysis. The IgG mAb completely inhibited the binding of Stx1B to Burkitt’s lymphoma cell line Ramos, while the inhibition by the IgA mAb was only partial. The IgG mAb was able to neutralize the cytotoxicity of Stx1 holotoxin towards Vero cells, whereas the IgA mAb was not. The binding affinity of each binding site was compared by means of surface plasmon resonance analysis involving a capture method, with which the binding of soluble Stx1B to immobilized mAb was detected. The association rate was similar but the dissociation rate was twofold faster in the case of the IgA mAb, resulting in twofold higher affinity of the IgG mAb. These results suggest that one can obtain high affinity IgA mAb but toxin neutralization is another challenge as to therapeutic antibodies of the IgA class.

Thieme R, Rakosy-Tican E, Gavrilenko T, Antonova O, Schubert J, Nachtigall M, Heimbach U, Thieme T (2008) Novel somatic hybrids (Solanum tuberosum L.+Solanum tarnii) and their fertile BC1 progenies express extreme resistance to potato virus Y and late blight. Theor Appl Genet 116 :691-700


Solanum tarnii, a wild diploid, tuber-bearing Mexican species belonging to the series Pinnatisecta is highly resistant to Potato virus Y (PVY) and Colorado potato beetle and shows a strong hypersensitive reaction to Phytophthora infestans. Therefore, it could be a potential source of resistance to pathogens for potato breeders. S. tarnii (2n=2x=24) is reproductively isolated from tetraploid Solanum tuberosum and hence difficult to include in potato breeding programmes. In this study, interspecific somatic hybrids were produced for the first time by protoplast electrofusion of the cells of potato cv. Delikat (Solanum tuberosum L.) and Solanum tarnii. The hybrid nature of the regenerants was confirmed by simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers and by morphological analysis and flow cytometry. Selected somatic hybrids were successfully backcrossed with cv. Delikat. Parental lines, primary somatic hybrids and BC1 progeny were assessed for resistance to PVY by mechanical inoculation, grafting and exposure to viruliferous aphid vectors in the field, and resistance to late blight (P. infestans) by detached leaflet and whole tuber tests. The somatic hybrids showed no symptoms of viral infection and most of them displayed high levels of resistance to foliage blight. The BC1 progenies were highly resistant to PVY and a few were resistant to foliage blight. Selected hybrids and BC1 clones were evaluated in the field for tuber quality and tuber yield. Some BC1 clones produced yields of good quality tubers. The results confirm that both the resistance to PVY and to late blight of S. tarnii is expressed in somatic hybrids, and PVY resistance is transferred to BC1 progeny, whereas blight resistance is harder to transfer. Somatic hybridization again proved to be a valuable tool for producing pre-breeding material with increased genetic diversity.

Tijdens M, Hoogveld HL, Kamst-van Agterveld MP, Simis SG, Baudoux AC, Laanbroek HJ, Gons HJ (2008) Population dynamics and diversity of viruses, bacteria and phytoplankton in a shallow eutrophic lake. Microb Ecol 56 :29-42


We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 x 10(7) and 1.3 x 10(8) virus-like particles ml(-1) and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus-host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht.

Tomchuck SL, Zwezdaryk KJ, Coffelt SB, Waterman RS, Danka ES, Scandurro AB (2008) Toll-like receptors on human mesenchymal stem cells drive their migration and immunomodulating responses. Stem Cells 26 :99-107


Adult human bone marrow-derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll-like receptors (TLRs) mediate stress responses of other bone marrow-derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription-polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor kappaB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR-driven stress and immune modulating response for hMSCs that is critical to consider in the design of stem cell-based therapies.

Tomi N, Fukuyo Y, Arakawa S, Nakajima T (2008) Pro-inflammatory cytokine production from normal human fibroblasts is induced by Tannerella forsythia detaching factor. J Periodontal Res 43 :136-142


BACKGROUND AND OBJECTIVE : Tannerella forsythia is a periodontal pathogen. Recently, we have reported that the cytopathic component of T. forsythia contains two distinct factors. One arrests the cell cycle at the G2 phase and the other, named forsythia detaching factor, detaches adhesion-dependent immortalized human cells. In this study, we investigated the biological function of forsythia detaching factor using human normal fibroblasts. MATERIAL AND METHODS : A recombinant forsythia detaching factor, reported previously, was used. TIG-3 cells, cultured in the absence or presence of forsythia detaching factor, were lysed and the supernatant was analyzed by western blotting with polyclonal forsythia detaching factor antibodies. The cells were subsequently fractionated to isolate the cytoplasmic, mitochondrial and remaining fractions. In order to measure the activity of mitochondria using nicotinamide adenine dinucleotide-linked reductase, the water-soluble tetrazolium method was used. The mitochondrial oxidative membrane potential was estimated by measuring the oxidization-dependent fluorogenic conversion of dihydrotetramethylrosamine using flow cytometry. The concentration of interleukin-8 in the culture supernatant was assayed using a Human IL-8 ELISA kit. RESULTS : Forsythia detaching factor-treated cells detached from the substratum and aggregated from 3 to 24 h. Then, the detached cells resumed adhesion and proliferated after 48 h. The western blot analysis revealed that most forsythia detaching factor trans-located into the mitochondrial fraction. Forsythia detaching factor suppressed the nicotinamide adenine dinucleotide-linked reductase activity in a dose-dependent manner and consequently increased the mitochondrial oxidative membrane potential. The production of interleukin-8 was reinforced in forsythia detaching factor-treated cells at 72 h through an increase of the mitochondrial oxidative membrane potential. CONCLUSION : The forsythia detaching factor might be involved in the virulence of T. forsythia through induction of the pro-inflammatory cytokine interleukin-8.

Tong TZ, Zhang YY, Zheng SK, Yang J (2008) [Control of microbial communities achieved by pH adjustment and its influences on batch treatment of antibiotic wastewater]. Huan Jing Ke Xue 29 :338-343


Scanning electron microscope (SEM), Fluorescent in-situ hybridization (FISH)-Flow Cytometry (FCM) as well as Biolog method were used to discuss the effect of pH control during the batch treatment on the composition and catabolic diversity of the microbial communities obtained from antibiotic wastewater. The following results were obtained : 1) At the end of batch treatment, the percentages of yeast cells in three cultures amount to 88.20%, 54.43% and 1.75%, respectively, when pH levels are individually maintained at 4-5, 5-6 and 6.5-7.5 throughout three batch experiments. Correspondingly, the percentages of bacterial cells in three cultures increase with the increase of pH levels. 2) No significant differences are found among the catabolic diversity of three cultures while the yeast-predominant culture has slightly less catabolic activities in Biolog FF microplate. 3) When bacteria gradually develop to be the dominant species in the culture, gradually enhanced COD removals of 34.8%, 44.8% and 61.2%, respectively, are achieved.

Torres CE, Gibello A, Nande M, Martin M, Blanco A (2008) Fluorescent in situ hybridization and flow cytometry as tools to evaluate the treatments for the control of slime-forming enterobacteria in paper mills. Appl Microbiol Biotechnol 78 :889-897


Slime formation is a serious problem nowadays in the paper industry. Some enterobacteria are associated with the formation of slime deposits in paper and board mills. Detection and characterization of slime forming bacteria, belonging to the genus Enterobacter, Raoultella, and Klebsiella have been achieved by fluorescence in situ hybridization (FISH), using one probe based on the enterobacterial repetitive intergenic consensus sequence and other two rRNA targeted oligonucleotide probes. The effects of three kinds of antimicrobiological products (biocides, dispersants, and enzymes) on these enterobacterial cells were analyzed by flow cytometry (FC). Biocides B : utrol 1009 and 1072 were the most effective microbiocides against all enterobacterial cells analyzed, reaching 90% of dead bacteria after 24 h. However, the enzymatic treatment (Buzyme) was not equally efficient on enterobacteria and its microbiocide capacity varied depending on the type of microorganism. FISH and FC were effective tools to detect important slime forming enterobacteria and to select specific treatments to control microbial problems in the paper industry.

Tracy BP, Gaida SM, Papoutsakis ET (2008) Development and application of flow-cytometric techniques for analyzing and sorting endospore-forming clostridia. Appl Environ Microbiol 74 :7497-7506


The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.

Travers MA, Barbou A, Le Goic N, Huchette S, Paillard C, Koken M (2008) Construction of a stable GFP-tagged Vibrio harveyi strain for bacterial dynamics analysis of abalone infection. FEMS Microbiol Lett 289 :34-40


Vibrio harveyi is a bacterial marine pathogen that can cause fatal disease in a large range of vertebrates and invertebrates, including the commercially important marine gastropod, Haliotis tuberculata. Since 1997, strains of this bacterium have regularly been causing high mortalities in farmed and wild abalone populations. The way in which the pathogen enters into abalone and the disease transmission mechanisms are thus far unknown. Therefore, a pathogenic strain, ORM4, was green fluorescent protein-tagged and validated both for its growth characteristics and for its virulence as a genuine model for abalone disease. The strain allows V. harveyi quantification by flow cytometry in seawater and in abalone haemolymph as well as the in situ detection of the parasite inside abalone tissues.

Trnovsky J, Merz W, Della-Latta P, Wu F, Arendrup MC, Stender H (2008) Rapid and accurate identification of Candida albicans isolates by use of PNA FISHFlow. J Clin Microbiol 46 :1537-1540


We developed the simple, rapid (1 h), and accurate PNA FISH(Flow) method for the identification of Candida albicans. The method exploits unique in solution in situ hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques : flow cytometry and fluorescence microscopy.

Trotter-Mayo RN, Roberts MR (2008) Leptin acts in the periphery to protect thymocytes from glucocorticoid-mediated apoptosis in the absence of weight loss. Endocrinology 149 :5209-5218


Leptin is a member of the IL-6 cytokine family and is primarily produced by adipose tissue. At high enough concentration, leptin engages leptin receptors expressed in the hypothalamus that regulate a variety of functions, including induction of weight loss. Mice deficient in leptin (ob/ob) or leptin receptor (db/db) function exhibit thymic atrophy associated with a reduction in double-positive (DP) thymocytes. However, the mediator of such thymic atrophy remains to be identified, and the extent to which leptin acts in the periphery vs. the hypothalamus to promote thymocyte cellularity is unknown. In the present study, we first demonstrate that thymic cellularity and composition is fully restored in ob/ob mice subjected to adrenalectomy. Second, we observe that ob/ob mice treated with low-dose leptin peripherally but not centrally exhibit increased thymocyte cellularity in the absence of any weight loss or significant reduction in systemic corticosterone levels. Third, we demonstrate that reconstitution of db/db mice with wild-type bone marrow augments thymocyte cellularity and restores DP cell frequency despite elevated corticosterone levels. These and additional data support a mode of action whereby leptin acts in the periphery to reduce the sensitivity of DP thymocytes to glucocorticoid-mediated apoptosis in vivo. Strikingly, our data reveal that leptin’s actions on thymic cellularity in the periphery can be uncoupled from its anorectic actions in the hypothalamus.

Uyttendaele M, Rajkovic A, Van Houteghem N, Boon N, Thas O, Debevere J, Devlieghere F (2008) Multi-method approach indicates no presence of sub-lethally injured Listeria monocytogenes cells after mild heat treatment. Int J Food Microbiol 123 :262-268


Application of mild inactivation treatments follows an increasing trend in the food industry and is often combined with sub-optimal intrinsic product conditions to ensure appropriate level of microbial safety. Listeria monocytogenes was subjected to mild heat treatment (20 min at 60 degrees C) and subsequently exposed to various mild preservation conditions based on increased NaCl concentration and decreased pH. Recovery and resuscitation of L. monocytogenes cells were studied using various methods. Using 12-fold Most Probable Number (MPN) method no difference in the amount of recovered cells under adverse conditions was noted between heat-treated and non-treated L. monocytogenes cells. Time-to-detection method using on-line OD measurements showed that heat-treated L. monocytogenes cells reached detection limit faster in acidified media and NaCl supplemented media in comparison with non-heated control cells. Flow cytometry (FCM) analysis using 5-6-carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) staining showed presence of low numbers of viable cells. Overall, there was no indication of sub-lethal injury in L. monocytogenes cells after mild heat treatment.

Van de Walle GR, Peters ST, VanderVen BC, O’Callaghan DJ, Osterrieder N (2008) Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D. J Virol 82 :11859-11868


Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.

Velzeboer I, Hendriks AJ, Ragas AM, Van de Meent D (2008) Aquatic ecotoxicity tests of some nanomaterials. Environ Toxicol Chem 27 :1942-1947


Nanoparticles of TiO2, ZrO2, AL2O3, CeO2, fullerene (C60), single-walled carbon nanotubes, and polymethylmethacrylate were tested for ecotoxic effects using one or more ecotoxicity endpoints : Microtox (bacteria), pulse-amplitude modulation (algae), Chydotox (crustaceans), and Biolog (soil enzymes). No appreciable effects were observed at nominal concentrations of up to 100 mg/L. Dilution of nanoparticle suspensions, either in ultrapure (Milli-Q) water or in natural (pond) water, led to formation of larger particles, which settled easily. (Nano)particles in water were characterized by means of atomic force microscopy, energy-dispersive x-ray analysis, inductively coupled plasma-mass spectrometry, flow cytometry, and spectrophotometry. It is concluded that the absence of ecotoxicity is the result of low concentrations of free nanoparticles in the tests, and it is suggested that colloid (in)stability is of primary importance in explaining ecotoxic effects of nanoparticles in the natural environment.

Verkaik N, Brouwer E, Hooijkaas H, van Belkum A, van Wamel W (2008) Comparison of carboxylated and Penta-His microspheres for semi-quantitative measurement of antibody responses to His-tagged proteins. J Immunol Methods 335 :121-125


The Luminex system is a flow cytometry based tool that permits the simultaneous measurement of many analytes from just a single serum sample. The technology uses microspheres, which are available in different colors and can be coated with different kinds of biomolecules. For the immobilisation of His-tagged proteins, two types of beads can be used : chemically activated carboxylated beads or Penta-His beads, which have antibodies against His-tags on their surface. In this study, we compared carboxylated and Penta-His beads. For carboxylated as compared to Penta-His beads, the non-specific background is lower (Median Fluorescence Intensity ; MFI>250, 0% versus 15%), the specific signal intensity is higher (mean MFI 2860 versus 722) and not dependent on the configuration of the protein. Above all, the protein coupled carboxylated beads are useful over longer periods of time. Therefore, we conclude that for developing a multiplex assay for semi-quantitative measurement of antibody responses against His-tagged proteins the best microspheres to use are the carboxylated ones.

Vestergaard LS, Lusingu JP, Nielsen MA, Mmbando BP, Dodoo D, Akanmori BD, Alifrangis M, Bygbjerg IC, Lemnge MM, Staalsoe T, Hviid L, Theander TG (2008) Differences in human antibody reactivity to Plasmodium falciparum variant surface antigens are dependent on age and malaria transmission intensity in northeastern Tanzania. Infect Immun 76 :2706-2714


Plasmodium falciparum variant surface antigens (VSA) are involved in the pathogenesis of malaria. Immunoglobulin G (IgG) with specificity for VSA (anti-VSA IgG) is therefore considered important for acquired immunity. To better understand the nature and dynamics of variant-specific IgG responses at population level, we conducted an immunoepidemiological study in nearby communities in northeastern Tanzania, situated at different altitudes and therefore exposed to different levels of P. falciparum transmission intensity. Samples of plasma and infected red blood cells (IRBC) were collected from 759 individuals aged 0 to 19 years. Plasma levels of IgG with specificity for VSA expressed by a panel of different parasite isolates were measured by flow cytometry, while the ability of plasma to inhibit IRBC adhesion to CD36 was examined in cellular assays. The level and repertoire of the heterologous anti-VSA IgG response developed dramatically in individuals at 1 to 2 years of age in the high-transmission area, reaching a maximum level at around 10 years of age ; only a modest further increase was observed among older children and adults. In contrast, at lower levels of malaria transmission, anti-VSA IgG levels were lower and the repertoire was more narrow, and similar age- and transmission-dependent differences were observed with regard to the ability of the plasma samples to inhibit adhesion of IRBC to CD36. These differences indicate a strong and dynamic relationship between malaria exposure and functional characteristics of the variant-specific antibody response, which is likely to be important for protection against malaria.

Vital M, Hammes F, Egli T (2008) Escherichia coli O157 can grow in natural freshwater at low carbon concentrations. Environ Microbiol 10 :2387-2396


Whereas much information on the die-off of Escherichia coli in the aquatic environment is available, only few data support its growth under such conditions. We therefore investigated batch growth in microcosms containing different types of sterile freshwater. The water samples were inoculated with low starting cell concentrations of E. coli O157 (3 x 10(3) cells ml(-1)) and growth was followed using nucleic acid staining combined with flow cytometry. We demonstrated that E. coli O157 is able to grow in sterile freshwater at low carbon concentrations, which is against the common view that cell numbers decline over time when added to freshwater samples. A correlation between apparent assimilable organic carbon (AOC(app)) concentration and the final cell concentration reached by E. coli O157 was established (P < 0.01). A considerable fraction of the AOC(app) (34 +/- 13%) was used by E. coli O157 but the numerical cell yield was about five-times lower in comparison with the bacterial AOC-test community, which originated from natural freshwater. On average, the maximum specific growth rate (mu(max)) of E. coli O157 growing in sterile freshwater at 30 degrees C was 0.19 +/- 0.07 h(-1). Batch growth assays at five different temperatures revealed a positive influence of temperature on mu(max) of E. coli O157. The results give new information on the behaviour of this common pathogen in the aquatic environment and contribute to microbial risk assessment in order to prevent spreading of water-borne diseases.

Vlamakis H, Aguilar C, Losick R, Kolter R (2008) Control of cell fate by the formation of an architecturally complex bacterial community. Genes Dev 22 :945-953


Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells.

Vogl G, Lesiak I, Jensen DB, Perkhofer S, Eck R, Speth C, Lass-Florl C, Zipfel PF, Blom AM, Dierich MP, Wurzner R (2008) Immune evasion by acquisition of complement inhibitors : the mould Aspergillus binds both factor H and C4b binding protein. Mol Immunol 45 :1485-1493


Pathogenic fungi represent a major threat particularly to immunocompromised hosts, leading to severe, and often lethal, systemic opportunistic infections. Although the impaired immune status of the host is clearly the most important factor leading to disease, virulence factors of the fungus also play a role. Factor H (FH) and its splice product FHL-1 represent the major fluid phase inhibitors of the alternative pathway of complement, whereas C4b-binding protein (C4bp) is the main fluid phase inhibitor of the classical and lectin pathways. Both proteins can bind to the surface of various human pathogens conveying resistance to complement destruction and thus contribute to their pathogenic potential. We have recently shown that Candida albicans evades complement by binding both Factor H and C4bp. Here we show that moulds such as Aspergillus spp. bind Factor H, the splicing variant FHL-1 and also C4bp. Immunofluorescence and flow cytometry studies show that the binding of Factor H and C4bp to Aspergillus spp. appears to be even stronger than to Candida spp. and that different, albeit possibly nearby, binding moieties mediate this surface attachment.

Walker A, Parkhill J (2008) Single-cell genomics. Nat Rev Microbiol 6 :176-177


Walton JT, Hill DJ, Protheroe RG, Nevill A, Gibson H (2008) Investigation into the effect of detergents on disinfectant susceptibility of attached Escherichia coli and Listeria monocytogenes. J Appl Microbiol 105 :309-315


AIMS : Investigate the effect of detergent treatment on susceptibility of attached Escherichia coli and Listeria monocytogenes to subsequent disinfectant treatment. METHODS AND RESULTS : Plate counts show that E. coli attached to stainless steel surfaces became significantly more susceptible to benzalkonium chloride (BAC) after treatment with sodium alkyl sulfate (SAS) and fatty alcohol ethoxylate (FAE). No change in susceptibility was observed with Sodium dodecyl sulfate (SDS). L. monocytogenes became significantly less susceptible to BAC after treatment with SAS and SDS yet no change in susceptibility was observed with FAE. Flow cytometry using the fluoresceine propidium iodide revealed significant increases in cell membrane permeability of both organisms by SAS and FAE, although the effect was much greater in E. coli. No change was observed with SDS. Hydrophobic interaction chromatography showed that both organisms became less hydrophobic following treatment with SAS and SDS but FAE had no effect. CONCLUSIONS : In E. coli, detergents that increase susceptibility to BAC increase membrane permeability. In L. monocytogenes, detergents that reduce susceptibility to BAC lower cell surface hydrophobicity. SIGNIFICANCE AND IMPACT OF THE STUDY : Detergents can influence the sensitivity of pathogenic food borne micro-organisms to BAC.

Wang H, Li X, He Y, Xie B, Tang W, Du J (2008) [Construction and expression analysis of micro-linear vector as a new general gene therapy vector]. Sheng Wu Gong Cheng Xue Bao 24 :1333-1339


The most difficult field in gene therapy is that vector system should offer both a means of successful transfection and a maximum of safety for the patient. Viral vectors and plasmid vectors are traditional vectors ; they may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes. Our aim is to develop a new general gene therapy vector which is suggested to be called as Micro-Linear Vector. The gene expression cassette is capped by our designed cap, including promoter, enhancer, objective gene, and RNA-stabilizing sequence, so it can defend the exnuclease in the eukaryotic cell, at the same time, DNA not encoding the objective gene is reduced to a minimum. The GFP gene is separated from the pEGFP-N3 plasmid, and acts as a reporter gene to construct the Micro-Linear Vector, then both the new vector and the plasmid are transfected to cells, the results are tested by fluorescence microscope and flow cytometry. The results show that the Micro-Linear Vector has a high effective of transfection and safety in 293, 3T3, CNE2 and B95-8 cell lines, at the same time it is less toxicity than the plasmid. We can get the rudiments of conclusion that Micro-Linear Vector has high effection of the transfection and more safety than tradition plasmid in eukaryotic cell.

Wang Y, Hammes F, Duggelin M, Egli T (2008) Influence of size, shape, and flexibility on bacterial passage through micropore membrane filters. Environ Sci Technol 42 :6749-6754


Sterilization of fluids by means of microfiltration is commonly applied in research laboratories as well as in pharmaceutical and industrial processes. Sterile micropore filters are subject to microbiological validation, where Brevundimonas diminuta is used as a standard test organism. However, several recent reports on the ubiquitous presence of filterable bacteria in aquatic environments have cast doubt on the accuracy and validity of the standard filter-testing method. Six different bacterial species of various sizes and shapes (Hylemonella gracilis, Escherichia coli, Sphingopyxis alaskensis, Vibrio cholerae, Legionella pneumophila, and B. diminuta) were tested for their filterability through sterile micropore filters. In all cases, the slender spirillum-shaped Hylemonella gracilis cells showed a superior ability to pass through sterile membrane filters. Our results provide solid evidence that the overall shape (including flexibility), instead of biovolume, is the determining factor for the filterability of bacteria, whereas cultivation conditions also play a crucial role. Furthermore, the filtration volume has a more important effect on the passage percentage in comparison with other technical variables tested (including flux and filter material). Based on our findings, we recommend a re-evaluation of the grading system for sterile filters, and suggest that the species Hylemonella should be considered as an alternative filter-testing organism for the quality assessment of micropore filters.

Wang Y, Hammes F, Egli T (2008) The impact of industrial-scale cartridge filtration on the native microbial communities from groundwater. Water Res 42 :4319-4326


Groundwater is a major source for bottled water, which is increasingly consumed all over the world. Some categories of bottled water can be subjected to treatments such as disinfection prior to bottling. In the current study, we present the quantitative impact of industrial-scale micro-filtration (0.22 microm pore size) on native microbial communities of groundwater and evaluate subsequent microbial growth after bottling. Two separate groundwater aquifers were tested. Flow-cytometric total cell concentration (TCC) and total adenosine tri-phosphate (ATP) analysis were used to quantify microbial abundance. The TCC of the native microbial community in both aquifers was in the range of 10(3)-10(4) cells/ml. Up to 10% of the native microbial community was able to pass through the cartridge filtration units installed at both aquifers. In addition, all samples (either with or without 0.22 microm filtration) showed significant growth after bottling and storage, reaching average final concentrations of 1-3 x 10(5) cells/ml. However, less growth was observed in carbon-free glassware than in standard polyethylene terephthalate (PET) bottles. Furthermore, our results showed that filtration and bottling can alter the microbial community patterns as observed with flow cytometry. The current study established that industrial-scale micro-filtration cannot serve as an absolute barrier for the native microbial community and provided significant insight to the impact of filtration and bottling on microbial concentrations in bottled water.

Wei MK, Wu QP, Huang Q, Wu JL, Zhang JM (2008) Plasma membrane damage to Candida albicans caused by chlorine dioxide (ClO2). Lett Appl Microbiol 47 :67-73


AIM : To investigate the plasma membrane damage of chlorine dioxide (ClO(2)) to Candida albicans ATCC10231 at or below the minimal fungicidal concentration (MFC). METHODS AND RESULTS : ClO(2) at MFC or below was adopted to treat the cell suspensions of C. albicans ATCC10231. Using transmission electron microscopy, no visible physiological alteration of cell shape and plasma membrane occurred. Potassium (K(+)) leakages were significant ; likewise, it showed time- and dose-dependent increases. However, adenosine triphosphate (ATP) leakages were very slight. Research shows that when 99% of the cells were inactivated, the leakage was measured at 0.04% of total ATP. Compared with the mortality-specific fluorescent dye of DiBAC(4)(3), majority of the inactivated cells were poorly stained by propidium iodide, another mortality-specific fluorescent dye which can be traced by flow cytometry. CONCLUSION : At or below MFC, ClO(2) damages the plasma membranes of C. albicans mainly by permeabilization, rather than by the disruption of their integrity. K(+) leakage and the concomitant depolarization of the cell membrane are some of the critical events. SIGNIFICANCE AND IMPACT OF THE STUDY : These insights into membrane damages are helpful in understanding the action mode of ClO(2).

Wilcks A, Smidt L, Bahl MI, Hansen BM, Andrup L, Hendriksen NB, Licht TR (2008) Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats. J Appl Microbiol 104 :1252-1259


AIMS : To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. METHODS AND RESULTS : Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. CONCLUSIONS : Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY : Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.

Xiong A, Austin TW, Lagasse E, Uchida N, Tamaki S, Bordier BB, Weissman IL, Glenn JS, Millan MT (2008) Isolation of human fetal liver progenitors and their enhanced proliferation by three-dimensional coculture with endothelial cells. Tissue Eng Part A 14 :995-1006


Liver progenitor cells, characterized by the coexpression of biliary and hepatocyte lineage markers and the ability to form colonies in culture, were isolated by flow cytometry from primary human fetal livers. These prospectively isolated liver progenitor cells supported hepatitis D virus infection, expressed, and produced albumin and alpha-fetoprotein, as tracked by albumin- and alpha-fetoprotein-driven lentiviral promoter reporter constructs and measured by ELISA, respectively. Coculture in three-dimensional (3D) fibrin gel with endothelial cells resulted in the formation of vascular structures by the endothelial cells and increased proliferation of liver progenitors. The enhanced proliferation of liver progenitors that was observed when liver progenitors and endothelial cells were cultured in direct contact was not achieved when liver progenitors and endothelial cells were cultured on adjacent but separate matrices and when they were cultured across transwell membranes. In conclusion, coculture of liver progenitors and endothelial cells in three-dimensional matrix resulted in enhanced liver progenitor proliferation and function. This coculture methodology offers a novel coculture system that could be applied for the development of engineered liver tissues.

Xiong JW, Xiong CL, Li D, Chen SH, Li XL, Tao DD (2008) [Apoptosis of sperm induced by murine cytomegalovirus infection and its mechanism regulated by sperm mitochondria : experiment with mice]. Zhonghua Yi Xue Za Zhi 88 :1673-1675


OBJECTIVE : To study the effects of murine cytomegalovirus (MCMV) infection on mature sperm apoptosis in mice, and to explore the mechanism of MCMV-induced apoptosis regulated by mitochondria. METHODS : MCMV was inoculated into the testes of 15 BALB/c mice to establish acute MCMV infection models and 15 mice were used as controls. 1, 2, 4, 6, and 9 days after the infection 3 mice from each group were killed. Flow cytometry was used to observe the apoptosis of sperms. Laser scanning confocal microscopy was performed to examine the mitochondrial membrane potential (delta psi psi m) of sperm. The mitochondria ultrastructure of sperm was observed under electron microscope. RESULTS : The sperm apoptotic ratebeganto increase from day 2 after inoculation (D2 PI), peaked to the level of (39.3 +/- 1.0)% compared with that of the control group on D4 PI, and then fell-off (F = 362.822, P < 0.05). The delta psi m of sperm began to increase on D1 PI at the level of (74.0 +/- 1.4), began to decrease on D2 PI [( 63.0 +/- 2.2)], dropped to the minimum on D4 PI [(40.2 +/- 2.3)], then ascended slowly again (F = 32.257, P < 0.05). The mitochondria ultrastructure of sperm showed damage after MCMV infection that was especially severe from D2 PI to D6 PI. CONCLUSION : MCMV acute infection in reproductive organ induces apoptosis of mature sperm in the cauda epididymidis. Sperm mitochondria participate in and regulate initiatively the apoptosis of sperm.

Xiong Q, Bao W, Ge Y, Rikihisa Y (2008) Ehrlichia ewingii infection delays spontaneous neutrophil apoptosis through stabilization of mitochondria. J Infect Dis 197 :1110-1118


The uncultivable obligate intracellular bacterium Ehrlichia ewingii, previously known only as a canine pathogen, is the most recently recognized agent of human ehrlichiosis. E. ewingii is the only Ehrlichia species known to infect neutrophils. In the blood or in ex vivo culture, neutrophils generally have a short life span. In the present study, we investigated the effect of E. ewingii infection on spontaneous apoptosis of neutrophils. E. ewingii infection significantly delayed dog neutrophil apoptosis during ex vivo culture. The inhibitory effect on neutrophil apoptosis by E. ewingii was reversible on clearance of the organism. By using the fluorescent mitochondrial dyes Mitotracker Red 580 and JC-1, we found that E. ewingii infection stabilized mitochondrial integrity by maintaining mitochondrial membrane potential in neutrophils. These results suggest that E. ewingii delays spontaneous apoptosis of neutrophils via stabilization of host cell mitochondria.

Xu XF, Chen ZT, Zhang JL, Chen W, Wang JL, Tian YP, Gao N, An J (2008) Rab8, a vesicular traffic regulator, is involved in dengue virus infection in HepG2 cells. Intervirology 51 :182-188


OBJECTIVE : The pathogenesis of dengue virus (DV) has not been completely clarified. Rab8 regulates vesicular traffic from Golgi to plasma membrane where DV is matured and then delivered by exocytosis. In this study, involvement of Rab8 in DV serotype 2 (DV2) infection was investigated in HpeG2 cells. METHODS : Distributions of Rab8 and DV2, and the number of infection cells were observed by immunostaining. HepG2(Rab8AM) and HepG2(Rab8DN) cells were constructed to stably express a constitutively active mutant of Rab8 and a dominant negative mutant, respectively, which were assessed by flow cytometry. Production of infectious virions and the amounts of DV2 entry were detected by standard plaque assay. Viral RNA replication was detected by real-time RT-PCR. RESULTS : Rab8 showed high co-localization with DV2 in HpeG2 cells and the amount of DV antigen-positive cells decreased in HepG2(Rab8AM) and HepG2(Rab8DN) cells. Also, progeny virus released from those cells was drastically reduced. Infectious virions produced in cells were also significantly reduced, while the viral RNA replication was down-regulated by a different level. Furthermore, viral entry into those cells was reduced by about 80%. CONCLUSIONS : Our data suggest that the function of Rab8 is important for DV2 infection, and Rab8 may be involved in DV2 infection.

Yang S, Peng Q, San Francisco M, Wang Y, Zeng Q, Yang CH (2008) Type III secretion system genes of Dickeya dadantii 3937 are induced by plant phenolic acids. PLoS ONE 3 :e2973


BACKGROUND : Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified. METHODOLOGY/PRINCIPAL FINDINGS : In this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a sigma(54)-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA. CONCLUSION/SIGNIFICANCE : The induction of T3SS expression by OCA and TCA is moderated through the rsmB-RsmA pathway. This is the first report of plant phenolic compounds that induce the expression T3SS genes of plant pathogenic bacteria.

Zehr JP, Bench SR, Carter BJ, Hewson I, Niazi F, Shi T, Tripp HJ, Affourtit JP (2008) Globally distributed uncultivated oceanic N2-fixing cyanobacteria lack oxygenic photosystem II. Science 322 :1110-1112


Biological nitrogen (N2) fixation is important in controlling biological productivity and carbon flux in the oceans. Unicellular N2-fixing cyanobacteria have only recently been discovered and are widely distributed in tropical and subtropical seas. Metagenomic analysis of flow cytometry-sorted cells shows that unicellular N2-fixing cyanobacteria in "group A" (UCYN-A) lack genes for the oxygen-evolving photosystem II and for carbon fixation, which has implications for oceanic carbon and nitrogen cycling and raises questions regarding the evolution of photosynthesis and N2 fixation on Earth.

Zhang N, Cheng AC, Wang MS, Li CF, Chen XY (2008) [The cell apoptosis induced by duck reovirus in duck embryo fibroblasts]. Bing Du Xue Bao 24 :213-219


Cell apoptosis induced by duck reovirus (DRV)in duck embryo fibroblasts (DEF) was ascertained by light microscope and electron microscopy, DNA Ladder, flow cytometry and fluorescent microscopy. Typical morphological apoptotic features including cell shrinkage and condensation, margination of nuclear chromatin were observed under light microscope and the formation of apoptotic bodies by electron microscopy. DNA ladder was shown by DNA fragment analysis at 24-144h post infection. Flow cytometry showed that the cell apoptosis appeared at 24h and reached it’s crest-time at 72-96h, decreased at 144h. Fluorescent microscopy showed that the apoptotic cells which showed green fluorescence appeared at 24h, the number of dead cells which showed red fluorescence increased with the time went by. The results above confirmed that the apoptosis of DEF was successfully induced by DRV.

Zhao L, Li S, Ge J, Sun A, Zou Y, Zhang S (2008) Temporal changes in stem cells in the circulation and myocardium of mice with Coxsackie virus B3-induced myocarditis. Microvasc Res 75 :358-366


Our goal was to investigate temporal changes in stem cell in the circulation and myocardium of mice with Coxsackie virus B3-induced myocarditis. Groups of mice were administered Eagle’s minimal essential medium or virus solution. The animals were further divided into six subgroups based on the following time points post-inoculation : 1, 3, 7, 14, 21, and 28 days. Ten animals were studied in each subgroup. Circulating blood mononuclear cells were collected from the heart and analyzed using flow cytometry. Myocardial inflammation, stem cell expression, and cell proliferation were detected by histology and immunofluorescence. H&E staining revealed neutrophil infiltration and bleeding by day 3 post-infection. Myeloperoxidase and reactive oxygen species levels peaked by day 3 and were followed by myocyte loss and collagen deposition. Circulating mesenchymal stem cells also peaked by day 3. In contrast, hematopoietic stem cells remained sustained increase within day 14. Immunohistochemical microscopy also showed a marked increase in cardiac stem cells by day 14. The kinetics of this increase was consistent with a rise in proliferating cells expressing nuclear and cytoplasmic proteins that are typical of cardiomyocyte or vascular endothelial cells. These results demonstrate the rapid kinetics of progenitor cells during viral myocarditis and suggest that the optimal time to administer cell therapy to induce heart repair is within 2 weeks after viral infection.

Zhou GZ, Gui L, Li ZQ, Yuan XP, Zhang QY (2008) Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus. J Virol Methods 148 :205-210


Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection.

Zhuang H, Matsunami H (2008) Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nat Protoc 3 :1402-1413


A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput ’deorphanization’ of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.