vendredi 24 avril 2009
par   G. Grégori

Aertsen A, Van Houdt R, Michiels CW (2005) Construction and use of an stx1 transcriptional fusion to gfp. FEMS Microbiol Lett 245 :73-77


Shiga toxins (Stxs), also termed Vero toxins, are cytotoxic ribosome inactivating proteins that are produced by a number of gastrointestinal pathogens and that contribute to the severity of the associated diseases. In this work, we constructed and validated a transcriptional fusion of the stx1AB promoter to the gfp reporter gene. The cloned promoter region encompasses both the proximal and the distal promoter regions of stx1AB, mediating control by the host’s iron-responsive Fur repressor and the Stx prophage’s Q antiterminator protein, respectively. The probe was validated by demonstrating its responsiveness towards mitomycin C and EDTA, and the contribution of host and phage encoded factors could be separated by studying stx1 expression in either wild-type or isogenic lysogenic cells. Moreover, stx1AB expressing populations could be visualized by flow cytometry. The potential use of such a probe for non-destructive online detection of stx1AB expression and visualization of stx1AB expressing populations is further discussed.

Alonso M, Stein DA, Thomann E, Moulton HM, Leong JC, Iversen P, Mourich DV (2005) Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers. J Fish Dis 28 :399-410


Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3’ fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5’ end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5’ end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

Ananta E, Voigt D, Zenker M, Heinz V, Knorr D (2005) Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound. J Appl Microbiol 99 :271-278


AIMS : To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS : According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS : The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY : Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.

Atir-Lande A, Gildor T, Kornitzer D (2005) Role for the SCFCDC4 ubiquitin ligase in Candida albicans morphogenesis. Mol Biol Cell 16 :2772-2785


The ability of Candida albicans, a major fungal pathogen, to switch between a yeast form, and a hyphal (mold) form is recognized as being important for the ability of the organism to invade the host and cause disease. We found that a C. albicans mutant deleted for CaCDC4, a homologue of the Saccharomyces cerevisiae F-box protein component of the SCF(CDC4) ubiquitin ligase, is viable and displays constitutive filamentous, mostly hyphal, growth. The phenotype of the Cacdc4-/- mutant suggests that ubiquitin-mediated protein degradation is involved in the regulation of the dimorphic switch of C. albicans and that one or more regulators of the yeast-to-mold switch are among the substrates of SCF(CaCDC4). Epistasis analysis indicates that the Cacdc4-/- phenotype is largely independent of the filamentation-inducing transcription factors Efg1 and Cph1. We identify C. albicans Far1 and Sol1, homologues of the S. cerevisiae SCF(CDC4) substrates Far1 and Sic1, and show that Sol1 is a substrate of C. albicans Cdc4. Neither protein is essential for the hyphal phenotype of the Cacdc4-/- mutant. However, ectopic expression and deletion of SOL1 indicate a role for this gene in C. albicans morphogenesis.

Bahl MI, Hansen LH, Sorensen SJ (2005) Construction of an extended range whole-cell tetracycline biosensor by use of the tet(M) resistance gene. FEMS Microbiol Lett 253 :201-205


An extended range whole-cell tetracycline biosensor strain was constructed by insertion of the tet(M) gene, encoding tetracycline resistance by ribosomal protection, into plasmid pTGFP2, which contains a transcriptional fusion between a tetracycline regulated promoter and the green fluorescent protein gene. Tetracycline, oxytetracycline, chlortetracycline and minocycline all effectively induced the resulting Escherichia coli MC4100/pTGM biosensor and similar dose-response characteristics were recorded by flow cytometry for all four compounds. The novel tetracycline biosensor was responsive to drug concentrations ranging from below 5 ngml(-1) to 16 microgml(-1), which represents a significant improvement of the original version.

Balaban NQ (2005) Szilard’s dream. Nat Methods 2 :648-649


Ben-Amor K, Heilig H, Smidt H, Vaughan EE, Abee T, de Vos WM (2005) Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis. Appl Environ Microbiol 71 :4679-4689


A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations ; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.

Bohm E, Grillari J, Voglauer R, Gross S, Ernst W, Ferko B, Kunert R, Katinger H, Borth N (2005) Establishment of a strategy for the rapid generation of a monoclonal antibody against the human protein SNEV (hNMP200) by flow-cytometric cell sorting. J Immunol Methods 307 :13-23


The screening for antigen-specific hybridoma cells with adequate production rates is still a time-, labour- and money-consuming procedure. A reduction in cell culture testing by specifically selecting those fused cells that produce antibody could therefore make hybridoma technology more attractive, even for small research groups or for newly discovered proteins at an early stage of research. Additional problems, such as the requirement to produce sufficient amounts of the unknown protein at a purity that allows specific immunisation of mice and testing of the resulting hybridoma clones, also need to be overcome. Here we present a new strategy to isolate rapidly and efficiently monoclonal antibodies against new proteins, for which only sequence information at the DNA level is known. The strategy consists of fusion of the protein to a hexa-His-tag to allow easy purification, production in yeast and insect cells to reduce background immunisation with host cell proteins and the selection of IgG-producing hybridoma cells by flow-cytometric cell sorting using the affinity matrix secretion assay technique.

Bossios A, Psarras S, Gourgiotis D, Skevaki CL, Constantopoulos AG, Saxoni-Papageorgiou P, Papadopoulos NG (2005) Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells. Respir Res 6 :114


BACKGROUND : Human rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro. METHODS : Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry. RESULTS : RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation. CONCLUSION : RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.

Brandl H, Bachofen R, Bischoff M (2005) Generation of bioaerosols during manual mail unpacking and sorting. J Appl Microbiol 99 :1099-1107


AIMS : The dynamics of bioaerosol generation in specific occupational environments where mail is manually unpacked and sorted was investigated. METHODS AND RESULTS : Total number of airborne particles was determined in four different size classes (0.3-0.5, 0.5-1, 1-5 and >5 microm) by laser particle counting. Time dependent formation of bioaerosols was monitored by culturing methods and by specific staining followed by flow cytometry. Besides handling of regular mail, specially prepared letters (’spiked letters’) were added to the mailbags to deliberately release powdered materials from letters and to simulate high impact loads. These letters contained various dry powdered biological and nonbiological materials such as milk powder, mushrooms, herbs and cat litter. Regarding the four size classes, particulate aerosol composition before mail handling was determined as 83.2 +/- 1.0, 15.2 +/- 0.7, 1.7 +/- 0.4 and 0.04 +/- 0.02%, respectively, whereas the composition changed during sorting to 66.8 +/- 7.9, 22.3 +/- 3.6, 10.4 +/- 4.0 and 0.57 +/- 0.27%, respectively. Mail processing resulted in an increase in culturable airborne bacteria and fungi. Maximum concentrations of bacteria reached 450 CFU m(-3), whereas 270 CFU of fungi were detected. CONCLUSIONS : Indoor particle concentrations steadily increased during mail handling mostly associated with particles of diameters >1 microm. However, it was not possible to distinguish spiked letters from nonspiked by simple particle counting and CFU determinations. SIGNIFICANCE AND IMPACT OF STUDY : The dynamics of bioaerosol generation have to be addressed when monitoring specific occupational environments (such as mail sorting facilities) regarding the occurrence of biological particles.

Burmolle M, Hansen LH, Sorensen SJ (2005) Use of a whole-cell biosensor and flow cytometry to detect AHL production by an indigenous soil community during decomposition of litter. Microb Ecol 50 :221-229


Quorum sensing, mediated by acylated homoserine lactones (AHLs), is well described for pure culture bacteria, but few studies report detection of AHL compounds in natural bacterial habitats. In this study, we detect AHL production during a degradation process in soil by use of whole-cell biosensor technology and flow cytometry analysis. An indigenous soil bacterium, belonging to the family of Enterobacteriaceae, was isolated and transformed with a low-copy plasmid harboring a gene encoding an unstable variant of the green fluorescent protein (gfpASV) fused to the AHL-regulated P(luxI) promoter originating from Vibrio fischeri. This resulted in a whole-cell biosensor, responding to the presence of AHL compounds. The biosensor was introduced to compost soil microcosms amended with nettle leaves. After 3 days of incubation, cells were extracted and analyzed by flow cytometry. All microcosms contained induced biosensors. From these microcosms, AHL producers were isolated and further identified as species previously shown to produce AHLs. The results demonstrate that AHL compounds are produced during degradation of litter in soil, indicating the presence of AHL-mediated quorum sensing in this environment.

Carvalho HM, Teel LD, Kokai-Kun JF, O’Brien AD (2005) Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization. Infect Immun 73 :2541-2546


The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin alpha.

Chamsartra S, Hewitt CJ, Nienow AW (2005) The impact of fluid mechanical stress on Corynebacterium glutamicum during continuous cultivation in an agitated bioreactor. Biotechnol Lett 27 :693-700


The effect of mechanical stresses generated by an extreme agitation intensity or a high aeration rate on growth parameters and cell physiology were studied during continuous cultivation of the Gram-positive bacterium Corynebacterium glutamicum. It is concluded that variations in agitation, aeration rate, or dO2 concentrations down to about 1% of saturation do not damage the bacterial cells or cause a significant change in physiological response, as measured by flow cytometry, even though the cell size was slightly reduced.

Chang SC, Anderson TI, Bahrman SE, Gruden CL, Khijniak AI, Adriaens P (2005) Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids. J Ind Microbiol Biotechnol 32 :629-638


The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

Cheeran MC, Hu S, Ni HT, Sheng W, Palmquist JM, Peterson PK, Lokensgard JR (2005) Neural precursor cell susceptibility to human cytomegalovirus diverges along glial or neuronal differentiation pathways. J Neurosci Res 82 :839-850


Cytomegalovirus (CMV) is a major cause of congenital brain disease, and its neuropathogenesis may be related to viral infection of rapidly dividing, susceptible neural precursor cells (NPCs). In the present study, we evaluated the susceptibility of human fetal brain-derived NPCs (nestin(+), A2B5(+), CD133(+)) to infection with CMV. Data derived from these studies demonstrated that undifferentiated NPCs supported productive viral replication. After differentiation in the presence of serum, a treatment that promotes development of an astroglial cell phenotype (GFAP(+), nestin(-), A2B5(-)), viral expression was retained. However, differentiation of NPCs in medium containing platelet-derived growth factor and brain-derived neurotropic factor, conditions that support the development of neurons (Tuj-1(+), nestin(-), A2B5(-)), resulted in reduced viral expression, with corresponding decreased CMV major immediate-early promoter (MIEP) activity relative to undifferentiated cells. Further experiments showed that cellular differentiation into a neuronal phenotype was associated with elevated levels of various CCAAT/enhancer binding protein beta (C/EBP)-beta isoforms, which suppressed MIEP activity in cotransfected NPCs. Taken together, these data demonstrate that the susceptibility of primary human NPCs to CMV is retained concomitantly with differentiation into glial cells but is actively repressed following differentiation into neurons.

Chen J, Miao L, Li JM, Li YY, Zhu QY, Zhou CL, Fang HQ, Chen HP (2005) Receptor-binding domain of SARS-Cov spike protein : soluble expression in E. coli, purification and functional characterization. World J Gastroenterol 11 :6159-6164


AIM : Spike protein of coronavirus is responsible for virus binding, fusion and entry, and is a major inducer of neutralizing antibodies. This paper was to find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS : Three fusion tags (glutathione S-transferase, GST ; thioredoxin, Trx ; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS : RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST/RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD’s binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST/RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION : In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21(DE3) ; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Chen PS, Li CS (2005) Bioaerosol characterization by flow cytometry with fluorochrome. J Environ Monit 7 :950-959


Traditional culture and microscopy methods for evaluation of bioaerosols are slow, tedious, and rather imprecise. In this study, the application of flow cytometry that was combined with a fluorescent technique (FCM/FL) was evaluated as a technique to quickly and accurately determine and quantify the total concentration and viability of bioaerosols. The optimal conditions of five fluorescent dyes [acridine orange (AO), SYTO-13, propidium iodide (PI), YOPRO-1, and 5-cyano-2,3-ditolytetrazolium chloride (CTC)] used in FCM/FL were determined for laboratory samples of bacterial aerosols (Escherichia coli, and endospores of Bacillus subtilis) and fungal aerosols (Candida famata and Penicillium citrinum spores). Based on the measured cell concentration, fluorescence intensity, and staining efficiency as indicators for dye performance evaluation, SYTO-13 was found to be the most suitable fluorescent dye for determining the total concentration of the bioaerosols, as well as YOPRO-1 was the most suitable for determining viability. Moreover, the established optimal FCM/FL with dyes was validated for characterizing microorganism profiles from both air and water samples from the aeration tank of hospital wastewater treatment plant. In conclusion, the FCM/FL successfully assessed the total concentration and viability for bacterial and fungal microorganisms in environmental field samples.

Christaki U, Vazquez-Dominguez E, Courties C, Lebaron P (2005) Grazing impact of different heterotrophic nanoflagellates on eukaryotic (Ostreococcus tauri) and prokaryotic picoautotrophs (Prochlorococcus and Synechococcus). Environ Microbiol 7 :1200-1210


Autotrophic picoplankton (<3 microm) composed of both prokaryotes and eukaryotes are the most abundant primary producers on Earth. In this study we examined the ingestion of the picoeukaryote Ostreococcus tauri by different marine heterotrophic nanoflagellates (HNF) with various morphologies, swimming and feeding behaviours. Cultures of specific bacterivorous nanoflagellates (Rhynchomonas nasuta, Jakoba libera, and a culture of Cafeteria sp./Monosiga sp.) and natural nanoflagellate populations were used as grazers. For comparison with Ostreococcus, we used similar-sized prokaryotes as prey, Prochlorococcus and Synechococcus. We observed large species-specific differences in terms of : use of picoautotrophs among nanoflagellates, time lag between prey addition and prey consumption (0-196 h), grazing rate (0-0.12 h(-1)), growth rate (0-0.3 h(-1)) and maximum abundance of HNF reached in experimental bottles (e.g. from 10(4) to 10(5) cells ml(-1), for a natural coastal population and a Cafeteria sp./Monosiga sp. culture feeding Ostreococcus respectively). Overall, this study shows that the nanoflagellate community composition is conclusive for picoautotrophic community structure and, vice versa, the picoautotrophic community structure favours or inhibits the growth of some nanoflagellate groups.

Clavel T, Henderson G, Alpert CA, Philippe C, Rigottier-Gois L, Dore J, Blaut M (2005) Intestinal bacterial communities that produce active estrogen-like compounds enterodiol and enterolactone in humans. Appl Environ Microbiol 71 :6077-6085


Lignans are dietary diphenolic compounds which require activation by intestinal bacteria to exert possible beneficial health effects. The intestinal ecosystem plays a crucial role in lignan metabolism, but the organisms involved are poorly described. To characterize the bacterial communities responsible for secoisolariciresinol (SECO) activation, i.e., the communities that produce the enterolignans enterodiol (ED) and enterolactone (EL), a study with 24 human subjects was undertaken. SECO activation was detected in all tested fecal samples. The intestinal bacteria involved in ED production were part of the dominant microbiota (6 x 10(8) CFU g(-1)), as revealed by most-probable-number enumerations. Conversely, organisms that catalyzed the formation of EL occurred at a mean concentration of approximately 3 x 10(5) CFU g(-1). Women tended to have higher concentrations of both ED- and EL-producing organisms than men. Significantly larger amounts of EL were produced by fecal dilutions from individuals with moderate to high concentrations of EL-producing bacteria. Two organisms able to demethylate and dehydroxylate SECO were isolated from human feces. Based on 16S rRNA gene sequence analyses, they were named Peptostreptococcus productus SECO-Mt75m3 and Eggerthella lenta SECO-Mt75m2. A new 16S rRNA-targeted oligonucleotide probe specific for P. productus and related species was designed and further used in fluorescent in situ hybridization experiments, along with five additional group-specific probes. Significantly higher proportions of P. productus and related species (P = 0.012), as well as bacteria belonging to the Atopobium group (P = 0.035), were typical of individuals with moderate to high concentrations of EL-producing communities.

Cools I, D’Haese E, Uyttendaele M, Storms E, Nelis HJ, Debevere J (2005) Solid phase cytometry as a tool to detect viable but non-culturable cells of Campylobacter jejuni. J Microbiol Methods 63 :107-114


Solid phase cytometry (SPC) in conjunction with fluorescent viability staining has been investigated as a tool to detect viable but non-culturable Campylobacter jejuni in drinking water. Inoculated water samples were filtered over a polyester membrane filter and the retained cells were stained using a carboxyfluorescein ester as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device (ChemScan) in 3 min. In parallel, the plate count was determined on Columbia Blood Agar. The number of culturable cells decreased below the detection limit of plate counting in less than 50 days. In contrast, the number of fluorescent bacteria remained at its initial level for at least 85 days. The discrepancy between the two results can be attributed to the transition of culturable C. jejuni cells into VBNC C. jejuni cells. Furthermore, as SPC can distinguish between low numbers of dividing and non-dividing cells of Campylobacter it has the potential to monitor attempts to resuscitate VBNC cells.

Corradi A, Ferrari M, Cantoni AM, Robotti C, Alborali L, Lecce RD, Candotti P, Sandri GP, Borghetti P (2005) Study on the virulence, cell-mediated immune response and histolesivity of three field PRRSV strains with an ORF 5 genetic variation. Vet Res Commun 29 Suppl 2 :241-243


Cotter MJ, Zaiss AK, Muruve DA (2005) Neutrophils interact with adenovirus vectors via Fc receptors and complement receptor 1. J Virol 79 :14622-14631


Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of Fc gammaRI (CD64), Fc gammaRII (CD32), and Fc gammaRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors.

Cox SE, Staalsoe T, Arthur P, Bulmer JN, Hviid L, Yeboah-Antwi K, Kirkwood BR, Riley EM (2005) Rapid acquisition of isolate-specific antibodies to chondroitin sulfate A-adherent plasmodium falciparum isolates in Ghanaian primigravidae. Infect Immun 73 :2841-2847


Recent evidence suggests that pregnancy-associated malaria (PAM), associated with maternal anemia and low birth weight, results from preferential sequestration of parasitized red blood cells (pRBC) in the placenta via binding of variant surface antigens (VSA) expressed on the surface of pRBC to chondroitin sulfate A (CSA). The VSA mediating CSA binding (VSA(CSA)) and thus sequestration of pRBC in the placenta are antigenically distinct from those that mediate pRBC sequestration elsewhere in the body, and it has been suggested that VSA(CSA) are relatively conserved and may thus constitute an attractive target for vaccination against PAM. Using flow cytometry, levels of antibody to VSA and VSA(CSA) expressed on the surface of red blood cells infected with Plasmodium falciparum isolates were measured during pregnancy and lactation in Ghanaian primigravid women enrolled in a trial of maternal vitamin A supplementation. Antibody responses to VSA(CSA) were detected within the first trimester of pregnancy and increased with increasing duration of pregnancy, and they seemed to be isolate specific, indicating that different CSA-adherent parasite lines express antigenically distinct VSA and thus may not be as antigenically conserved as has been previously suggested. Levels of anti-VSA(CSA) were not significantly associated with placental malarial infection determined by histology, indicating that primary immune responses to VSA(CSA) may not be sufficient to eradicate placental parasitemia in primigravidae.

da Silva TL, Reis A, Kent CA, Roseiro JC, Hewitt CJ (2005) The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations. Biotechnol Bioeng 92 :568-578


The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.

Daley JK, Gechman LA, Skipworth J, Rall GF (2005) Poliovirus replication and spread in primary neuron cultures. Virology 340 :10-20


While some neurotropic viruses cause rapid central nervous system (CNS) disease upon entry into the brain parenchyma, other viruses that are cytolytic in the periphery either result in little neuropathology or are associated with a protracted course of CNS disease consistent with persistent infection. One such virus, poliovirus (PV), is an extremely lytic RNA virus that requires the expression of CD155, the poliovirus receptor (PVR), for infection. To compare the kinetics of PV infection in neuronal and non-neuronal cell types, primary hippocampal neurons and fibroblasts were isolated from CD155+ transgenic embryos and infected with the Mahoney and Sabin strains of PV. Despite similar levels of infection in these ex vivo cultures, PV-infected neurons produced 100-fold fewer infectious particles as compared to fibroblasts throughout infection, and death of PV-infected neurons was delayed approximately 48 h. Spread in neurons occurred primarily by trans-synaptic transmission and was CD155-dependent. Together, these results demonstrate that the magnitude and speed with which PV replication, spread, and subsequent cell death occur in neurons is decreased as compared to non-neuronal cells, implicating cell-specific effects on replication that may then influence viral pathogenesis.

Dassanayake RP, Zhou Y, Hinkley S, Stryker CJ, Plauche G, Borda JT, Sestak K, Duhamel GE (2005) Characterization of cytolethal distending toxin of campylobacter species isolated from captive macaque monkeys. J Clin Microbiol 43 :641-649


An association between certain Campylobacter species and enterocolitis in humans and nonhuman primates is well established, but the association between cytolethal distending toxin and disease is incompletely understood. The purpose of the present study was to examine Campylobacter species isolated from captive conventionally raised macaque monkeys for the presence of the cdtB gene and for cytolethal distending toxin activity. The identity of each isolate was confirmed on the basis of phenotypic and genotypic analyses. The presence of cytolethal distending toxin was confirmed on the basis of characteristic morphological changes in HeLa cells incubated with filter-sterilized whole-cell lysates of reference and monkey Campylobacter isolates and examinations by light microscopy, confocal microscopy, and flow cytometry. Although cdtB gene sequences were found in both Campylobacter jejuni and Campylobacter coli, the production of cytolethal distending toxin correlated positively (P < 0.0001) only with C. jejuni. We concluded that cytolethal distending toxin activity is a characteristic of C. jejuni. Our C. jejuni cdtB gene-specific PCR assay might be of assistance for differentiating toxigenic C. jejuni from C. coli in clinical laboratories.

d’Avila-Levy CM, Silva BA, Hayashi EA, Vermelho AB, Alviano CS, Saraiva EM, Branquinha MH, Santos AL (2005) Influence of the endosymbiont of Blastocrithidia culicis and Crithidia deanei on the glycoconjugate expression and on Aedes aegypti interaction. FEMS Microbiol Lett 252 :279-286


Blastocrithidia culicis and Crithidia deanei are trypanosomatid protozoa of insects that normally contain intracellular symbiotic bacteria. The protozoa can be rid of their endosymbionts by antibiotics, producing a cured cell line. Here, we analyzed the glycoconjugate profiles of endosymbiont-harboring and cured strains of B. culicis and C. deanei by Western blotting and flow cytometry analyses using lectins that recognize specifically sialic acid and mannose-like residues. The absence of the endosymbiont increased the intensity of the lectins binding on both trypanosomatids. In addition, wild and cured strain-specific glycoconjugate bands were identified. The role of the surface saccharide residues on the interaction with explanted guts from Aedes aegypti gut was assessed. The aposymbiotic strains of B. culicis and C. deanei presented interaction rates 3.3- and 2.3-fold lower with the insect gut, respectively, when compared with the endosymbiont-bearing strains. The interaction rate of sialidase-treated cells of the wild and cured strains of B. culicis and C. deanei was reduced in at least 90% in relation to the control. The interaction of B. culicis (wild strain) with explanted guts was inhibited in the presence of mucin (56%), fetuin (62%), sialyllactose (64%) and alpha-methyl-D-mannoside (80%), while in C. deanei (wild strain) the inhibition was 53%, 56%, 79% and 34%, respectively. Collectively, our results suggest a possible involvement of sialomolecules and mannose-rich glycoconjugates in the interaction between insect trypanosomatids and the invertebrate host.

DeCoster DJ, Vena RM, Callister SM, Schell RF (2005) Susceptibility testing of Mycobacterium tuberculosis : comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method. Clin Microbiol Infect 11 :372-378


Tuberculosis is a leading cause of morbidity and mortality worldwide. Susceptibility testing of the causative agent, Mycobacterium tuberculosis, is critical for control of the disease. This study compared the flow cytometric susceptibility assay with the proportion method and the BACTEC TB-460 system. There was agreement between the flow cytometric and proportion methods for 73 (94%) of 78 isoniazid tests, and complete agreement for 26 ethambutol and rifampicin tests. In contrast, the proportion and BACTEC methods failed to agree for 22%, 15% and 8% of isoniazid, ethambutol and rifampicin tests, respectively. These findings indicated that susceptibility testing by the flow cytometric assay is accurate, with results available within 24 h of initiation of the testing procedure.

Devilla RA, Brown MT, Donkin M, Readman JW (2005) The effects of a PSII inhibitor on phytoplankton community structure as assessed by HPLC pigment analyses, microscopy and flow cytometry. Aquat Toxicol 71 :25-38


Measurements of the stress imposed by a PSII inhibiting herbicide (Irgarol 1051) on the composition of a phytoplankton community was investigated by comparing chemotaxonomy, as determined by high performance liquid chromatography (HPLC), optical microscopy and analytical flow cytometry (AFC). Changes in community structure were induced in microcosms containing a natural marine phytoplankton community exposed to different concentrations of Irgarol 1051 (0.5 and 1.0 microgl-1). Microcosms were maintained under controlled laboratory conditions in semi-continuous culture over 120 h. Class-specific phytoplankton biomass (chlorophyll a) was estimated using CHEMTAX analyses of pigment concentrations. Microscopic identification and carbon content estimates were cross-correlated with CHEMTAX and also with AFC enumeration/size classifications of major phytoplankton groups. CHEMTAX-HPLC analyses and microscopy results demonstrated that prasinophytes and prymnesiophytes were the most affected groups following exposure to Irgarol 1051. The selective reductions in both classes as estimated by both techniques revealed similar trends. Results for chlorophytes and dinoflagellates showed these groups to be most tolerant to Irgarol 1051. Indeed, class-specific biomass for chlorophytes as determined by CHEMTAX and microscopy were correlated (R2=0.53) which demonstrated an increase in both abundance and carbon content following exposures to Irgarol 1051. Abundances of nanoeukaryotes as determined by microscopy afforded good agreement with results from AFC (R2=0.8), although for picoeukaryotes, abundances were underestimated by microscopy (R2=0.43). The relative performance of the selected techniques is discussed.

Devos L, Clymans K, Boon N, Verstraete W (2005) Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples. J Appl Microbiol 99 :916-925


AIMS : To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples. METHODS AND RESULTS : The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples. CONCLUSIONS : The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples. SIGNIFICANCE AND IMPACT OF THE STUDY : This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).

Dixon BR, Bussey JM, Parrington LJ, Parenteau M (2005) Detection of Cyclospora cayetanensis oocysts in human fecal specimens by flow cytometry. J Clin Microbiol 43 :2375-2379


A diagnosis of cyclosporiasis typically involves stool examinations for the presence of Cyclospora oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of Cyclospora oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for Cyclospora by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analyst’s levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for Cyclospora oocysts, such as in an outbreak situation.

Dorsey CW, Laarakker MC, Humphries AD, Weening EH, Baumler AJ (2005) Salmonella enterica serotype Typhimurium MisL is an intestinal colonization factor that binds fibronectin. Mol Microbiol 57 :196-211


MisL is an autotransporter protein encoded by Salmonella pathogenicity island 3 (SPI3). To investigate the role of MisL in Salmonella enterica serotype Typhimurium (S. Typhimurium) pathogenesis, we characterized its function during infection of mice and identified a host receptor for this adhesin. In a mouse model of S. Typhimurium intestinal persistence, a misL mutant was shed with the faeces in significantly lower numbers than the wild type and was impaired in its ability to colonize the cecum. Previous studies have implicated binding of extracellular matrix proteins as a possible mechanism for S. Typhimurium intestinal persistence. A gluthathione-S-transferase (GST) fusion protein to the MisL passenger domain (GST-MisL(29-281)) was constructed to investigate binding to extracellular matrix proteins. In a solid-phase binding assay the purified GST-MisL(29-281) fusion protein bound to fibronectin and collagen IV, but not to collagen I. MisL expression was not detected by Western blot in S. Typhimurium grown under standard laboratory conditions. However, when expression of the cloned misL gene was driven by the Escherichia coli arabinose promoter, MisL could be detected in the S. Typhimurium outer membrane by Western blot and on the bacterial cell surface by flow cytometry. Expression of MisL enabled S. Typhimurium to bind fibronectin to its cell surface, resulting in attachment to fibronectin-coated glass slides and in increased invasiveness for human epithelial cells derived from colonic carcinoma (T84 cells). These data identify MisL as an extracellular matrix adhesin involved in intestinal colonization.

During RL, Li W, Hao B, Koenig JM, Stephens DS, Quinn CP, Southwick FS (2005) Anthrax lethal toxin paralyzes neutrophil actin-based motility. J Infect Dis 192 :837-845


Bacillus anthracis causes high-level bacteremia, strongly suggesting paralysis of the innate immune system. We have examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly. Polymorphonuclear neutrophils (PMNs) treated with a sublethal concentration of LT (50 ng/mL) for 2 h demonstrated insignificant apoptosis or necrosis. However, this same concentration slowed human PMN formylmethionylleucylphenylalanine (FMLP)-stimulated chemokinesis by >60%, markedly reduced polar morphology, and rendered PMNs incapable of responding to a chemotactic gradient. These changes were accompanied by a >50% reduction in FMLP-induced actin filament assembly. One hour of exposure to LT failed to impair polarity or actin assembly, and the effects of LT were independent of mitogen-activated protein kinase kinase 1 inhibition. We conclude that 2 h of exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis.

Eda S, Elliott B, Scott MC, Waters WR, Bannantine JP, Whitlock RH, Speer CA (2005) New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne’s disease) by flow cytometry. Foodborne Pathog Dis 2 :250-262


Johne’s disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups : (1) sera from a JD-free farm ; (2) sera from JD-positive farms that had tested negative by ELISA ; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.

Emmett SR, Dove B, Mahoney L, Wurm T, Hiscox JA (2005) The cell cycle and virus infection. Methods Mol Biol 296 :197-218


A number of different viruses interact with the cell cycle in order to subvert host-cell function and increase the efficiency of virus replication ; examples can be found from DNA, retro, and RNA viruses. The majority of studies have been conducted on DNA and retroviruses whose primary site of replication is the nucleus, but increasingly a number of researchers are demonstrating that RNA viruses, whose primary site of replication is normally the cytoplasm, also interfere with the cell cycle. Viral interference with the cell cycle can have a myriad of different effects to improve virus infection, for example to promote replication of viral DNA genomes, or to delay the cell cycle to allow sufficient time for RNA virus assembly. Although cell cycle control is fairly well characterized in terms of checkpoints and control molecules (e.g., cyclins), in recent years several researchers have demonstrated that the nucleolus is also involved in cell cycle control. The nucleolus and associated subnuclear structures can sequester cell cycle regulatory complexes, and nucleolar proteins also have a direct and indirect effect on the cycling cell. Viruses also interact with the nucleolus. In order to study the interactions between a virus and the cell cycle and vice versa we have developed and adapted a number of different approaches and strategies. These include determinations of virus yield and measurements of virus replication to flow cytometry and confocal analysis of the host cell. Increasingly we have found that proteomic approaches allow the rapid analysis of a whole plethora of cell cycle proteins that may be affected by virus infection.

Engelbert M, Gilmore MS (2005) Fas ligand but not complement is critical for control of experimental Staphylococcus aureus Endophthalmitis. Invest Ophthalmol Vis Sci 46 :2479-2486


PURPOSE : To determine the role of complement and Fas Ligand (FasL) in the host defense against Staphylococcus aureus endophthalmitis. METHODS : C3-/-, FasL defective gld, and C57/BL6 (wild-type [WT]) mice were infected intravitreally with 500 and 5000 CFU S. aureus, and the course of infection was followed by determining the intraocular bacteria counts, retinal function by ERG, and morphologic damage and inflammation by histopathology and flow cytometry. RESULTS : In WT eyes injected with 500 CFU, S. aureus grew to 1 x 10(7) CFU/mL by 24 hours, but was cleared by 96 hours. In the WT eyes injected with 5000 CFU, S. aureus grew to 2 x 10(9) CFU/mL by 72 hours, resulting in corneal perforation. C3-/- eyes injected with 500 CFU reached transiently higher levels than their WT counterparts (P < 0.001), but eventually followed a similar course. Bacterial counts in gld eyes infected with 500 CFU were similar to those in WT eyes infected with 5000 CFU. In WT and C3-/- eyes injected with 500 CFU, retinal function decreased only transiently and recovered to 66% in 72 hours. In WT eyes injected with 5000 CFU and gld eyes infected with 500 CFU, retinal function was completely lost by 24 hours. By 24 hours, WT and C3-/- eyes injected with 500 CFU were infiltrated with a similar number of granulocytes, but recruitment was significantly impaired in gld eyes (P < 0.005). Cell counts in WT and C3-/- eyes decreased thereafter without overt retinal disease. In eyes injected with 5000 CFU and gld eyes infected with 500 CFU, inflammatory cells completely filled the intraocular space by 48 hours. Retinal and uveal tissue was destroyed by that time. CONCLUSIONS : The tipping point for a good versus a bad outcome in this murine model of endophthalmitis lies between 500 and 5000 CFU S. aureus. This point is identical in animals deficient in complement activation, suggesting that complement does not play a significant role in the ocular defense against intraocular bacteria. In contrast, FasL was found to be critical for clearance, since animals deficient in FasL signaling were unable to control infection with 500 CFU.

French AR, Pingel JT, Kim S, Yang L, Yokoyama WM (2005) Rapid emergence of escape mutants following infection with murine cytomegalovirus in immunodeficient mice. Clin Immunol 115 :61-69


Natural killer (NK) cells play a crucial role in the initial host defense against pathogens such as murine cytomegalovirus (MCMV). They respond rapidly and effectively control pathogen replication while the adaptive immune system is being activated. However, in the absence of an adaptive immune system, an effective initial NK cell response is not sufficient for long-term pathogen control as demonstrated by the late recrudescence of disease and mortality in immunodeficient mice infected with MCMV. In this setting, NK cells suppress the initial infection but exert enough selective pressure to drive the outgrowth of MCMV mutants that escape recognition by NK cells. Herein, we characterize the rapid emergence of escape mutants following infection with a plaque-purified MCMV isolate and demonstrate that these mutant viruses are no longer effectively controlled by NK cells. These findings suggest that late recrudescence of viral infections in certain clinical settings may also be due to viral escape from NK cells or other components of innate immunity.

Fritsch M, Starruss J, Loesche A, Mueller S, Bley T (2005) Cell cycle synchronization of Cupriavidus necator by continuous phasing measured via flow cytometry. Biotechnol Bioeng 92 :635-642


The continuous phasing technique was successfully used to obtain a high degree of cell cycle synchrony in cultures of the model organism Ralstonia eutropha JMP 134 (today reclassified into Cupriavidus necator). The responses of the organism were evaluated with flow cytometric determinations of DNA contents and cell size (by fluorescence and forward scatter measurements, respectively, after staining with the DNA-binding dye 4’,6-diamidino-2’-phenylindole, DAPI), and cell concentration, after staining with the nucleic acid binding dye LDS-751. The strain was cultivated on a mineral medium with pyruvic acid sodium salt as the limiting carbon and energy source. Famine conditions, and thus cell dormancy, were achieved in every cycle. The best synchronization, according to the determination of DNA contents, was induced with phasing cycle durations of at least 4 h. The method allows the induction of synchrony for an indefinite period if the medium is exchanged rapidly and precisely. The results show that the time required for a complete cell cycle of Cupriavidus necator JMP 134 is independent of the chosen phasing cycle duration, provided that each process cycle lasts at least 3 h which is much longer than the time needed for a single DNA replication cycle. With shorter cycling periods DNA-synthesis is carried out in an uncoupled manner and only weak cell cycle synchrony can be attained. The results also show that DNA-synthesis can only be undertaken by cells when they have exceeded a critical size.

Gabier AC, Gourdon P, Reitz J, Leveau JY, Bouix M (2005) Intracellular physiological events of yeast Rhodotorula glutinis during storage at +4 degrees C. Int J Food Microbiol 105 :97-109


Samples of the cheese yeast Rhodotorula glutinis were analysed during storage at +4 degrees C for cultivability, viability, vitality (metabolic activity), membrane potential state, intracellular pH, and carbohydrate content. The results have allowed to describe cellular events occurring during storage. The loss of vitality came with the decrease of carbohydrate content. The fall of trehalose content under a threshold value induced the deterioration of the membrane potential. Later, when all the cells were depolarised, the intracellular pH decreased and the cultivability dropped, whereas viable cells still decreased slowly. Then, it led to an intermediate physiological state similar to the viable but non-cultivable state. Finally, the fall of viability dropped. In this work, we have defined rapid methods relevant to describe the sequence of intracellular events in the cheese yeast R. glutinis during storage, and we applied them to understand the weak vitality without fall of viability of yeast samples. These methods might allow to rapidly test yeast sample quality before use and to predict, at the moment of the harvesting, the conservation of the yeast.

Gamalero E, Lingua G, Tombolini R, Avidano L, Pivato B, Berta G (2005) Colonization of tomato root seedling by Pseudomonas fluorescens 92 rkG5 : spatio-temporal dynamics, localization, organization, viability, and culturability. Microb Ecol 50 :289-297


The localization, viability, and culturability of Pseudomonas fluorescens 92 rkG5 were analyzed on three morphological root zones (root tip + elongation, root hair, and collar) of 3-, 5-, and 7-day-old tomato plants. Qualitative information about the localization and viability was collected by confocal laser scanning microscopy. Quantitative data concerning the distribution, viability, and culturability were obtained through combined dilution plating and flow cytometry. Colonization by P. fluorescens affected root development in a complex way, causing a general increase in the length of the collar and early stimulation of the primary root growth (3rd day), followed by a reduction in length (7th day). The three root zones showed different distribution, organization, and viability of the bacterial cells, but the distribution pattern within each zone did not change with time. Root tips were always devoid of bacteria, whereas with increasing distance from the apex, microcolonies or strings of cells became more and more prominent. Viability was high in the elongation zone, but it declined in the older parts of the roots. The so-called viable but not culturable cells were observed on the root, and their proportion in the distal (root tip + elongation) zone dramatically increased with time. These results suggest the existence of a specific temporal and spatial pattern of root colonization, related to cell viability and culturability, expressed by the plant-beneficial strain P. fluorescens 92 rkG5.

Garcia-Mediavilla MV, Sanchez-Campos S, Gonzalez-Perez P, Gomez-Gonzalo M, Majano PL, Lopez-Cabrera M, Clemente G, Garcia-Monzon C, Gonzalez-Gallego J (2005) Differential contribution of hepatitis C virus NS5A and core proteins to the induction of oxidative and nitrosative stress in human hepatocyte-derived cells. J Hepatol 43 :606-613


BACKGROUND/AIMS : We aimed to explore the effects of hepatitis C virus (HCV) core and NS5A proteins on reactive oxygen (ROS) and nitrogen species (RNS) formation and on gene expression profile of iNOS in human hepatocyte-derived cells. METHODS : Production of ROS and RNS and nitrotyrosine residues accumulation were determined by flow cytometry and fluorescent microscopy as well as by Western blot, respectively, in NS5A- and core-transfected cells. Northern blot, Western blot, real-time PCR, and luciferase assays were used to assess iNOS gene expression in both transfectants. RESULTS : Cytokine-activated NS5A- and core-transfected cells induced ROS and RNS production but an earlier and more marked increase was observed in NS5A-expressing cells. Superoxide production was also augmented, showing a similar temporal pattern of appearance in both NS5A- and core-transfected cells. Although both NS5A and core HCV proteins were able to up-regulate iNOS gene expression, accompanied by a nitrotyrosine-containing proteins accumulation, an earlier iNOS overexpression was observed in NS5A-expressing cells, suggesting a different time course of iNOS activation pattern for core and NS5A HCV proteins. CONCLUSIONS : Our results indicate a differential contribution of both HCV proteins to oxidative and nitrosative stress generation.

Garmendia J, Frankel G (2005) Operon structure and gene expression of the espJ—tccP locus of enterohaemorrhagic Escherichia coli O157:H7. FEMS Microbiol Lett 247 :137-145


In this study, we investigated the genetic organisation and expression of the espJ—tccP locus of enterohaemorrhagic Escherichia coli (EHEC O157:H7), which encodes the type III secretion system effector proteins EspJ and TccP. Using flow cytometry and fluorescence microscopy, we found that espJ and tccP constitute an operon, whose expression in vitro is affected by the composition of the medium and environmental signals such as temperature, pH, osmolarity and O(2) pressure. Moreover, expression of the espJ—tccP operon is not regulated by Ler, a LEE-encoded transcriptional regulator, but by an as yet unidentified regulatory system(s).

Garrido D, Suau A, Pochart P, Cruchet S, Gotteland M (2005) Modulation of the fecal microbiota by the intake of a Lactobacillus johnsonii La1-containing product in human volunteers. FEMS Microbiol Lett 248 :249-256


Lactobacillus johnsonii La1 (La1) is a probiotic strain capable of stimulating the immune system of the host and interfering with gastrointestinal pathogens. This study evaluates how the ingestion of different amounts of La1 influences the main bacterial populations of the fecal microbiota. Eight asymptomatic volunteers participated in the study. After a basal period, they ingested daily 100 mL of a product containing 10(8) CFU mL(-1) of La1 during the first week, 200 mL during the second week and 500 mL during the third week. Fecal samples were obtained at the end of each period and subsequently during 7 weeks. Lactobacilli were determined by culture on MRS agar and La1 colonies were confirmed by ERIC-PCR. The main populations of fecal bacteria were identified by FISH and flow cytometry. At baseline, 37.7% of the total fluorescent bacteria were Eubacterium rectale, 18.3% Fusobacterium prausnitzii, 13.2% Bacteroides, 8.6% Atopobium, 2.30%, Clostridium histolyticum, 2.05% Bifidobacterium and 0.95% Lactobacillus. Fecal excretion of La1 increased during the intake period and decreased during the post-ingestion period, so that no La1 was observed in the stools of the volunteers seven weeks after the intake product has been finished. La1 intake increased the populations of C. histolyticum (p=0.049), Lactobacillus (p=0.056) and Bifidobacterium (p=0.067), and decreased those of F. prausnitzii (p=0.005) while it did not affect Bacteroides, E. rectale and Atopobium populations. These bacterial populations returned to their baseline levels during the post-ingestion period. The regular intake of a La1-containing product beneficially affects the homeostasis of the human fecal microbiota, probably contributing to the health-promoting effects of this probiotic.

Gonzalez CF, Ackerley DF, Lynch SV, Matin A (2005) ChrR, a soluble quinone reductase of Pseudomonas putida that defends against H2O2. J Biol Chem 280 :22590-22595


Most bacteria contain soluble quinone-reducing flavoenzymes. However, no biological benefit for this activity has previously been demonstrated. ChrR of Pseudomonas putida is one such enzyme that has also been characterized as a chromate reductase ; yet we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H(2)O(2). Expression of chrR was induced by H(2)O(2), and levels of chrR expression in overexpressing, wild type, and knock-out mutant strains correlated with the H(2)O(2) tolerance and scavenging ability of each strain. The chrR expression level also correlated with intracellular H(2)O(2) levels as measured by protein carbonylation assays and fluorescence-activated cell scanning analysis with the H(2)O(2)-responsive dye H(2)DCFDA. Thus, enhancing the activity of ChrR in a chromate-remediating bacterial strain may not only increase the rate of chromate transformation, it may also augment the capacity of these cells to withstand the unavoidable production of H(2)O(2) that accompanies chromate reduction.

Green CB, Zhao X, Yeater KM, Hoyer LL (2005) Construction and real-time RT-PCR validation of Candida albicans PALS-GFP reporter strains and their use in flow cytometry analysis of ALS gene expression in budding and filamenting cells. Microbiology 151 :1051-1060


The gene encoding yeast-enhanced green fluorescent protein (GFP) was placed under control of ALS gene promoters in Candida albicans. The PALS-GFP reporter strains were validated using various techniques including a new real-time RT-PCR assay to quantify ALS gene expression. The PALS-GFP reporter strains were grown in media that promoted yeast or germ tube forms, and the resulting fluorescence was measured by flow cytometry. In addition to results that indicate differences in ALS gene expression due to growth medium, growth stage and developmental programme, new data show large differences in transcriptional level among the ALS genes. Expression of ALS1 was associated with transfer of the PALS1-GFP strain to fresh growth medium. ALS3 expression increased markedly when germ tubes were visible microscopically and ALS7 expression exhibited a transient peak between 2 and 3 h following inoculation into fresh YPD medium. Transcription from the ALS1 and ALS3 promoters was strongest among those tested and contrasted markedly with the weaker promoter strength at the ALS5, ALS6, ALS7 and ALS9 loci. These weaker transcriptional responses were also observed using real-time RT-PCR measurements on wild-type C. albicans cells. Assuming a positive correlation between transcriptional level and protein production, these results suggest that some Als proteins are abundant on the C. albicans cell surface while others are produced at a much lower level.

Groisman A, Lobo C, Cho H, Campbell JK, Dufour YS, Stevens AM, Levchenko A (2005) A microfluidic chemostat for experiments with bacterial and yeast cells. Nat Methods 2 :685-689


Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer.

Guseva NV, Dessus-Babus SC, Whittimore JD, Moore CG, Wyrick PB (2005) Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis. Microbes Infect 7 :1469-1481


Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) >> MCF-7 (57%)>Ishikawa (51%) >> HCC-1806 (20%). The cell line ER isoform composition was re-defined as : ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa ; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.

Guzman CA, Cebolla A, Beltrametti F, Staender LH, de Lorenzo V (2005) Physiological stress of intracellular Shigella flexneri visualized with a metabolic sensor fused to a surface-reporter system. FEBS Lett 579 :813-818


When deleted of its N-terminal signal-reception domain, the broad host range sigma54-dependent transcriptional regulator XylR, along with its cognate promoter Pu, becomes a sensor of the metabolic stress of the carrier bacteria. We have employed a surface reporter system to visualize the physiological status of intracellular Shigella flexneri during infection of Henle 407 cells in culture. To this end, the xylRDeltaA gene has been engineered adjacent to a bicistronic transcriptional fusion of Pu to a lamB variant tagged with a short viral sequence (cor) and beta-galactosidase (lacZ). The accessibility of the cor epitope to the externalmost medium and the expression of Pu in the bacterial population was confirmed, respectively, with immunomagnetic beads and the sorting of Escherichia coli cells treated with a fluorescent antibody. Intracellular Shigella cells expressed the Pu-lamB/cor-lacZ reporter at high levels, suggesting that infectious cells endure a considerable metabolic constraint during the invasion process.

Hammes FA, Egli T (2005) New method for assimilable organic carbon determination using flow-cytometric enumeration and a natural microbial consortium as inoculum. Environ Sci Technol 39 :3289-3294


The concentration of easily assimilable organic carbon (AOC) largely determines the microbiological stability of drinking water. However, AOC determination is often neglected in practice due to the complex and tedious nature of the conventional bioassay. The three major drawbacks of the conventional method are (1) a long assay time of 9-12 days, (2) the use of a labor-intensive enumeration technique (plating on growth media), and (3) limited information supplied by the use of selected pure cultures (Pseudomonas fluorescens P-17 and Spirillum NOX) for measuring a complex pool of natural bioavailable carbon compounds. A new method is proposed here, in which plating was replaced with fluorescence staining of total nucleic acids combined with flow cytometry as a rapid and straightforward growth enumeration method. This approach also allowed for the detection of inactive and/or unculturable microorganisms. Hence, the conventionally used pure cultures were replaced in the new AOC assay with a natural microbial consortium. It was shown that the flow-cytometric enumeration method could be used to establish complete growth curves for a natural microbial consortium growing on AOC. Compared to the end-point measurements of the conventional method, such kinetic data provide much clearer insight into the actual growth potential of a water.

Hartmann H, Stender H, Schafer A, Autenrieth IB, Kempf VA (2005) Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry. J Clin Microbiol 43 :4855-4857


Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

He X, Gonzalez V, Tsang A, Thompson J, Tsang TC, Harris DT (2005) Differential gene expression profiling of CD34+ CD133+ umbilical cord blood hematopoietic stem progenitor cells. Stem Cells Dev 14 :188-198


Umbilical cord blood (CB)-derived primitive hematopoietic stem progenitor cells (HSPC) are a promising source for stem cell-based gene therapy due to the reduced incidence and severity of graftversus- host disease (GVHD) after human leukocyte antigen (HLA)-disparate CB transplantation. Cell-surface markers such as CD34 and CD133 have been used in combination to enrich primitive HSPC for research and clinical applications. To understand the molecular characteristics of the CB HSPC, we compared the global gene expression of freshly isolated CB CD34+ CD133+ cells with their progenies using a cDNA microarray containing 22,000 human cDNA clones printed on a single chip. A total of 139 genes were differentially expressed between CB HSPC and their progenies. These transcripts included a number of known genes that might play roles in key functions of CB HSPC as well as many genes of unknown function. Among the genes showing the greatest differential expression levels in HSPC were : psoriasin 1, CRHBP, HDAC3, MLLT3, HBEX2, SPINK2, c-kit, H2BFQ, CD133, HHEX, TCF4, ALDH1A1, and FHL1. These data provide more information on the molecular phenotype of CB HSPC and may lead to the identification of new genes critical to stem cell function.

Heaney J, Cosby SL, Barrett T (2005) Inhibition of host peripheral blood mononuclear cell proliferation ex vivo by Rinderpest virus. J Gen Virol 86 :3349-3355


Rinderpest, or cattle plague, is caused by Rinderpest virus (RPV), which is related most closely to human Measles virus (MV), both being members of the genus Morbillivirus, a group of viruses known to have strong immunosuppressive effects in vitro and in vivo. Here, it was shown that peripheral blood mononuclear cells (PBMCs) isolated from cattle experimentally infected with either wild-type or vaccine strains of RPV impaired the proliferation of PBMCs derived from uninfected animals ; however, in contrast to either mild or virulent strains of wild-type virus, the inhibition induced by the vaccine was both weak and transient. Flow-cytometric analysis of PBMCs obtained from cattle infected with different strains of RPV showed that the proportion of infected cells was virus dose-dependent and correlated with lymphoproliferative suppression.

Hewson CA, Jardine A, Edwards MR, Laza-Stanca V, Johnston SL (2005) Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells. J Virol 79 :12273-12279


Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.

Ho LJ, Hung LF, Weng CY, Wu WL, Chou P, Lin YL, Chang DM, Tai TY, Lai JH (2005) Dengue virus type 2 antagonizes IFN-alpha but not IFN-gamma antiviral effect via down-regulating Tyk2-STAT signaling in the human dendritic cell. J Immunol 174 :8163-8172


The immunopathogenesis mechanism of dengue virus (DV) infection remains elusive. We previously showed that the target of DV in humans is dendritic cells (DCs), the primary sentinels of immune system. We also observed that despite the significant amount of IFN-alpha induced ; DV particles remain massively produced from infected DCs. It suggests that DV may antagonize the antiviral effect of IFN-alpha. Recent work in animal studies demonstrated the differential critical roles of antiviral cytokines, namely IFN-alpha/IFN-beta and IFN-gamma, in blocking early viral production and in preventing viral-mediated disease, respectively. In this study, we examined the effects of IFN-alpha and IFN-gamma in DV infection of monocyte-derived DCs. We showed that the preinfection treatment with either IFN-alpha or IFN-gamma effectively armed DCs and limited viral production in infected cells. However, after infection, DV developed mechanisms to counteract the protection from lately added IFN-alpha, but not IFN-gamma. Such a selective antagonism on antiviral effect of IFN-alpha, but not IFN-gamma, correlated with down-regulated tyrosine-phosphorylation and DNA-binding activities of STAT1 and STAT3 transcription factors by DV. Furthermore, subsequent studies into the underlying mechanisms revealed that DV attenuated IFN-alpha-induced tyrosine-phosphorylation of Tyk2, an upstream molecule of STAT activation, but had no effect on expression of both IFN-alpha receptor 1 and IFN-alpha receptor 2. Moreover, DV infection by itself could activate STAT1 and STAT3 through IFN-alpha-dependent and both IFN-alpha-dependent and IFN-alpha-independent mechanisms, respectively. These observations provide very useful messages with physiological significance in investigation of the pathogenesis, the defense mechanisms of human hosts and the therapeutic considerations in DV infection.

Ho YC, Chung YC, Hwang SM, Wang KC, Hu YC (2005) Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells. J Gene Med 7 :860-868


BACKGROUND : Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS : A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS : In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS : These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.

Hoefel D, Monis PT, Grooby WL, Andrews S, Saint CP (2005) Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system. Appl Environ Microbiol 71 :6479-6488


Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 10(5) bacteria ml(-1). The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene ; 3.43 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) active AOB ml(-1) were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml(-1) detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.

Hoefel D, Monis PT, Grooby WL, Andrews S, Saint CP (2005) Profiling bacterial survival through a water treatment process and subsequent distribution system. J Appl Microbiol 99 :175-186


AIMS : To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. METHODS AND RESULTS : Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. CONCLUSIONS : Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. SIGNIFICANCE AND IMPACT OF THE STUDY : Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.

Hung SL, Cheng YY, Peng JL, Chang LY, Liu TY, Chen YT (2005) Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils. J Periodontol 76 :373-379


BACKGROUND : Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS : Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS : At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS : Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.

Hunt ME, Brown DR (2005) Mycoplasma alligatoris infection promotes CD95 (FasR) expression and apoptosis of primary cardiac fibroblasts. Clin Diagn Lab Immunol 12 :1370-1377


Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P<0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P<0.16) in 5-bromo-2’-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P<0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway.

Jugeau S, Tenaud I, Knol AC, Jarrousse V, Quereux G, Khammari A, Dreno B (2005) Induction of toll-like receptors by Propionibacterium acnes. Br J Dermatol 153 :1105-1113


BACKGROUND : The bacterium Propionibacterium acnes is involved in the induction and maintenance of the inflammatory phase of acne. Recent studies have found that keratinocytes express toll-like receptors (TLRs) implicated in immediate immunity. No studies have, to date, been carried out on the action of P. acnes upon TLR activation in keratinocytes. OBJECTIVES : Focusing on the inflammatory phase of acne, to clarify the role of P. acnes in immediate immunity by inducing expression of TLR-2 and TLR-4 by keratinocytes. We also studied how the secretion and expression of matrix metalloproteinase (MMP)-9 is induced by P. acnes. METHODS : The work was carried out on two levels : in vivo with the study of the expression of TLR-2 and TLR-4 proteins in biopsies of acne lesions and in vitro on cultured keratinocyte monolayers to study the modulating effects of P. acnes on the expression of TLR-2 and TLR-4 and also on the expression and secretion of MMP-9. RESULTS : Our findings reveal that in vivo TLR-2 and TLR-4 expression is increased in the epidermis of acne lesions. In vitro, an increase in TLR-2 and TLR-4 expression by human keratinocytes occurred in the first hours of incubation with bacterial fractions as well as an increase of the expression and secretion by the keratinocytes of MMP-9, which plays a role in inflammation. CONCLUSIONS : This work demonstrates that P. acnes induces TLR expression and that this mechanism could play an essential role in acne-linked inflammation. These receptors could be involved notably in acute acne.

Julia O, Vives-Rego J (2005) Skew-Laplace distribution in Gram-negative bacterial axenic cultures : new insights into intrinsic cellular heterogeneity. Microbiology 151 :749-755


The application of flow cytometry and skew-Laplace statistical analysis to assess cellular heterogeneity in Gram-negative axenic cultures is reported. In particular, fit to the log-skew-Laplace distribution for cellular side scatter or ’granulosity’ is reported, and a number of theoretical and applied issues are considered in relation to the biological significance of this fit.

Kalvegren H, Bylin H, Leanderson P, Richter A, Grenegard M, Bengtsson T (2005) Chlamydia pneumoniae induces nitric oxide synthase and lipoxygenase-dependent production of reactive oxygen species in platelets. Effects on oxidation of low density lipoproteins. Thromb Haemost 94 :327-335


There is increasing evidence that Chlamydia pneumoniae is linked to atherosclerosis and thrombosis. In this regard, we have recently shown that C. pneumoniae stimulates platelet aggregation and secretion, which may play an important role in the progress of atherosclerosis and in thrombotic vascular occlusion. The aims of the present study were to investigate the effects of C. pneumoniae on platelet-mediated formation of reactive oxygen species (ROS) and oxidation of low-density lipoprotein (LDL) in vitro. ROS production was registered as changes in 2’,7’-dichlorofluorescin- fluorescence in platelets with flow cytometry. LDL-oxidation was determined by measuring thiobarbituric acid reactive substances (TBARs). We found that C. pneumoniae stimulated platelet production of ROS. Polymyxin B treatment of C. pneumoniae, but not elevated temperature, abolished the stimulatory effects on platelet ROS-production, which suggests that chlamydial lipopolysaccharide has an important role. Inhibition of nitric oxide synthase with nitro-L-arginine, lipoxygenase with 5,8,11-eicosatriynoic acid and protein kinase C with GF 109203X significantly lowered the production of radicals. In contrast, inhibition of NADPH-oxidase with di-phenyleneiodonium (DPI) did not affect the C. pneumoniae induced ROS-production. These findings suggest that the activities of nitric oxide synthase and lipoxygenase are the sources for ROS and that the generation is dependent of the activity of protein kinase C. The C. pneumoniae-induced ROS-production in platelets was associated with an extensive oxidation of LDL, which was significantly higher compared to the effect obtained by separate exposure of LDL to C. pneumoniae or platelets. In conclusion, C. pneumoniae interaction with platelets leading to aggregation, ROS-production and oxidative damage on LDL, may play a crucial role in the development of atherosclerotic cardiovascular disease.

Kampen AH, Tollersrud T, Lund A (2005) Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro. Infect Immun 73 :1578-1583


Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P <0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P <0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.

Kanno F, Korostoff J, Volgina A, DiRienzo JM (2005) Resistance of human periodontal ligament fibroblasts to the cytolethal distending toxin of Actinobacillus actinomycetemcomitans. J Periodontol 76 :1189-1201


BACKGROUND : The cytolethal distending toxin (CDT) of Actinobacillus actinomycetemcomitans is a typical member of this Gram-negative bacterium holotoxin family that targets a wide spectrum of eukarytotic cells, typically causing cell cycle arrest at either the G(1) or G(2)/M phase of the cell cycle. In view of the possible role of the CDT as a prominent A. actinomycetemcomitans virulence factor in periodontal diseases, we have examined the effects of the toxin on primary cultures of human periodontal ligament fibroblasts (HPLF). METHODS : HPLF and an immortalized human gingival epithelial cell line, GMSM-K, were exposed to recombinant A. actinomycetemcomitans CDT. Effects of the toxin on cell proliferation and cell cycle were assessed by a cell viability assay and flow cytometry, respectively. Double-strand DNA damage was detected by pulsed field gel electrophoresis. Binding of the toxin and its individual subunits to HPLF was examined by immunofluorescence microscopy. RESULTS : Viability of HPLF was not reduced following prolonged exposure to the CDT. There was no indication of cell cycle arrest or double-strand DNA damage. GMSM-K cells exhibited morphological alterations and a rapid decrease in cell viability within 6 and 12 hours, respectively, following exposure to the toxin for 5 minutes. These effects were dependent on toxin dose and age of the cultures and occurred more rapidly compared to CDT-treated HeLa cells. CDT-treated GMSM-K cells displayed cell cycle arrest at the S phase of growth and double-strand DNA damage was observed by 6 hours post-intoxication. Holotoxin and the CdtA subunit were detected on the surface of both HPLF and epithelial cells. CONCLUSIONS : These results demonstrate that HPLF are resistant to the cytotoxic effects of the A. actinomycetemcomitans CDT. The mechanism of resistance is not known but may be related to the inability of the toxin to cause DNA damage. The difference in sensitivities of HPLF and oral epithelial cells to the CDT has important implications for the role of this putative microbial virulence factor in periodontal pathogenesis.

Karunakaran R, Mauchline TH, Hosie AH, Poole PS (2005) A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria. Microbiology 151 :3249-3256


A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Kim BJ, Choi CH, Lee CH, Jeong SY, Kim JS, Kim BY, Yim HS, Kang SO (2005) Glutathione is required for growth and prespore cell differentiation in Dictyostelium. Dev Biol 284 :387-398


Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.

Kippin TE, Martens DJ, van der Kooy D (2005) p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity. Genes Dev 19 :756-767


Adult stem cells in various tissues are relatively quiescent. The cell cycle inhibitor p21cip1/waf1 (p21) has been shown to be important for maintaining hematopoietic stem cell quiescence and self-renewal. We examined the role of p21 in the regulation of adult mammalian forebrain neural stem cells (NSCs). We found that p21-/- mice between post-natal age 60-240 d have more NSCs than wild-type (+/+) controls due to higher proliferation rates of p21-/- NSCs. Thereafter, NSCs in p21-/- mice decline and are reduced in number at 16 mo relative to p21+/+ mice. Similarly, both p21-/- and p21+/+ NSCs display self-renewal in vitro ; however, p21-/- NSCs display limited in vitro self-renewal (surviving a few passages, then exhausting). Thus, p21 contributes to adult NSC relative quiescence, which we propose is necessary for the life-long maintenance of NSC self-renewal because NSCs may be limited to a finite number of divisions.

Krarup A, Sorensen UB, Matsushita M, Jensenius JC, Thiel S (2005) Effect of capsulation of opportunistic pathogenic bacteria on binding of the pattern recognition molecules mannan-binding lectin, L-ficolin, and H-ficolin. Infect Immun 73 :1052-1060


Mannan-binding lectin (MBL), L-ficolin, and H-ficolin are pattern recognition molecules of the innate immune system. We investigated their ability to bind to different serotypes and noncapsulated variants of two gram-positive bacterial species, Streptococcus pneumoniae and Staphylococcus aureus. MBL did not bind to capsulated S. aureus or capsulated S. pneumoniae but did bind to a noncapsulated S. aureus variant (Wood). L-ficolin bound to some capsulated S. aureus serotypes (serotypes 1, 8, 9, 11, and 12) and capsulated S. pneumoniae serotypes (11A, 11D, and 11F) but not to noncapsulated strains. H-ficolin did not bind to any of the S. pneumoniae and S. aureus serotypes included in this study but did bind to one strain of Aerococcus viridans. The concentrations of the three proteins in 97 plasma samples were estimated. The median concentrations were 0.8 mug per ml for MBL, 3.3 mug per ml for L-ficolin, and 18.4 mug per ml for H-ficolin.

Lacroix-Gueu P, Briandet R, Leveque-Fort S, Bellon-Fontaine MN, Fontaine-Aupart MP (2005) In situ measurements of viral particles diffusion inside mucoid biofilms. C R Biol 328 :1065-1072


Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.

Lambeth CR, White LJ, Johnston RE, de Silva AM (2005) Flow cytometry-based assay for titrating dengue virus. J Clin Microbiol 43 :3267-3272


Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.

Lay C, Rigottier-Gois L, Holmstrom K, Rajilic M, Vaughan EE, de Vos WM, Collins MD, Thiel R, Namsolleck P, Blaut M, Dore J (2005) Colonic microbiota signatures across five northern European countries. Appl Environ Microbiol 71 :4153-4155


The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.

Lay C, Sutren M, Rochet V, Saunier K, Dore J, Rigottier-Gois L (2005) Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota. Environ Microbiol 7 :933-946


Among human faecal bacteria, many members of the Clostridium leptum subgroup are fibrolytic and butyrate producing microorganisms thereby contributing to processes important to colonic health. Yet this phylogenetic subgroup remains poorly described to date. To improve detection and description of members of the C. leptum subgroup, the Clep 866 group probe was developed. Its association with probes targeting the Clostridium viride cluster (Cvir 1414) and Eubacterium desmolans species (Edes 635) allowed for the first time the detection of all members found in this phylogenetic group in human faecal microbiota. A species-specific probe was also designed to detect members of the Ruminococcus callidus species (Rcal 733). The design of signature regions was based on alignment of 16S rRNA sequences isolated from faeces of five healthy adults. Furthermore, an oligonucleotide competitor strategy was developed in order to improve the specificity of the probes formerly validated or designed in this study. The oligonucleotide probes were tested using a collection of target and non-target strains using FISH combined with flow cytometry. These new probes were added to a panel of 18 phylogenetic probes selected to describe faecal microbiota composition in 21 human faeces of healthy adults. Clostridium leptum subgroup represented 22% of the total faecal bacteria and codominated with members of Clostridium coccoides group. The cluster Faecalibacterium prausnitzii was the dominant component of the C. leptum subgroup and 20% of the latter subgroup remained unidentified at the species level.

Lee GC, Lee DG, Choi SM, Yoo JH, Park SH, Choi JH, Min WS, Cho OH, Lee CH, Shin WS (2005) Use of time-saving flow cytometry for rapid determination of resistance of human cytomegalovirus to ganciclovir. J Clin Microbiol 43 :5003-5008


There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV) : one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL 97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL 54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC(50)) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC(50) values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV ; one was GCV resistant due to the mutation M 460 V, and the GCV IC(50) results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.

Lee JC, Yokota K, Arimitsu H, Hwang HJ, Sakaguchi Y, Cui J, Takeshi K, Watanabe T, Ohyama T, Oguma K (2005) Production of anti-neurotoxin antibody is enhanced by two subcomponents, HA1 and HA3b, of Clostridium botulinum type B 16S toxin-haemagglutinin. Microbiology 151 :3739-3747


Clostridium botulinum type B strain produces two forms of progenitor toxin, 16S and 12S. The 12S toxin is formed by association of a neurotoxin (NTX) and a non-toxic non-haemagglutinin (NTNH), and the 16S toxin is formed by conjugation of the 12S toxin with a haemagglutinin (HA). HA consists of four subcomponents designated HA1, HA2, HA3a and HA3b. When mice were immunized with formalin-detoxified NTX, 12S or 16S, a significantly greater amount of anti-NTX antibody (Ab) was produced in the mice injected with 16S than in NTX- or 12S-injected mice. Immunization with NTX mixed with HA1 and/or HA3b also increased the anti-NTX Ab production, whereas NTX mixed with HA2 did not, indicating that HA1 and HA3b have adjuvant activity. This was further confirmed by immunizing mice with human albumin (Alb) alone or Alb mixed with either HA1 or HA3b. When mouse-spleen cells were stimulated with NTX, 16S or different HA subcomponents, 16S, HA1, HA3b and the mixture of HA1 and HA3 significantly increased interleukin 6 (IL6) production compared with NTX alone. Transcription of IL6 mRNA was low after stimulation with NTX alone, but increased to 16S-stimulation levels when NTX was mixed with HA1 or HA3b. In flow cytometry using labelled Abs against CD3 and CD19, the percentage of CD19 cells was higher following stimulation with 16S or NTX mixed with HA1 or HA3b compared with stimulation with NTX. The percentage of CD3 cells remained unchanged. These results suggest strongly that HA1 and HA3b demonstrate adjuvant activity via increasing IL6 production.

Lemar KM, Passa O, Aon MA, Cortassa S, Muller CT, Plummer S, O’Rourke B, Lloyd D (2005) Allyl alcohol and garlic (Allium sativum) extract produce oxidative stress in Candida albicans. Microbiology 151 :3257-3265


Both the growth and respiration of Candida albicans are sensitive to extracts of Allium sativum and investigations into the anticandidal activities are now focussing on the purified constituents to determine the targets of inhibition. Of particular interest is allyl alcohol (AA), a metabolic product that accumulates after trituration of garlic cloves. Putative targets for AA were investigated by monitoring changes in intracellular responses after exposure of C. albicans cells to AA or a commercially available garlic extract. Two-photon laser scanning microscopy and other techniques were used. Changes typical of oxidative stress—NADH oxidation and glutathione depletion, and increased reactive oxygen species—were observed microscopically and by flow cytometry. Known targets for AA are alcohol dehydrogenases Adh1 and 2 (in the cytosol) and Adh3 (mitochondrial), although the significant decrease in NAD(P)H after addition of AA is indicative of another mechanism of action.

Lennartz K, Lu M, Flasshove M, Moritz T, Kirstein U (2005) Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet. Cytometry A 66 :119-127


BACKGROUND : The jet-in-air cell sorters currently available are not very suitable for sorting potentially biohazardous material under optimal conditions because they do not protect operators and samples as recommended in the guidelines for safe biotechnology. To solve this problem we have adapted a cell sorting system to a special biosafety cabinet that satisfies the requirements for class II cabinets. With aid of this unit, sorting can be performed in conformance with the recommendations for biosafety level 2. METHODS : After integrating a modified fluorescence-activated cell sorter (FACS) Vantage into a special biosafety cabinet, we investigated the influence of the laminar air flow (LAF) inside the cabinet on side stream stability and the analytical precision of the cell sorter. In addition to the routine electronic counting of microparticles, we carried out tests on the containment of aerosols, using T4 bacteriophage as indicators, to demonstrate the efficiency of the biosafety cabinet for sorting experiments performed under biosafety level 2 conditions. RESULTS : The experiments showed that LAF, which is necessary to build up sterile conditions in a biosafety cabinet, does not influence the conditions for side stream stability or the analytical precision of the FACS Vantage cell sorting system. In addition, tests performed to assess aerosol containment during operation of the special biosafety cabinet demonstrated that the cabinet-integrated FACS Vantage unit (CIF) satisfies the conditions for class II cabinets. In the context of gene transfer experiments, the CIF facility was used to sort hematopoietic progenitor cells under biosafety level 2 conditions. CONCLUSIONS : The newly designed biosafety cabinet offers a practical modality for improving biosafety for operators and samples during cell sorting procedures. It can thus also be used for sorting experiments with genetically modified organisms in conformance with current biosafety regulations and guidelines.

Leong CM, Hibma MH (2005) A flow cytometry-based assay for the measurement of protein regulation of E-cadherin-mediated adhesion. J Immunol Methods 302 :116-124


Epithelial (E)-cadherin is a transmembrane protein that mediates calcium-dependent cell adhesion. E-cadherin has significant roles in tissue development, adhesion between keratinocytes and retention of Langerhans cells in the epidermis, and its loss on tumour cells is frequently associated with metastasis. Here we describe a simple, flow cytometric adhesion assay to measure the effects of potential regulators of cell surface E-cadherin expression on E-cadherin-mediated adhesion between cells. In this assay, cells that have been transiently transfected to express the protein of interest are enzymatically treated to remove cell surface E-cadherin. Cells are then incubated in low attachment plates, during which time the E-cadherin is re-expressed and E-cadherin-mediated aggregation occurs. The effect of the protein of interest on the percentage of E-cadherin-mediated aggregates that form during incubation is measured flow cytometrically, following staining with an E-cadherin specific antibody. A major advantage of this assay is that a potential regulatory protein of interest can be tested in a transient expression system by co-expression with green fluorescent protein and analysis of adhesion conducted on green fluorescent cells only. We have applied this assay to measure the regulatory effects of E6 protein from human papillomavirus type 16 on E-cadherin-mediated adhesion but this assay potentially has broad applicability for testing the effects of other proteins on E-cadherin-mediated adhesion in an accurate and highly reproducible manner.

Li B, Feng DY, Cheng RX, He QQ, Hu ZL, Zheng H, Wen JF (2005) [The effects of hepatitis C virus core protein on biological behaviors of human hepatocytes]. Zhonghua Yi Xue Za Zhi 85 :1243-1248


OBJECTIVE : To investigate the effects of hepatitis C virus (HCV) core protein on the biological behaviors of human hepatocytes and their underlying mechanism. METHODS : A cell line expressing stably HCV core protein-QSG7701/core was constructed by transfecting the plasmid pcDNA3.1-core (expressing HCV core protein) into the human immortalized hepatocytes of the line QSG7701. The biological behaviors of these transfected cells were observed through plating-efficiency test, growth curve and flow cytometry (FCM). The association between HCV core protein and the expression of activated caspase-3 protein was evaluated by immunocytochemistry. The phosphorylation of mitogen-activate protein kinases (MAPKs) was detected with Western blotting. The activation of nuclear transcriptors AP-1, important effector molecule of MAPKs, and nuclear factor-kappa binding (NF-kappaB) were evaluated with luciferase assays and electrophoretic mobility shift assay (EMSA). RESULTS : HCV core protein was expressed in the QSG7701/core cells and not in the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. There were no significant differences in the expression levels of total P44/42(MAPK), p38(MAPK) and JNK among the QSG7701/core cells, QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. The expression levels of phophorylated P44/42(MAPK), p38(MAPK) and JNK in the QSG7701/core cells were significantly weaker than those in the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. Plating efficiency test showed that the clone formation rate of the QSG7701/core cells was 32.25%, significantly lower than those of QSG7701/pcDNA3.1 and untransfected QSG7701 cells (47.5% and 42.5% respectively, both P < 0.01). The growth curve showed that the multiplication time of the QSG7701/core cells was 36 hours, significantly longer than those of the QSG7701/pcDNA3.1 and untransfected QSG7701 cells (27 and 28 hours respectively). FCM showed that the apoptotic rate of the QSG770/1core was 1.04%, lower than those of the QSG7701/pcDNA3.1 and untransfected QSG7701 cells (1.68% and 3.7% respectively), and that the percentage of theQSG770/1core cells at the G(0)/G(1) stage increased and those in the S stage decreased. Immunocytochemistry showed that the expression intensity of caspase-3 in the QSG7701/core cells was significantly weaker than those of the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. CONCLUSION : HCV core protein suppresses cell proliferation and apoptosis by downregmicrolating the phosphorylation of MAPKs and activating the transcriptors AP-1 and NF-kappaB, thus promoting the persistency of HCV infection which leads to chronic hepatitis C and hepatocellular cancer.

Lin J, Yan XJ, Zheng L, Ma HH, Chen HM (2005) Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites. J Appl Microbiol 99 :1373-1382


AIMS : To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS : After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS : Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY : Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.

Liu CZ, Shih MH, Tsai PJ (2005) ClfA(221-550), a fibrinogen-binding segment of Staphylococcus aureus clumping factor A, disrupts fibrinogen function. Thromb Haemost 94 :286-294


Clumping factor A (ClfA) is a surface protein of Staphylococcus aureus bacteria known for its ability to bind the C-terminus of plasma fibrinogen gamma chain, which participates in mediating fibrinogen-platelet interaction and fibrin cross-linking, resulting in thrombus formation. With an aim to develop agents that block fibrinogen gamma chain C-terminus, the fibrinogen-binding segment of ClfA locating at residues 221-550 was produced by recombinant technology and tested for its ability to inhibit platelet functions and fibrin clot formation. Recombinant ClfA(221-550) bound fibrinogen and blocked fibrinogen-platelet interaction, resulting in the inhibition of both ADP- and collagen-induced platelet aggregations. ClfA(221-550) also affected fibrin clot formation, in which factor XIIIa-mediated cross-linking of fibrinogen gamma chains was abrogated by ClfA(221-550) leaving the release of fibrinopeptides A and B from fibrinogen by thrombin unaltered, indicating that ClfA(221-550) interfered with fibrin clot formation without affecting thrombin’s catalytic activity. Platelet-mediated clot retraction depends on both platelet-fibrinogen interaction and fibrin clot formation, which makes platelet thrombus less susceptible to fibrinolysis. At the concentration that reduced platelet aggregation by 40%, ClfA(221-550) prevented platelet-mediated clot retraction, whereas the glycoprotein IIb/IIIa antagonist tirofiban needed a higher concentration in inhibiting clot retraction than inhibiting platelet aggregation. By virtue of the multiple effects of ClfA(221-550) on platelet aggregation, fibrin clot formation and platelet-mediated clot retraction, the binding of ClfA(221-550) to fibrinogen merits further investigation for its potential as a new antithrombotic agent.

Looser V, Hammes F, Keller M, Berney M, Kovar K, Egli T (2005) Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation. Biotechnol Bioeng 92 :69-78


The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.

Martin AP, Zubkov MV, Burkill PH, Holland RJ (2005) Extreme spatial variability in marine picoplankton and its consequences for interpreting Eulerian time-series. Biol Lett 1 :366-369


A high-resolution mesoscale spatial survey of picoplankton in the Celtic Sea, using flow cytometry, reveals cell concentrations of Synechococcus spp. cyanobacteria and heterotrophic bacteria that vary up to 50-fold over distances as short as 12 km. Furthermore, the range of abundances is comparable to that typically found on seasonal scales at a single location. Advection of such spatial variability through a time-series site would therefore constitute a major source of ’error’. Consequently, attempts to model and to investigate the ecology of these globally important organisms in situ must take into account and quantify the hitherto ignored local spatial variability as a matter of necessity.

Martin SF, Chatterjee S, Parinandi N, Alevriadou BR (2005) Rac1 inhibition protects against hypoxia/reoxygenation-induced lipid peroxidation in human vascular endothelial cells. Vascul Pharmacol 43 :148-156


Both in vivo models of ischemia/reperfusion and in vitro models of hypoxia (H)/reoxygenation (R) have demonstrated the crucial role of the Rac1-regulated NADPH oxidase in the production of injurious reactive oxygen species (ROS) by vascular endothelial cells (ECs). Since membrane lipid peroxidation has been established as one of the mechanisms leading to cell death, we examined lipid peroxidation in H/R-exposed cultured human umbilical vein ECs (HUVECs) and the role of Rac1 in this process. H (24 h at 1% O2)/R (5 min) caused an increase in intracellular ROS production compared to a normoxic control, as measured by dichlorofluorescin fluorescence. Nutrient deprivation (ND ; 24 h), a component of H, was sufficient to induce a similar increase in ROS under normoxia. Either H(24 h)/R (2 h) or ND (24 h) induced increases in lipid peroxidation of similar magnitude as measured by flow cytometry of diphenyl-1-pyrenylphosphine-loaded HUVECs and Western blotting analysis of 4-hydroxy-2-nonenal-modified proteins in cell lysates. In cells infected with a control adenovirus, H (24 h)/R (2 h) and ND (24 h) resulted in increases in NADPH-dependent superoxide production by 5- and 9-fold, respectively, as measured by lucigenin chemiluminescence. Infection of HUVECs with an adenovirus that encodes the dominant-negative allele of Rac1 (Rac1N17) abolished these increases. Rac1N17 expression also suppressed the H/R- and ND-induced increases in lipid peroxidation. In conclusion, ROS generated via the Rac1-dependent pathway are major contributors to the H/R-induced lipid peroxidation in HUVECs, and ND is able to induce Rac1-dependent ROS production and lipid peroxidation of at least the same magnitude as H/R.

McDonald CP, Colvin J, Robbins S, Barbara JA (2005) Use of a solid-phase fluorescent cytometric technique for the detection of bacteria in platelet concentrates. Transfus Med 15 :175-183


Blood services worldwide are now striving to reduce the risk of transmission of bacteria by transfusion. The BacT/ALERT microbial detection system (bioMerieux, Basingstoke, Hants, UK) is currently regarded as the ’gold standard’ for bacterial screening of platelet concentrates. The BacT/ALERT is a culture system and will not generate an ’instant’ (within 2 h) determination. We report on the Scansystem (Hemosystem, Marseille, France), a solid-phase fluorescent cytometric technique, which enables the rapid detection of bacteria (within 90 min) in platelet concentrates. The study was performed in two parts - one involving the routine screening of platelet concentrates and the other determining the sensitivity of the system. In both arms of the study, the BacT/ALERT was used for comparative purposes. In total, 900 platelet concentrates were screened (63 apheresis and 837 buffy coat pooled). No bacteria were detected in any of the platelet concentrates tested by means of either the Scansystem or the BacT/ALERT. The sensitivity of the Scansystem was in the order of 10(3) cfu mL(-1). Escherichia coli and Staphylococcus aureus were detected by using the Scansystem at 1 cfu mL(-1). The BacT/ALERT detected all organisms tested (n = 6) at 1 cfu mL(-1). The Scansystem offers a sensitive alternative technology to bacterial culture, with the benefit of a rapid test time.

Milhau N, Renson P, Dreesen I, Greenland T, Bellaton C, Guiguen F, Mornex JF, Le Jan C (2005) Viral expression and leukocyte adhesion after in vitro infection of goat mammary gland cells with caprine arthritis-encephalitis virus. Vet Immunol Immunopathol 103 :93-99


A characteristic lesion in goats infected by the lentivirus CAEV is mastitis with lymphoid hyperplasia. In order to investigate the mechanism of lesion formation, cultures highly enriched in microvascular endothelial cells, mature and immature luminal epithelial cells, fibroblasts and myoepithelial cells were established from goat mammary gland biopsies. Their susceptibility to in vitro infection with two distinct types of CAEV was investigated by PCR, antigen expression and cytopathy. The capacity of infected mammary gland cells to bind uninfected caprine leukocytes was determined by flow cytometry. All cell types tested were susceptible to CAEV infection in vitro, with different levels of sensitivity according to cell phenotype. Our results suggest that the limited extent of natural infection of mammary gland cells reflects a protective local immune response, and that the myoepithelial cell could act as a reservoir cell. After infection, the mature luminal cell acquires the capacity to bind leukocytes in vitro, which could indicate a facilitation of cellular interactions. The distinct reactions of the different cell types to CAEV infection may be correlated with events leading to progressive lesion development during the natural infection.

Mitchell M, Hudspeth M, Wright A (2005) Flow cytometry susceptibility testing for the antifungal caspofungin. J Clin Microbiol 43 :2586-2589


Rapid antifungal susceptibility testing for the antifungal agent caspofungin can be performed using flow cytometry (FC). An FC procedure using acridine orange provided minimum inhibitory concentration (MIC) results within 7 to 9 h which were compared with results obtained using the NCCLS M27-A2 protocol. To evaluate the consistency of this method, susceptibility testing using caspofungin was performed using 73 isolates of eight different species of Candida from various clinical samples in Central California. Macrotiter or microdilution tests were performed according to the NCCLS M27-A2 protocol, and the MICs were compared to those provided by our flow cytometry method. All isolates tested had results within the sensitive interpretive category, and 90% of the results compared within 1 dilution, showing excellent agreement between the methods. The MIC at which 50% of the isolates tested were inhibited (MIC50) and the MIC90 of caspofungin for all eight Candida species were within 1 dilution. This flow cytometer 7-h protocol for testing the antifungal susceptibility of Candida species to caspofungin provided results equivalent to those obtained with the M27-A2 protocol.

Miteva VI, Brenchley JE (2005) Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core. Appl Environ Microbiol 71 :7806-7818


The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 microm3) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5 degrees C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-microm, 0.2-microm, and even 0.1-microm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-microm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria ; Cytophaga-Flavobacteria-Bacteroides ; high-G+C gram-positive ; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.

Morris RJ, Chong LK, Wilkinson GW, Wang EC (2005) A high-efficiency system of natural killer cell cloning. J Immunol Methods 307 :24-33


The culture of human natural killer (NK) cell clones has traditionally been a long, laborious process with an efficiency of only 1-2%. Recently, a stem cell growth medium (SCGM) has been described to expand preferentially polyclonal NK cells from peripheral blood. We have tested SCGM in a single cell sorting system and shown a 4-5 fold increase in the number of proliferating NK clones compared to standard RPMI media. The cloning efficiency was further enhanced by the provision of irradiated feeder cells derived from multiple donors combined with the addition of the anti-CD3 antibody, OKT3. The combination of SCGM, single cell sorting and these multiple optimisations enhanced NK cloning efficiency by more than tenfold to greater than 20% for short-term cultures when deriving 10(5) cells and as high as 10% for longer term cultures when deriving more than 2 x 10(6) cells. This novel system thus facilitates the generation of NK clones and allows larger scale studies of NK function that were beyond the scope of previous methodology.

Mou X, Moran MA, Stepanauskas R, Gonzalez JM, Hodson RE (2005) Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations. Appl Environ Microbiol 71 :1405-1416


Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 microM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, alpha-Proteobacteria) ; Caulobacter (alpha-Proteobacteria) ; and Brachymonas and Xenophilus (beta-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, alpha-Proteobacteria) and novel gamma-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.

Narayan K, Dail D, Li L, Cadavid D, Amrute S, Fitzgerald-Bocarsly P, Pachner AR (2005) The nervous system as ectopic germinal center : CXCL13 and IgG in lyme neuroborreliosis. Ann Neurol 57 :813-823


Lyme neuroborreliosis (LNB) is a chronic infection in which B-cell activation, plasma cell infiltration, and enhanced Ig production in infected tissue are prominent feature. However, little is known about how B cells and plasma cells invade and persist in target organs. To assess this issue, we developed real-time PCR measurements of IgG and CXCL13 production. We used these RNA assays and specific enzyme-linked immunosorbent assays for protein and demonstrated that human peripheral blood mononuclear cells (PBMCs), stimulated by Borrelia burgdorferi sonicate, produced CXCL13 and IgG. Magnetic separation of PBMC populations and flow cytometry showed that CXCL13 is produced by dendritic cells. We then measure the expression of CXCL13 and IgG in tissues and correlated the expression of these host genes with spirochetal load. We also measured expression of dbpA and BBK32, two spirochetal genes important in chronic infection. There was a strong correlation between host immune response gene expression (CXCL13 and IgG) and spirochetal load. Immunohistochemistry of infected nonhuman primates tissue confirmed that CXCL13 is expressed in the nervous system. We conclude that persistent production of CXCL13 and IgG within infected tissue, two characteristics of ectopic germinal centers, are definitive features of LNB.

Narimatsu R, Patterson BK (2005) High-throughput cervical cancer screening using intracellular human papillomavirus E6 and E7 mRNA quantification by flow cytometry. Am J Clin Pathol 123 :716-723


The Papanicolaou (Pap) test, based solely on the morphologic examination of exfoliated cells from the cervix, has reduced deaths due to cervical cancer by 74% in the United States during the past 40 years. During that time, the molecular mechanisms of cervical cancer have largely been elucidated. Taken together, these observations have identified a need for a high-throughput cervical cancer screening assay. We report the development of a high-throughput assay consisting of simultaneous immunophenotyping and ultrasensitive in situ hybridization for human papillomavirus (HPV) E6 and E7 messenger RNA (mRNA). This assay can be performed in less than 3 hours directly from liquid-based cervical cytology specimens. Overall, HPV fluorescence in situ hybridization (FISH) for E6 and E7 mRNA demonstrated 83.3% sensitivity and 91.3% specificity for high-grade squamous intraepithelial lesions compared with the Pap test in 231 liquid-based cytology samples from 2 cohorts. In a subset of these samples, HPV FISH demonstrated higher sensitivity and specificity than Hybrid Capture (Digene, Gaithersburg, MD) for high-risk genotypes.

Nasirudeen AM, Tan KS (2005) Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways. Biochimie 87 :489-497


We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.

Naumova ES, Naumov GI, Masneuf-Pomarede I, Aigle M, Dubourdieu D (2005) Molecular genetic study of introgression between Saccharomyces bayanus and S. cerevisiae. Yeast 22 :1099-1115


The genomic constitution of different S. bayanus strains and natural interspecific Saccharomyces hybrids has been studied by genetic and molecular methods. Unlike S. bayanus var. uvarum, some S. bayanus var. bayanus strains (the type culture CBS 380, CBS 378, CBS 425, CBS 1548) harbour a number of S. cerevisiae subtelomeric sequences : Y’, pEL50, SUC, RTM and MAL. The two varieties, having 86-100% nDNA-nDNA reassociation, are partly genetically isolated from one another but completely isolated from S. cerevisiae. Genetic and molecular data support the maintaining of var. bayanus and var. uvarum strains in the species S. bayanus. Using Southern hybridization with species-specific molecular markers, RFLP of the MET2 gene and flow cytometry analysis, we showed that the non-S. cerevisiae parents are different in lager brewing yeasts and in wine hybrid strains. Our results suggest that S. pastorianus is a hybrid between S. cerevisiae and S. bayanus var. bayanus, while S. bayanus var. uvarum contributed to the formation of the wine hybrids S6U and CID1. According to the partial sequence of ACT1 gene and flow cytometry analysis, strain CID1 is a triple hybrid between S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum.

Neel BD, Aouacheria A, Nouvion AL, Ronot X, Gillet G (2005) Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells. Exp Cell Res 311 :106-116


Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

Nielsen MA, Grevstad B, TM AE, Kurtzhals JA, Giha H, Staalsoe T, Hviid L, Theander TG (2005) Differential induction of immunoglobulin G to Plasmodium falciparum variant surface antigens during the transmission season in Daraweesh, Sudan. J Infect Dis 192 :520-527


BACKGROUND : The acquisition of immunoglobulin (Ig) G to variant surface antigens (VSAs) seems important for the development of protective immunity against malaria. Unlike VSAs expressed by parasite isolates associated with uncomplicated malaria, VSAs expressed by parasite isolates associated with severe malaria (VSA(SM)) are frequently recognized by IgG. METHODS : We analyzed levels of anti-VSA IgG in 57 individuals in Daraweesh, Sudan, before and after the transmission season. IgG responses to 79 Plasmodium falciparum isolates from children with defined malaria syndromes and exposed to high transmission in a different part of Africa were also analyzed. RESULTS : After the transmission season, individuals with malaria had an increase in IgG recognition to 25.8% (95% confidence interval [CI], 19.9%-31.7%) and a decrease in IgG recognition to 7.6% (95% CI, 4.4%-10.8%) of 79 parasite isolates, and individuals without malaria had an increase in IgG recognition to 8.1% (95% CI, 6.0%-10.2%) and a decrease in IgG recognition to 11.9% (95% CI, 7.0%-16.8%) of 79 parasite isolates. Most newly acquired IgG responses were against parasite isolates expressing VSAs(SM) that are frequently recognized by IgG. CONCLUSIONS : Anti-VSA IgG levels decrease in the absence of infection, and an episode of clinical malaria induces IgG against a range of VSAs, particularly VSAs(SM).

Nilsson M, Weineisen M, Andersson T, Truedsson L, Sjobring U (2005) Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum. Eur J Immunol 35 :1472-1481


During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.

Nishimura Y, Kim C, Nagata T (2005) Vertical and seasonal variations of bacterioplankton subgroups with different nucleic Acid contents : possible regulation by phosphorus. Appl Environ Microbiol 71 :5828-5836


We used flow cytometry to examine seasonal variations in basin-scale distributions of bacterioplankton in Lake Biwa, Japan, a large mesotrophic freshwater lake with an oxygenated hypolimnion. The bacterial communities were divided into three subgroups : bacteria with very high nucleic acid contents (VHNA bacteria), bacteria with high nucleic acid contents (HNA bacteria), and bacteria with low nucleic acid contents (LNA bacteria). During the thermal stratification period, the relative abundance of VHNA bacteria (%VHNA) increased with depth, while the reverse trend was evident for LNA bacteria. Seasonally, the %VHNA was strongly positively correlated (r = 0.87 ; P < 0.001) with the concentration of dissolved inorganic phosphorus, but not with the concentration of chlorophyll a. The growth of VHNA bacteria was significantly enhanced by addition of phosphate or phosphate plus glucose but not by addition of glucose alone. Although the growth of VHNA and HNA bacteria generally exceeded that of LNA bacteria, our data also revealed that LNA bacteria grew faster than and were grazed as fast as VHNA bacteria in late August, when nutrient limitation was presumably severe. Based on these results, we hypothesize that in severely P-limited environments such as Lake Biwa, P limitation exerts more severe constraints on the growth of bacterial groups with higher nucleic acid contents, which allows LNA bacteria to be competitive and become an important component of the microbial loop.

Okuda N, Seya T (2005) [Immunologic tests : C3 receptor (CR1, 2)]. Nippon Rinsho 63 Suppl 7 :67-70


Olicard C, Didier Y, Marty C, Bourgougnon N, Renault T (2005) In vitro research of anti-HSV-1 activity in different extracts from Pacific oysters Crassostrea gigas. Dis Aquat Organ 67 :141-147


Mortalities related to the detection of Ostreid Herpesvirus 1 (OsHV-1) have been previously reported in France among larvae and spat of the Pacific oyster Crassostrea gigas. Adult oysters appear less sensitive to herpesvirus infections, although OsHV-1 has been detected in adults without signs of disease or mortality. This suggests that the virus is able to persist in its host and that adult oysters may be able to control OsHV-1 infection. Little is known about antiviral substances in invertebrates. The present work concerns the research of antiviral substances in adult oyster C. gigas, where putative antiviral activities were monitored using 3 strategies : (1) in metabolites with variable polarity, (2) in peptidic extracts and (3) in crude haemolymph. In vitro antiviral assays were based on inhibition of Herpes simplex virus type 1 (HSV-1) replication in Vero cell monolayers. All extracts presented no cytotoxicity. Antiviral activity was detected in the fresh filtered haemolymph (EC50:425 microg ml(-1)) and seasonal variation of the haemolymph antiviral activity was monitored.

Olsen PA, Randol M, Krauss S (2005) Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides. Gene Ther 12 :546-551


Oligonucleotide-based sequence alteration in living cells is a substantial methodological challenge in gene therapy. Here, we demonstrate that using corrective single-stranded oligonucleotides (ssODN), high and reproducible sequence correction rates can be obtained. CHO cell lines with chromosomally integrated multiple copy EGFP reporter genes routinely show rates of 4.5% targeted sequence correction after transfection with ssODN. We demonstrate that the cell cycle influences the rates of targeted sequence correction in vivo, with a peak in the early S phase during ssODN exposure. After cell division, the altered genomic sequence is predominantly passed to one daughter cell, indicating that targeted sequence alteration occurs after the replication fork has passed over the targeted site. Although high initial correction rates can be obtained by this method, we show that a majority of the corrected cells arrest in the G2/M cell cycle phase, although 1-2% of the corrected cells form viable colonies. The G2/M arrest observed after targeted sequence correction can be partially released by caffeine, pentoxifylline or Go6976 exposure. Despite substantial remaining challenges, targeted sequence alteration based on ssODN increasingly promises to become a powerful tool for functional gene alterations.

Olsen PA, Randol M, Luna L, Brown T, Krauss S (2005) Genomic sequence correction by single-stranded DNA oligonucleotides : role of DNA synthesis and chemical modifications of the oligonucleotide ends. J Gene Med 7 :1534-1544


BACKGROUND : Single-stranded oligonucleotides (ssODN) can induce site-specific genetic alterations in selected mammalian cells, but the involved mechanisms are not known. METHODS : We corroborate the potential of genomic sequence correction by ssODN using chromosomally integrated mutated enhanced green fluorescent protein (mEGFP) reporter genes in CHO cell lines. The role of integration site was studied in a panel of cell clones with randomly integrated reporters and in cell lines with site-specific single copy integration of the mEGFP reporter in opposite orientations. Involvement of end modification was examined on ssODN with unprotected or phosphorothioate (PS) protected ends. Also ssODN containing octyl or hexaethylene glycol (HEG) end blocking groups were tested. The significance of DNA synthesis was investigated by cell cycle analysis and by the DNA polymerases alpha, delta and epsilon inhibitor aphidicolin. RESULTS : Correction rates of up to 5% were observed upon a single transfection of ssODN. Independent of the mEGFP chromosomal integration site and of its orientation towards the replication fork, antisense ssODN were more effective than sense ssODN. When ssODN ends were blocked by either octyl or HEG groups, correction rates were reduced. Finally, we demonstrate a dependence of the process on DNA synthesis. CONCLUSIONS : We show that, on a chromosomal level, the orientation of the replication fork towards the targeted locus is not central in the strand bias of ssODN-based targeted sequence correction. We demonstrate the importance of accessible ssODN ends for sequence alteration. Finally, we provide evidence for the involvement of DNA synthesis in the process.

Paes MV, Pinhao AT, Barreto DF, Costa SM, Oliveira MP, Nogueira AC, Takiya CM, Farias-Filho JC, Schatzmayr HG, Alves AM, Barth OM (2005) Liver injury and viremia in mice infected with dengue-2 virus. Virology 338 :236-246


The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.

Page BT, Kurtzman CP (2005) Rapid identification of Candida species and other clinically important yeast species by flow cytometry. J Clin Microbiol 43 :4507-4514


Two rapid diagnostic assays, utilizing two different Luminex flow cytometry methods, were developed for identification of clinically important ascomycetous yeast species. Direct hybridization and allele-specific primer extension methods were both successful in establishing a DNA-based assay that can rapidly and accurately identify Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis as well as other clinical species. The direct hybridization assay was designed to identify a total of 19 ascomycetous yeast species, and the allele-specific primer extension assay was designed to identify a total of 34 species. Probes were validated against 438 strains representing 303 species. From culture to identification, the allele-specific primer extension method takes 8 h and the direct hybridization method takes less than 5 h to complete. These assays represent comprehensive, rapid tests that are well suited for the clinical laboratory.

Pagnini U, Montagnaro S, Sanfelice di Monteforte E, Pacelli F, De Martino L, Roperto S, Florio S, Iovane G (2005) Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells. Vet Immunol Immunopathol 103 :283-293


Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.

Park HS, Schumacher R, Kilbane JJ, 2nd (2005) New method to characterize microbial diversity using flow cytometry. J Ind Microbiol Biotechnol 32 :94-102


The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.

Park SG, Jeong YJ, Lee YY, Kim IJ, Seo SK, Kim EJ, Jung HC, Pan JG, Park SJ, Lee YJ, Kim IS, Choi IH (2005) Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naive antibody phage library. Antiviral Res 68 :109-115


We report the construction of a large nonimmunized human phage antibody library in single-chain variable region fragment (scFv) format, which allowed the selection of antibodies that neutralize hepatitis B virus (HBV) in vitro. We generated 1.1 x 10(10) independent scFv clones using the cDNA of functional variable (V) gene segments of heavy and light chains purified from the peripheral blood mononuclear cells of 50 nonimmunized human donors. Using BIAcore, we selected two clones that recognized pre-S1 and neutralized pre-S1 and HBV binding to Chang liver cells. Clone G10 had the highest affinity (K(D)=1.69 x 10(-7)M), which was higher than that of clone 1E4 that was generated previously from a heavy chain-shuffled immune library. The off-rates of clones were within 10(-3)s(-1) as determined by BIAcore and were comparable to those of antibodies derived from a normal secondary immune response. In the inhibition assays of pre-S1 and virus binding to Chang liver cells using flow cytometry and the polymerase chain reaction, G10 had better neutralizing activity than 1E4. The new phage library may be a valuable source of antibodies with reasonable affinities to different targets, and the anti-pre-S1 G10 may be a good candidate for immunoprophylaxis against HBV infection.

Persson F, Langmark J, Heinicke G, Hedberg T, Tobiason J, Stenstrom TA, Hermansson M (2005) Characterisation of the behaviour of particles in biofilters for pre-treatment of drinking water. Water Res 39 :3791-3800


Biofiltration of surface water was examined using granular activated carbon (GAC) and expanded clay (EC). Particle removal was 60-90%, measured by flow cytometry, which enabled discrimination between total- and autofluorescent particles (microalgae) in size ranges of 0.4-1 and 1-15 microm, and measured by on-line particle counting. Total particles were removed at a higher degree than autofluorescent particles. The biofilters were also challenged with 1 microm fluorescent microspheres with hydrophobic and hydrophilic surface characteristics and bacteriophages (Salmonella typhimurium 28B). Added microspheres were removed at 97-99% (hydrophobic) and 85-89% (hydrophilic) after 5 hydraulic residence times (HRT) and microspheres retained in the biofilter media were slowly detaching into the filtrate for a long time after the addition. Removal of bacteriophages (5 HRT) was considerably lower at 40-59%, and no long-lasting detachment was observed. A comparison of experimental data with theoretical predictions for removal of particles in clean granular media filters revealed a similar or higher removal of particles around 1 microm in size than predicted, while bacteriophages were removed at a similar or lesser extent than predicted. The results highlight the selectivity and dynamic behaviour of the particle removal processes and have implications for operation and microbial risk assessment of a treatment train with biofilters as pre-treatment.

Phe MH, Dossot M, Guilloteau H, Block JC (2005) Nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria. Water Res 39 :3618-3628


Flow cytometry (FCM), combined with staining using two fluorochromes (propidium iodide, PI, or SYBR Green II RNA gel stain, SYBR-II), was used to assess nucleic acid injuries to chlorinated drinking water bacteria. Highly fluorescent SYBR-II-stained bacteria were converted to bacteria with low fluorescence after chlorination. PI staining of bacteria exposed to different doses of chlorine showed membrane permeabilisation ([Cl2] < 0.2 mg L(-1)) and nucleic acid damage at higher doses ([Cl2] > 0.3 mg L(-1)). Above a threshold dose (between 1.5 and 3 mg Cl2 L(-1)), nucleic acids appeared severely damaged and incapable of being stained by PI or SYBR-II. These results constitute evidence that FCM is a promising tool for assessing drinking water bacteria injuries and for controlling chlorine disinfection efficiency much more rapidly than the standard sensitive but time-consuming heterotrophic plate count method.

Pianetti A, Falcioni T, Bruscolini F, Sabatini L, Sisti E, Papa S (2005) Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods. Appl Environ Microbiol 71 :7948-7954


The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods ; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG (2005) Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16. J Med Microbiol 54 :77-81


The time needed to obtain susceptibility results of Mycobacterium tuberculosis using classical methodologies is still too long, and flow cytometry is a promising technique in the setting of the clinical laboratory, giving fast results. A safe, reliable and rapid method to study susceptibility to streptomycin, isoniazide, rifampicin and ethambutol is described. Isolates of mycobacteria, grown for 72 h in the absence or presence of antimycobacterial drugs in the mycobacteria growth indicator tube (MGIT), were heat-killed, stained with SYTO 16 (a nucleic acid fluorescent stain that only penetrates cells with severe lesion of the membrane) and then analysed by flow cytometry. Sixteen strains with different susceptibility patterns were tested and an excellent correlation with the BACTEC MGIT 960 protocol for susceptibility was shown. In contrast to resistant strains, sensitive strains lose their cellular integrity after incubation with antimycobacterial drugs, allowing SYTO 16 to penetrate the cells. Comparing the intensity of fluorescence of Mycobacterium cells incubated with antimycobacterial drugs with that of drug-free cells, after staining with SYTO 16, it was possible to distinguish between sensitive, intermediate and resistant phenotypes. Other cytometric assays have been described for mycobacteria susceptibility testing but these have lower accuracy and safety. The described flow cytometric assay is a simple, fast, safe and accurate way to determine susceptibility of M. tuberculosis.

Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG, Espinel-Ingroff A (2005) Comparison of two probes for testing susceptibilities of pathogenic yeasts to voriconazole, itraconazole, and caspofungin by flow cytometry. J Clin Microbiol 43 :4674-4679


A cytometric approach to determine the susceptibilities of Candida spp. and Cryptococcus neoformans to voriconazole, itraconazole, and caspofungin is described. A total of 63 clinical isolates with different susceptibility patterns were exposed for 1, 2, 4, and 6 h to serial concentrations of each antifungal agent, followed by staining with two fluorescent probes : propidium iodide (PI) and FUN-1. FUN-1 was able to identify the susceptibility patterns of the assayed strains to the three agents after 1 h. PI penetrated a maximum of 50% of the cells treated with PI, at the highest concentration of caspofungin, 16 mug/ml, after 6 h of incubation (this percentage varied with the strain and was drug concentration and time of incubation dependent) and did not stain cells treated with high concentrations of either azole after 6 h. The use of FUN-1 appears to be an excellent fast and reliable alternative to the classical dilution method for determining the susceptibility of Candida spp. and C. neoformans to these three antifungal agents.

Pina-Vaz C, Rodrigues AG, Costa-de-Oliveira S, Ricardo E, Mardh PA (2005) Potent synergic effect between ibuprofen and azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry. J Antimicrob Chemother 56 :678-685


OBJECTIVES : Resistance to antifungals often relates to efflux pumps exporting drugs ; several modulators may block them, reverting resistance. Verapamil, beta-oestradiol and progesterone, known efflux pump inhibitors of human neoplastic cells, and ibuprofen were tested as potential modulators of resistance of Candida spp. METHODS : Forty-two clinical isolates of Candida (38 fluconazole-resistant), two ATCC type strains and two C. albicans strains with known mechanisms of fluconazole resistance were incubated with subinhibitory concentrations of the modulators. After exposure, MICs of fluconazole, itraconazole and voriconazole were re-determined. Simultaneously, yeasts exposed to modulators were stained with FUN-1 and analysed by flow cytometry. 3H-labelled itraconazole was also used to study efflux in the presence and absence of modulators. RESULTS : Fluconazole MICs decreased in most strains after exposure to modulators, including control strains with documented efflux overexpression. No significant MIC variation was noticed for : all C. krusei strains tested, for the resistant strain by target change, for susceptible strains, and for a very few other clinical isolates. Reverted resistant phenotypes showed cross-resistance to itraconazole and to voriconazole, which was also reverted by the modulators. For these strains, an increase in FUN-1 staining and increased accumulation of 3H-labelled itraconazole were noticed after incubation with modulators. CONCLUSIONS : Resistance related to overexpression of efflux pumps was common among clinical isolates and could be reverted by the assayed modulators, particularly ibuprofen. The mechanism of resistance in all tested C. krusei and in a few other strains seems, however, to be of a different nature. Ibuprofen is a promising compound in association with azoles, deserving future clinical trials. FUN-1 proved to be a good marker of efflux in Candida.

Possemiers S, Van Camp J, Bolca S, Verstraete W (2005) Characterization of the bactericidal effect of dietary sphingosine and its activity under intestinal conditions. Int J Food Microbiol 105 :59-70


Sphingosine is known as a natural antimicrobial agent, protecting the human skin from bacterial colonization and possibly affecting the intestinal microbial community after ingestion. In this study we further investigated the antibacterial spectrum of dietary d-eythro-sphingosine in saline towards three intestinal pathogens and to the health promoting lactobacilli and bifidobacteria. The degree of bactericidal effect was studied using plate counts and Live/Dead analysis combined with flow cytometry. To assess activity under complex intestinal conditions, sphingosine was dosed to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) for a period of 11 days. Finally, we tried to elucidate the factors influencing the activity and the mode of action of sphingosine. In all performed experiments, high correlation occurred between plate counts and Live/Dead analysis. In saline a strong antibacterial effect was seen to all tested species, Gram-negative and Gram-positive, and sphingosine not selectively acted against pathogens, as health promoting bacteria were also affected. Under simulated intestinal conditions however, no shifts in bacterial concentrations were detected. Experiments with individual medium components thought that the effect of sphingosine is very easily neutralized by BSA, stearic acid and surfactants. Based on our results, d-erythro-sphingosine would only be active when protonated and its mode of action would imply electrostatic attraction to the bacteria and disruption of membrane integrity. In conclusion, the application of sphingosine is limited to specific environments, as activity was very sensitive to inhibition. Yet, because of its broad spectrum membrane disrupting activity, it could be very useful under controlled conditions.

Provvedi R, Maggi T, Oggioni MR, Manganelli R, Pozzi G (2005) Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria. BMC Biotechnol 5 :3


BACKGROUND : In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. RESULTS : The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5’ end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion. CONCLUSION : We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.

Qin QW, Gin KY, Lee LY, Gedaria AI, Zhang S (2005) Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture. J Virol Methods 125 :49-54


A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.

Quin LR, Carmicle S, Dave S, Pangburn MK, Evenhuis JP, McDaniel LS (2005) In vivo binding of complement regulator factor H by Streptococcus pneumoniae. J Infect Dis 192 :1996-2003


Pneumococcal surface protein C (PspC) binds to the complement regulatory protein factor H (FH), which inhibits alternative pathway activation. In the present study, using a mouse model of systemic infection and flow-cytometric analyses, we demonstrated an in vivo interaction between FH and pneumococci and showed differential FH binding during bacteremia. Flow-cytometric analyses of pneumococci harvested after intraperitoneal (ip) challenge demonstrated increased binding of FH, compared with that after intravenous (iv) challenge. Real-time polymerase chain reaction analyses of PspC mRNA showed that, relative to pneumococci grown in vitro, those recovered from the blood of mice 24 h after iv challenge exhibited 23-fold higher mRNA levels ; however, after ip challenge, PspC mRNA induction was increased 870-fold. A subsequent increase in PspC expression was detected by flow cytometry using a monoclonal antibody against PspC. Furthermore, pneumococci with FH bound to complement before exposure had increased proliferation, compared with pneumococci not pretreated with FH. These results suggest that the interaction between PspC and FH contributes to pneumococcal virulence.

Reinhardt B, Mertens T, Mayr-Beyrle U, Frank H, Luske A, Schierling K, Waltenberger J (2005) HCMV infection of human vascular smooth muscle cells leads to enhanced expression of functionally intact PDGF beta-receptor. Cardiovasc Res 67 :151-160


BACKGROUND : Cytomegaloviruses have been shown to promote atherogenesis in animal models. In humans, several epidemiological and clinical studies suggest involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. HCMV is suspected to be associated with an enhanced restenosis rate and the occurrence of vasculopathies after solid organ transplantation. However, knowledge about the cellular and molecular bases of these findings is very limited. METHODS AND RESULTS : Human coronary artery smooth muscle cells (HCASMC) were successfully infected with HCMV in vitro. Infection of HCASMC with all HCMV strains analyzed resulted in a substantial upregulation of the beta-receptor of platelet-derived growth factor (PDGFR-beta) expression as demonstrated by immunohistochemistry, immunofluorescence, FACS, and Western blot analysis. The amount of PDGFR-beta protein present in HCASMC rapidly increased after 12 h of infection and this difference persisted for 72 h post-infection. We showed by quantitative FACS analysis that the extent of PDGFR-beta upregulation differed significantly between the HCMV strains TB40E, Toledo, and AD169. The expression of insulin-like growth factor receptors as well as hepatocyte growth factor receptors, however, was down-modulated in HCMV-infected HCASMC. Most importantly, the HCMV-associated upregulation of PDGFR-beta protein resulted in functionally intact receptors. A significantly higher increase of proliferative activity following stimulation with PDGF-BB was observed in HCMV-infected HCASMC compared to the uninfected control. CONCLUSIONS : Our data suggest that HCMV directly activates the PDGF system, which could promote atherogenesis and restenosis by activation of smooth muscle cell proliferation and neointima formation.

Reis A, da Silva TL, Kent CA, Kosseva M, Roseiro JC, Hewitt CJ (2005) Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry. J Biotechnol 115 :199-210


Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3’-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3) ; cells or spores with a depolarised cytoplasmic membrane (B), no staining ; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI ; and permeablised cells with a disrupted cytoplasmic membrane ’ghost cells’ (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting ’ghost cells’ which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.

Rezcallah MS, Hodges K, Gill DB, Atkinson JP, Wang B, Cleary PP (2005) Engagement of CD46 and alpha5beta1 integrin by group A streptococci is required for efficient invasion of epithelial cells. Cell Microbiol 7 :645-653


Membrane cofactor protein (MCP or CD46), a widely distributed complement regulatory human protein, is a cell surface receptor for many pathogens including group A streptococci (GAS). The surface M protein of GAS binds CD46 and mediates GAS adherence to keratinocytes. In the present study, we studied the role of CD46 in GAS invasion of human lung epithelial cells, A549. Anti-CD46 antibody which specifically blocks the domain to which M protein binds inhibited adherence to and invasion of A549 cells by GAS. Moreover, downregulation of CD46 expression on A549 by RNA interference resulted in reduced invasion of these cells by GAS. A mutant form of CD46 with a deletion in the cytoplasmic domain was overexpressed in A549 cells, which resulted in partial inhibition of invasion. This indicates that the cytoplasmic tail is required for CD46 to promote invasion by GAS. Invasion assays with Lactococcus lactis that express M protein demonstrated the dependence of CD46-promoted invasion on interaction with M protein. In addition, CD46-mediated invasion was also found to be dependent on the extracellular matrix protein fibronectin.

Robbins SH, Tessmer MS, Van Kaer L, Brossay L (2005) Direct effects of T-bet and MHC class I expression, but not STAT1, on peripheral NK cell maturation. Eur J Immunol 35 :757-765


The homeostatic maturation of NK cells is severely impaired in mice lacking the transcription factor T-bet, and the expression of the NK cell maturation marker killer cell lectin-like receptor G1 (KLRG1) has been shown to be dependent on MHC class I molecules. Interferon (IFN)-gamma signaling via the signal transducer and activator of transcription (STAT)1 is vital for T-bet and MHC class I induction. Here we investigated the relationship between STAT1, T-bet, and MHC class I molecules with regard to the phenotypic maturation of peripheral NK cells. We demonstrate that, to varying degrees, the maturation status of peripheral NK cells is impaired in naive mice with deficiencies in STAT1, T-bet, or MHC class I molecules. We find that in naive animals, the expression of wild-type levels of MHC class I molecules in trans is sufficient to restore the maturation profiles of STAT1(-/-) NK cells in vivo. In contrast, expression of T-bet is required in cis for normal NK cell maturation to occur. Additionally, we demonstrate that the activation-induced maturation of NK cells during the course of murine cytomegalovirus (MCMV) infection does not require expression of MHC class I molecules or STAT1 but is severely delayed in the absence of T-bet.

Robertson BH, Nicholson JK (2005) New microbiology tools for public health and their implications. Annu Rev Public Health 26 :281-302


The realm of diagnostic assays for detection of acute infections is rapidly changing from antibody detection to pathogen detection, from clinical laboratory based to point-of-care based, from single analyte detection to multiple analyte detection, and is more focused on detection using less invasive approaches for collecting biological samples. New assays are typically more sensitive than are conventional assays and have the capability of providing more information that characterizes the pathogen or the host response to the pathogen. From a public health perspective, the advent of molecular epidemiology, which allows tracking of pathogens based on unique genetic sequences or antigenic properties, has revolutionized how epidemiologists investigate and evaluate epidemics and assess endemic diseases. In addition, the use of point-of-care (POC) devices can impact the detection and surveillance of infections and will enhance our ability to accurately identify the causes of illnesses.

Rodriguez S, Alonso M, Perez-Prietol SI (2005) Comparison of two birnavirus-rhabdovirus coinfections in fish cell lines. Dis Aquat Organ 67 :183-190


Aquabirnaviruses, such as the infectious pancreatic necrosis virus (IPNV), Novirhabdoviruses, such as the infectious hematopoiteic necrosis virus (IHNV) and the viral hemorrhagic septicemia virus (VHSV), cause considerable losses to the salmonid industry worldwide. Coinfections of 2 viruses have been described, but the interactions between rhabdoviruses and birnaviruses have not been examined closely. Using virus titration, flow cytometry and RT-PCR assays, we compared the effect of IPNV on the replication of IHNV and VHSV in tissue culture cells. RT-PCR assays indicated that simultaneous infection of IPNV with VHSV does not affect the replication of the rhabdovirus either in the first or successive passages ; the infective titers were similar in single and double infections. In contrast, coinfection of IPNV with IHNV induced a fall in infectivity, with reduced expression of IHNV viral antigens in BF-2 cells from Lepomis macrochirus and a loss of 4.5 log10 units of the infective titer after 3 successive passages. It was possible to stimulate BF-2 cells to produce significant interferon-like activity against IHNV but not against VHSV.

Rood PP, Hara H, Ezzelarab M, Busch J, Zhu X, Ibrahim Z, Ball S, Ayares D, Awwad M, Cooper DK (2005) Preformed antibodies to alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig cells in humans, baboons, and monkeys : implications for xenotransplantation. Transplant Proc 37 :3514-3515


OBJECTIVE : The aim of our study was to determine the prevalence and cytotoxicity of primate antibodies directed to antigens other than Galalpha1,3Gal (Gal), termed nonGal antigens. METHODS : Sera from human, baboon, and cynomolgus monkeys were tested by flow cytometry for IgM and IgG binding to both wild-type (WT) and GT-KO pig peripheral mononuclear cells (PBMC). Also, complement-dependent cytotoxicity assays were performed. RESULTS : All species demonstrated significantly higher antibody binding and cytotoxicity to WT cells compared to GT-KO cells (P < .01). Cynomolgus monkeys had significantly higher IgM binding to WT and GT-KO cells than did baboons or humans (P < .01). Furthermore, approximately 50% of both human and baboon sera proved to be lytic to GT-KO cells, compared to 76% of monkey sera (P < .01). CONCLUSIONS : We confirm the advantage of using GT-KO pig grafts over WT pig grafts. However, our results suggest that, compared to the cynomolgus monkey, the baboon may be a more suitable model to study antibody-mediated rejection of GT-KO pig grafts.

Rubinsztein-Dunlop S, Guy B, Lissolo L, Fischer H (2005) Identification of two new Helicobacter pylori surface proteins involved in attachment to epithelial cell lines. J Med Microbiol 54 :427-434


Helicobacter pylori causes the development of gastritis, gastric ulcers and adenocarcinomas in humans. The establishment of infection is influenced by adherence to the gastric epithelium, and several bacterial adhesins and host cell receptors have been identified. H. pylori recognize the Lewis(b) receptor through the BabA adhesin but also readily adhere to epithelia in the absence of the Lewis(b) epitope, demonstrating the relevance of additional adhesive interactions. This study presents a novel method of identifying bacterial adhesins. Nickel beads were coated with H. pylori-derived, recombinantly expressed ORF proteins, and epithelial cells from the human stomach, intestine or urinary tract were allowed to adhere to those beads. The binding of epithelial cells to the protein-coated nickel beads was confirmed by electron microscopy or flow cytometry using antibodies directed towards the His-tags. Among the five ORFs tested, two new adhesive proteins (HP1188 and HP1430) were identified. Both were expressed on the surface of virulent H. pylori, with the HP1188 protein being most abundant. The purified HP1188 and HP1430 proteins bound more strongly to gastric than to other epithelial cell lines, suggesting that they may be involved in the colonization of the human gastric mucosa. In conclusion, this method facilitates the identification of ORFs of microbial origin involved in cellular interactions such as adherence.

Rudensky B, Broidie E, Yinnon AM, Weitzman T, Paz E, Keller N, Raveh D (2005) Rapid flow-cytometric susceptibility testing of Candida species. J Antimicrob Chemother 55 :106-109


OBJECTIVES : To develop a rapid flow-cytometric antifungal susceptibility test and to compare results with the standard methods. METHODS : Reference and laboratory strains of Candida were tested for susceptibility to fluconazole and echinocandin by fluorescent flow cytometry using Acridine Orange as indicator of viability. Flow cytometry results were compared with MICs as determined by macrodilution and/or Etest. RESULTS : Seventy Candida strains were tested for susceptibility to fluconazole, and 74 strains for susceptibility to echinocandin. Minimal concentration of fluconazole causing 40% cell damage, as determined by flow cytometry, showed excellent association with MIC, as determined by other methods. The flow method, completed within 5 h, had excellent sensitivity and specificity to distinguish between sensitive, susceptible dose-dependent and resistant strains. The flow cytometry method for echinocandin was completed within 3 h, and minimal concentration causing 50% cell damage was associated with MIC as determined by macrodilution. CONCLUSIONS : Antifungal susceptibility testing by FACS is a reliable, rapid method for determining susceptibility of Candida to fluconazole and echinocandin. The method allows same-day results, assisting in the selection of appropriate antifungal therapy.

Sachidanandham R, Gin KY, Poh CL (2005) Monitoring of active but non-culturable bacterial cells by flow cytometry. Biotechnol Bioeng 89 :24-31


Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states : cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.

Sakamoto C, Yamaguchi N, Nasu M (2005) Rapid and simple quantification of bacterial cells by using a microfluidic device. Appl Environ Microbiol 71 :1117-1121


This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.

Sakurai F, Kawabata K, Yamaguchi T, Hayakawa T, Mizuguchi H (2005) Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors : comparison of promoter activities. Gene Ther 12 :1424-1433


Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1alpha promoter (EF1alpha promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/beta-actin promoter with beta-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+ CD38(low/-) and CD34+ AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

Saunier K, Rouge C, Lay C, Rigottier-Gois L, Dore J (2005) Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides. Syst Appl Microbiol 28 :454-464


Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.

Serada K, Moritomo T, Teshirogi K, Itou T, Shibashi T, Inoue Y, Nakanishi T (2005) Comparison of respiratory burst activity of inflammatory neutrophils in ayu (Plecoglossus altivelis) and carp (Cyprinus carpio). Fish Shellfish Immunol 19 :363-373


Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

Sermon J, Vanoirbeek K, De Spiegeleer P, Van Houdt R, Aertsen A, Michiels CW (2005) Unique stress response to the lactoperoxidase-thiocyanate enzyme system in Escherichia coli. Res Microbiol 156 :225-232


Using a differential fluorescence induction approach, we screened a promoter trap library constructed in a vector with a promoterless gfp gene for Escherichia coli MG1655 promoters that are induced upon challenge with the antimicrobial lactoperoxidase-thiocyanate enzyme system. None of the thirteen identified lactoperoxidase-inducible open reading frames was inducible by H(2)O(2) or by the superoxide generator plumbagin. However, analysis of specific promoters of known stress genes showed some of these, including recA, dnaK and sodA, to be inducible by the lactoperoxidase-thiocyanate enzyme system. The results show that the lactoperoxidase-thiocyanate enzyme system elicits a distinct stress response different from but partly overlapping other oxidative stress responses. Several of the induced genes or pathways may be involved in bacterial defense against the toxic effects of the lactoperoxidase-thiocyanate enzyme system.

Sheehan A, O’Loughlin C, O’Cuinn G, Fitzgerald RJ, Wilkinson MG (2005) Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry : implications for intracellular enzyme accessibility. J Appl Microbiol 99 :1007-1018


AIMS : To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS : Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains ; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS : Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY : This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.

Shin DJ, Choy HE, Hong Y (2005) Use of Clostridium septicum alpha toxins for isolation of various glycosylphosphatidylinositol-deficient cells. J Microbiol 43 :266-271


In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.

Shon W, Lim S, Bae KS, Baek S, Lee W (2005) The expression of alpha4 integrins by human polymorphonuclear neutrophils in response to sonicated extracts of Enterococcus faecalis. J Endod 31 :369-372


This study was undertaken to investigate immunopathologic mechanism of Enterococcus faecalis in relation to persistent apical periodontitis. We monitored the expression levels of alpha4 integrin in human polymorphonuclear neutophils (PMNs) after stimulated with sonicated extracts of E. faecalis (SEF) and compared with lipopolycaccarides (LPS) of Escherichia coli for various incubation time. Venous blood was collected from healthy volunteers and then PMNs were isolated and cultured with various concentrations of SEF for different periods of time. The levels of alpha4 integrin were measured by flow cytometry analysis. E. coli LPS group was used as a positive control and untreated PMNs as a negative control. Results showed that the expressions levels of alpha4 integrin were increased in human PMNs stimulated with E. coli LPS in comparison with unstimulated control cells (p < 0.05). In case of SEF stimulated group, the expression levels were decreased in time-dependent manner in comparison to E. coli LPS group (p < 0.05). Notably, after 12 h for incubation with SEF, the expression of alpha4 integrin was decreased in dose-dependent manner (p < 0.05). These findings suggest that E. faecalis seem to suppress PMNs recruiting activity by down-regulating alpha4 integrin expression, providing the possible mechanism that E. faecalis may play a crucial role in persistent apical periodontitis.

Singh JC, Cruickshank SM, Newton DJ, Wakenshaw L, Graham A, Lan J, Lodge JP, Felsburg PJ, Carding SR (2005) Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis. Am J Physiol Gastrointest Liver Physiol 288 :G514-524


The interleukin-2-deficient (IL-2(-/-)) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2(-/-) mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2(-/-) mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-kappaB (p65) activation with the inhibitory p50 form of NF-kappaB predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

Soboleski MR, Oaks J, Halford WP (2005) Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. Faseb J 19 :440-442


Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence support this conclusion : GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.

Sommaruga R, Hofer JS, Alonso-Saez L, Gasol JM (2005) Differential sunlight sensitivity of picophytoplankton from surface Mediterranean Coastal Waters. Appl Environ Microbiol 71 :2154-2157


We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.

Sommer U, Costello CE, Hayes GR, Beach DH, Gilbert RO, Lucas JJ, Singh BN (2005) Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J Biol Chem 280 :23853-23860


A secreted cysteine protease (CP) fraction from Trichomonas vaginalis is shown here to induce apoptosis in human vaginal epithelial cells (HVEC) and is analyzed by mass spectrometry. The trichomonad parasite T. vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, are shown to induce HVEC apoptosis, as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis is correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. SDS-PAGE analysis of the CP fraction reveals triplet bands around 30 kDa, and matrix-assisted laser desorption ionization time-of-flight MS indicates two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs results in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. The present results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention.

Song XD, Wang L, Ji B (2005) [Effect of qihong capsule in inhibiting cell apoptosis induced by Coxsackie virus B]. Zhongguo Zhong Xi Yi Jie He Za Zhi 25 :511-515


OBJECTIVE : To investigate whether the cell apoptosis could be induced by Coxsackie virus B (Cox B) and Qihong capsule (QHC) has the inhibition on the cell apoptosis. METHODS : Cultured cells were divided into 4 groups, the Cox B infected group, the QHC treated and the Cox B infected group, the QHC control group and the normal control group. The cells apoptosis was determined by TUNEL labeled in situ, Hoechst 33258 staining and Annexin-V/PI staining, the apoptotic incidence was assayed by flow cytometry, and the change in expression of apoptotic related cytokines was measured with RT-PCR. RESULTS : The Cox B infected cell nucleus displayed strong blue fluorescence by Hoechst 33258 staining, and typical change of apoptotic cells could be detected. HeLa cell membrane showed strong green fluorescence and nucleus showed strong red fluorescence by Annexin-V/PI staining. Flow cytometric observation on DNA of PI stained cells showed obviously an apoptotic peak in the Cox B infected group. QHC could decrease the apoptosis incidence, while there was no cell apoptosis occurred in the normal control group. Besides, QHC could regulate the expression of apoptotic related cytokines. CONCLUSION : QHC has effect in inhibiting cell apoptosis induced by Cox B.

Steichen CT, Kearney JF, Turnbough CL, Jr. (2005) Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis. J Bacteriol 187 :5868-5876


Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated ; however, our results indicate that it is not a glycoprotein. We showed that DeltabxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the DeltabxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of DeltabxpB spores and their resistance properties were similar to those of wild-type spores. However, DeltabxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from DeltabxpB spores of a hair-like nap or of enzymes that degrade germinants.

Stepanauskas R, Glenn TC, Jagoe CH, Tuckfield RC, Lindell AH, McArthur JV (2005) Elevated microbial tolerance to metals and antibiotics in metal-contaminated industrial environments. Environ Sci Technol 39 :3671-3678


To test the hypothesis that industrial metal contaminants select for microorganisms tolerant to unrelated agents, such as antibiotics, we analyzed metal and antibiotic tolerance patterns in microbial communities in the intake and discharge of ash settling basins (ASBs) of three coal-fired power plants. High-throughput flow-cytometric analyses using cell viability probes were employed to determine tolerances of entire bacterioplankton communities, avoiding bias toward culturable versus nonculturable bacteria. We found that bacterioplankton collected in ASB discharges were significantly more tolerant to metal and antibiotic exposures than bacterioplankton collected in ASB intakes. Optical properties of microorganisms collected in ASB discharges indicated no defensive physiological adaptations such as formation of resting stages or excessive production of exopolymers. Thus, it is likely that the elevated frequency of metal and antibiotic tolerances in bacterioplankton in ASB discharges were caused by shifts in microbial community composition, resulting from the selective pressure imposed by elevated metal concentrations or organic toxicants present in ASBs.

Stitz J, Krutzik PO, Nolan GP (2005) Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades. Nucleic Acids Res 33 :e39


We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- gamma (IFN-gamma)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-gamma, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the beta-chain of the IFN-gamma receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.

Sundh I, Bastviken D, Tranvik LJ (2005) Abundance, activity, and community structure of pelagic methane-oxidizing bacteria in temperate lakes. Appl Environ Microbiol 71 :6746-6752


The abundance and activity of methane-oxidizing bacteria (MOB) in the water column were investigated in three lakes with different contents of nutrients and humic substances. The abundance of MOB was determined by analysis of group-specific phospholipid fatty acids from type I and type II MOB, and in situ activity was measured with a 14CH4 transformation method. The fatty acid analyses indicated that type I MOB most similar to species of Methylomonas, Methylomicrobium, and Methylosarcina made a substantial contribution (up to 41%) to the total bacterial biomass, whereas fatty acids from type II MOB generally had very low concentrations. The MOB biomass and oxidation activity were positively correlated and were highest in the hypo- and metalimnion during summer stratification, whereas under ice during winter, maxima occurred close to the sediments. The methanotroph biomass-specific oxidation rate (V) ranged from 0.001 to 2.77 mg CH4-C mg(-1) C day(-1) and was positively correlated with methane concentration, suggesting that methane supply largely determined the activity and biomass distribution of MOB. Our results demonstrate that type I MOB often are a large component of pelagic bacterial communities in temperate lakes. They represent a potentially important pathway for reentry of carbon and energy into pelagic food webs that would otherwise be lost as evasion of CH4.

Tadonleke RD, Planas D, Lucotte M (2005) Microbial food webs in boreal humic lakes and reservoirs : ciliates as a major factor related to the dynamics of the most active bacteria. Microb Ecol 49 :325-341


In order to assess the factors that determine the dynamics of bacteria with high nucleic acid content in aquatic systems, we (i) conducted 24-h in situ dialysis experiments, involving different fractions of plankton and unfiltered water and (ii) examined empirical relationships between bacteria and both abiotic factors and protists, in boreal humic freshwaters (reservoir and lakes) in the James Bay region (Quebec, Canada). Bacteria were subdivided into two subgroups on the basis of their nucleic acid content assessed by flow cytometry. The abundance of bacteria with the highest nucleic acid content and high light scatter (HNA-hs) was significantly correlated, across sites, to bacterial production, whereas bacteria with lower nucleic acid content (LNA) and total bacteria were not. In addition, HNA-hs growth was higher and more variable than LNA growth, indicating that HNA-hs were the most dynamic bacteria. Heterotrophic nanoflagellate and ciliate biomass represented, on average, 5 and 13% of bacterial biomass, respectively. Both in ambient waters and in experiments, ciliates were significantly and negatively correlated with bacteria, whereas heterotrophic nanoflagellates, likely under the grazing pressure from ciliates and metazooplankton, were not. Among ciliates, Cyclidium glaucoma appeared to play an important role. Its growth was significantly and negatively correlated to that of HNA-hs but not to that of LNA. In ambient waters, the abundance of this species explained 56% of the variations in HNA-hs abundance and only 27% of those for LNA. The abundances of total bacteria and LNA significantly increased with chlorophyll a, whereas those of HNA-hs did not. In addition, during the experiments, the estimated potential losses of HNA-hs significantly increased with the initial abundance of C. glaucoma. These results suggest selective removal of the most dynamic bacteria by C. glaucoma and indicate that ciliates may play an important role in the dynamics of active bacteria in natural waters. These findings suggest the existence, within the aquatic microbial food webs, of keystone species that are very important in regulating the activity structure of bacteria.

Tang YZ, Gin KY, Lim TH (2005) High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry. Appl Environ Microbiol 71 :8157-8164


Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity) : (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

Tobella LM, Bunster M, Pooley A, Becerra J, Godoy F, Martinez MA (2005) Biosynthesis of poly-beta-hydroxyalkanoates by Sphingopyxis chilensis S37 and Wautersia sp. PZK cultured in cellulose pulp mill effluents containing 2,4,6-trichlorophenol. J Ind Microbiol Biotechnol 32 :397-401


Poly-beta-hydroxyalkanoates (PHA) polymer is synthesized by different bacterial species. There has been considerable interest in the development and production of biodegradable polymers ; however, the high cost of PHA production has restricted its applications. Kraft cellulose industry effluents containing 2,4,6-trichlorophenol (10 or 20 microg ml(-1)) were used by the bacteria Sphingopyxis chilensis S37 and Wautersia sp. PZK to synthesize PHA. In this condition, S. chilensis S37 was able to grow and degrade 2,4,6-trichlorophenol (ca. 60%) and 80% of these cells accumulated PHA. Wautersia PZK completely degraded 2,4,6-TCP and more than 90% of the cells accumulated PHA in 72 h. The PHA detection was performed by flow cytometry and polyester composition was characterized by gas chromatography-mass spectroscopy (GC-MS), indicating that these polymers are made by 3-hydroxybutyric acid and 3-hydroxyhexadecanoic acid for S37 and PZK strains, respectively. Results demonstrated that strains’ growth and PHA production and composition are not modified in cellulose effluents with or without 2,4,6-TCP (10-20 microg ml(-1)). Therefore, our results indicate that S. chilensis S37 and Wautersia sp. PZK are able to degrade a toxic compound such as a 2,4,6-TCP and simultaneously produce a valuable biopolymer using low-value substrates.

Tsugawa H, Ono T, Murakami H, Okawa Y (2005) Invasive phenotype and apoptosis induction of Plesiomonas shigelloides P-1 strain to Caco-2 cells. J Appl Microbiol 99 :1435-1443


AIMS : The mechanism of the host cell invasion of Plesiomonas shigelloides and its capability to induce apoptosis were investigated. METHODS AND RESULTS : We performed a time course experiment on the bacterial adherence and invasion of the P. shigelloides P-1 strain into Caco-2 cells using an invasion assay and flow cytometry. The adherence of P. shigelloides to the Caco-2 cells was almost completed within 10 min after the infection. Thereafter, P. shigelloides starts internalization within the Caco-2 cells, which was completed within 60 min after the infection. Based on the invasion assay using nocodazole, cytochalasin D, and genistein, it became clear that the mechanism of the internalization depended on the signal transduction followed by the rearrangement of the cytoskeletal protein. Based on the DNA laddering and TUNEL methods, the cytotoxicity of the Caco-2 cells by the invasion of P. shigelloides occurred through the induction of apoptosis. CONCLUSIONS : This work demonstrated that the mechanism of invasion of P. shigelloides into Caco-2 cells and the invasion of P. shigelloides induces apoptotic cell death. SIGNIFICANCE AND IMPACT OF THE STUDY : This work revealed the virulence factor, which may be important for understanding of the pathogenesis of P. shigelloides.

Tzeng L, Singer M (2005) DNA replication during sporulation in Myxococcus xanthus fruiting bodies. Proc Natl Acad Sci U S A 102 :14428-14433


During the developmental process of the Gram-negative soil bacterium Myxococcus xanthus, vegetatively growing rod cells differentiate to ultimately become metabolically quiescent and environmentally resistant myxospores encased within fruiting bodies. This program, initiated by nutrient deprivation, is propagated by both cell-autonomous and cell-nonautonomous signals. Our goal was to determine whether M. xanthus, like many other developmental systems, uses cell-cycle cues to regulate and control its developmental program. To address this question, the DNA replication cycle was used as a marker to monitor progression through the cell cycle in vegetative, stationary, and developing M. xanthus populations. Using flow cytometry, quantitative fluorescence microscopy, and FISH to establish the chromosome copy number of myxospores, it was determined that vegetatively growing cells contain one to two copies of the genome, but upon entry into stationary phase, the chromosome copy number drops to a single copy. Of particular interest, fruiting body-derived myxospores contain a specific two-chromosome DNA complement with both origin and terminus regions localized to the periphery of the myxospore. We speculate that this duplication of genetic information in the myxospore would help assure viability during germination by providing a second copy of each gene. The results of this study imply that not only is DNA replication tightly regulated during the developmental process of M. xanthus, but that there are also regulatory mechanisms to ensure that all myxospores acquire two copies of the chromosome.

Uchiyama T, Abe T, Ikemura T, Watanabe K (2005) Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol 23 :88-93


Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.

Underhill GH, Zisoulis DG, Kolli KP, Ellies LG, Marth JD, Kansas GS (2005) A crucial role for T-bet in selectin ligand expression in T helper 1 (Th1) cells. Blood 106 :3867-3873


Proinflammatory T helper 1 (Th1) cells express high levels of carbohydrate ligands for the endothelial selectins, but the molecular basis for this phenotype is incompletely understood. We document here a significant role in selectin ligand formation for the recently described Th1 transcription factor T-bet. Th1 cells generated from T-bet-/- mice showed significantly lower levels of ligands for both E-selectin and P-selectin, compared with wild-type (WT) Th1 cells. Enforced expression of T-bet in WT Th0 cells only modestly up-regulated P-selectin ligands and had no effect on E-selectin ligands. To define a mechanism for the defects observed in T-bet-/- mice, we examined expression of glycosyltransferases involved in selectin ligand biosynthesis. T-bet-/- Th1 cells expressed significantly lower levels of core 2 beta1,6 N-acetylglucosaminyltransferase I (C2GlcNAcT-I), but no differences in levels of alpha 2,3-sialyltransferase IV (ST3Gal-IV). Further, we show that T-bet is responsible for the signal transducer and activator of transcription 4 (Stat4)-independent increase in Th1 cells of fucosyltransferase VII (FucT-VII). We also identify ST3Gal-VI, which is thought to play an important role in E- and P-selectin ligand formation, as an interleukin 12 (IL-12)-regulated, T-bet-dependent gene. These data show that T-bet controls selectin ligand formation in Th1 cells via control of expression of multiple key enzymes in response to IL-12 signaling and establishes an independent transcriptional pathway for control of Th1 cell traffic.

Usherwood EJ, Meadows SK, Crist SG, Bellfy SC, Sentman CL (2005) Control of murine gammaherpesvirus infection is independent of NK cells. Eur J Immunol 35 :2956-2961


Numerous studies have shown that NK cells are important in controlling the early stages of infection with alpha- or betaherpesviruses. In contrast, little is known about the impact of NK cells on gammaherpesvirus infections. We tested mice with defects in NK cells for their ability to resist murine gammaherpesvirus (MHV-68) infection. The depletion of NK cells had no effect on the control of the acute or latent stages of the infection. In addition, transgenic mice deficient in NK cells controlled the infection in a comparable manner to wild-type mice. We also showed that the antiviral CD8 T cell response was unaffected by the presence or absence NK cells. We conclude that NK cells contribute little to the control of MHV-68 infection, and therefore, NK cells are not essential for controlling all herpesvirus infections.

Vaahtovuo J, Korkeamaki M, Munukka E, Viljanen MK, Toivanen P (2005) Quantification of bacteria in human feces using 16S rRNA-hybridization, DNA-staining and flow cytometry. J Microbiol Methods 63 :276-286


Hybridization of bacteria with fluorescent probes targeting 16S rRNA and inspection of hybridized bacteria with fluorescence microscopy (microscopy-FISH, i.e. fluorescence in situ hybridization) have constituted an accessible method for the analysis of mixed bacterial samples such as feces. However, microscopy-FISH is a slow method and prone to errors. Flow cytometry (FCM) enables analysis of bacteria more rapidly, accurately and reliably than microscopy. In this study, a FCM method for the analysis of 16S rRNA-hybridized and DNA-stained fecal bacteria was developed. The results of FCM-FISH were comparable to those of microscopy-FISH, and the coefficients of variation of the FCM analyses were extraordinarily low. In previous FCM-FISH studies, the Eub 338 probe, which is supposed to hybridize all bacteria, has been used to detect all bacteria present in the sample. We found that Eub 338 did not bind to all bacteria, which could be detected by DNA-staining ; while SYTOX Orange DNA-stain detected all bacterial species tested and produced high fluorescence intensities enabling clear separation of bacteria from non-bacterial material. Thus, DNA-staining is a method of choice for the detection of all bacteria in FCM-FISH. We conclude that FCM of 16S rRNA-hybridized and DNA-stained bacteria is a rapid and reliable method for the analysis of mixed bacterial samples including feces.

Valli M, Sauer M, Branduardi P, Borth N, Porro D, Mattanovich D (2005) Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry. Appl Environ Microbiol 71 :1515-1521


Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.

Vitova M, Hendrychova J, Cepak V, Zachleder V (2005) Visualization of DNA-containing structures in various species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye. Folia Microbiol (Praha) 50 :333-340


We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time PCR), which enabled routine staining in vivo. Cells do not need to be fixed or treated chemically or physically before staining, thus the shape, size and position of DNA-containing structures are not affected. The fluorescence signal is sharp and reproducible. Examples of application of the method are shown in color microphotographs for representatives of eukaryotic algae from the taxa Chlorophyta, Rhodophyta and the prokaryotic Cyanophyta. The method is also useful for studying progress of the cell cycle in algal cells dividing by multiple fission, as shown by observation of changes in nuclear number during the cell cycle of the green alga Chlamydomonas reinhardtii and Scenedesmus quadricauda. Staining with SYBR Green I can be recommended as a fast, safe and efficient method for the detection of DNA-containing structures in vivo.

Vogt C, Losche A, Kleinsteuber S, Muller S (2005) Population profiles of a stable, commensalistic bacterial culture grown with toluene under sulphate-reducing conditions. Cytometry A 66 :91-102


BACKGROUND : Most bacteria present in nature are not culturable in pure culture by means of classic cultivation methods (Pace NR, 1997, Science 276:734-740 ; Amann RI et al., 1995, Microbiol Rev 59:143-169.). However, it was recently shown that most aerobic heterotrophic bacteria could grow only on artificial media when other micro-organisms are present (Kaeberlein T et al., 2002, Science 296:1127-1129). Because the sulphate reducer Desulfobacula toluolica DSM 7467 and a bacterium (strain MV1) identified as Cellulosimicrobium sp. were not culturable unaccompanied, flow cytometry was used to highlight the strains’ relation within the consortium. METHODS : DNA patterns were used to provide strain-specific information about population proliferation dynamics. Cells were grown anaerobically and fed with toluene under sulphate-reducing conditions. RESULTS : Oxidation of toluene occurred only in association with sulphate reduction and growth of D. toluolica. A characteristic chromosomal pattern, with at least six subpopulations of D. toluolica, appeared during the stationary phase, and asymmetric cell division was detected. The accompanying strain MV1 grew repeatedly to a high percentage of the culture only in certain growth phases of D. toluolica independently of the feeding substrate toluene. CONCLUSIONS : A commensalistic relation between the two strains is suggested. The repeated rapid and frequent changes of the quantities within the community subsets are indicative of very flexible adaptations to changing environmental conditions, reflecting the need for modulated cell states and the ability to use every available source of carbon and energy for survival.

Wallberg F, Sundstrom H, Ledung E, Hewitt CJ, Enfors SO (2005) Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry. Biotechnol Lett 27 :919-926


Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6-48 mg PMP/g CDW) whilst highlighting population heterogeneity.

Wehrman TS, Casipit CL, Gewertz NM, Blau HM (2005) Enzymatic detection of protein translocation. Nat Methods 2 :521-527


Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from beta-galactosidase and comprises one enzyme fragment, omega, which is localized to a particular subcellular region, and a small complementing peptide, alpha, which is fused to the protein of interest. The concentration of alpha in the immediate vicinity of omega correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.

Welch TJ, Wiens GD (2005) Construction of a virulent, green fluorescent protein-tagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge. Dis Aquat Organ 67 :267-272


A green fluorescent protein (GFP) expressing strain of Yersinia ruckeri was created by the transposition of a Tn10-GFP-kan cassette into the genome of Y. ruckeri Strain YRNC10. The derivative, YRNC10-gfp, was highly GFP fluorescent, retained the gfp-km marker in the absence of kanamycin selection, and exhibited in vitro growth kinetics similar to those of the wild type strain. YRNC10-gfp colonized and caused mortality in immersion and intraperitoneally challenged rainbow trout Oncorhynchus mykiss, although it was modestly attenuated compared to the wild type strain. The distribution and location of YRNC10-gfp in infected fish was visualized by epifluorescence microscopy. Abundant extracellular bacteria and a small number of intracellular bacteria were observed in the kidney, spleen and peripheral blood. To determine the percentage of trout cells containing intracellular bacteria, GFP fluorescence was measured by flow cytometry. A small population of GFP positive leukocytes was detected in peripheral blood (1.6%), spleen (1.1%) and anterior kidney (0.4%) tissues. In summary, this is the first report of the construction of a virulent, GFP-tagged Y. ruckeri, which may be a useful model for detecting and imaging the interactions between an aquatic pathogen and the natural salmonid host.

Winkler ME, Winkler M, Burian R, Hecker J, Loss M, Przemeck M, Lorenz R, Patience C, Karlas A, Sommer S, Denner J, Martin U (2005) Analysis of pig-to-human porcine endogenous retrovirus transmission in a triple-species kidney xenotransplantation model. Transpl Int 17 :848-858


Clinical pig-to-human xenotransplantation might be associated with the risk of transmission of xenozoonoses, especially porcine endogenous retroviruses (PERVs). We have established a pig-to-humanised-cynomolgus monkey xenotransplantation model allowing the analysis of potential PERV-transmission from normal or transgenic porcine organs to human vascular tissue. Pig-to-human kidney xenotransplantation was performed in cynomolgus monkeys. An interposition graft constructed from a human saphena vein replaced the porcine kidney vein. After graft rejection and/or death of the recipient (survival 2, 4, 6, 13, 16, 19 days), the human interposition grafts were removed. Human endothelial cells (huECs) were isolated from the interposition grafts and cultivated in vitro. Explanted human vascular tissue, isolated huECs, plasma and serum samples of the graft recipients were characterised by flow cytometry and immunohistochemistry and screened for indications of PERV transmission by quantitative polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR) and RT assay. PERV-specific immune response of recipients was analysed by Western blot. No evidence of PERV infection or PERV-specific immune response was detected.

Wolkowicz R, Jager GC, Nolan GP (2005) A random peptide library fused to CCR5 for selection of mimetopes expressed on the mammalian cell surface via retroviral vectors. J Biol Chem 280 :15195-15201


A random peptide library was expressed on the surface of a mammalian cell by applying retroviral vectors. The random sequence was fused to the CCR5 chemokine receptor, which served as a scaffold to present the library at the cell surface. We used this library to isolate an epitope mimetope in a proof of principle system. This approach can become a tool for rapid creation of peptidic expression domains in a eukaryotic environment. Applications include the creation of decoys for receptors in cell-cell interactions, screening for molecules that drive ligand expression on target cells in two-cell interaction screens, among other utilities.

Wu LT, Tsou MF, Ho CC, Chuang JY, Kuo HM, Chung JG (2005) Berberine inhibits arylamine N-acetyltransferase activity and gene expression in Salmonella typhi. Curr Microbiol 51 :255-261


The effects of berberine on growth, arylamine N-acetyltransferase (NAT) activity, and gene expression in Salmonella Typhi (Typhi) were described. The growth inhibition of Typhi was determined by measuring absorbance by optical density (OD at 650 nm). The NAT activity was determined by measuring the levels of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) by high-performance liquid chromatography. The results demonstrated that 24-h berberine treatment decreased bacteria growth and amounts of AAF in Typhi. Western blotting and flow cytometry were used for examining the levels of NAT after bacteria were cotreated with or without various concentrations of berberine, and results indicated that berberine decreased the levels of NAT in Typhi. Polymerase chain reaction was used for examining the gene expression of NAT (mRNA NAT), and results indicated that berberine affects mRNA NAT1 expression in Typhi.

Yamamoto N, Yamamoto N, Petroll MW, Cavanagh HD, Jester JV (2005) Internalization of Pseudomonas aeruginosa is mediated by lipid rafts in contact lens-wearing rabbit and cultured human corneal epithelial cells. Invest Ophthalmol Vis Sci 46 :1348-1355


PURPOSE : The internalization of Pseudomonas aeruginosa (PA) in nasal and tracheal epithelium has recently been shown to involve the formation of cholesterol- and sphingolipid-rich plasma membrane domains (lipid rafts). The purpose of this study was to investigate the role of lipid rafts in PA internalization by corneal epithelium in vivo, in vitro, and after contact lens wear. METHODS : Lipid raft formation was evaluated in rabbit corneas with and without contact lens wear and a human corneal epithelial (hTCEpi) cell line before and after PA infection with cornea-pathogenic strains by staining with FITC-conjugated cholera toxin beta-subunit, known to bind the lipid raft component GM1. Bacterial internalization was assessed by gentamicin survival assay. The role of lipid rafts in PA internalization was evaluated by pretreatment of hTCEpi cells with cholesterol metabolism inhibitors. The interaction of PA with lipid rafts was confirmed by flow cytometry. RESULTS : Contact lens wear in rabbits induced lipid raft formation in occasional surface corneal epithelial cells. Subsequent PA exposure showed preferential binding to lipid raft-forming cells, leading to lipid raft aggregation and PA internalization. A similar sequence of lipid raft formation and PA internalization was also observed in hTCEpi for all PA strains. Internalization of all PA strains was blocked by three cholesterol metabolism inhibitors (P < 0.01). Flow cytometry showed an association of PA with rafts. CONCLUSIONS : These findings demonstrate that contact-lens-mediated PA internalization involves lipid raft formation. Also, hTCEpi cells may be used as an experimental model for studying further the molecular mechanism(s) of PA infection in the corneal epithelium.

Yamate M, Yamashita M, Goto T, Tsuji S, Li YG, Warachit J, Yunoki M, Ikuta K (2005) Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus. Microbes Infect 7 :1530-1540


Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.

Yu J, Lin J, Benjamin WH, Jr., Waites KB, Lee CH, Nahm MH (2005) Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies. J Clin Microbiol 43 :156-162


We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes : serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.

Yu KO, Im JS, Molano A, Dutronc Y, Illarionov PA, Forestier C, Fujiwara N, Arias I, Miyake S, Yamamura T, Chang YT, Besra GS, Porcelli SA (2005) Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides. Proc Natl Acad Sci U S A 102 :3383-3388


A form of alpha-galactosylceramide, KRN7000, activates CD1d-restricted Valpha14-invariant (Valpha14i) natural killer (NK) T cells and initiates multiple downstream immune reactions. We report that substituting the C26:0 N-acyl chain of KRN7000 with shorter, unsaturated fatty acids modifies the outcome of Valpha14i NKT cell activation. One analogue containing a diunsaturated C20 fatty acid (C20:2) potently induced a T helper type 2-biased cytokine response, with diminished IFN-gamma production and reduced Valpha14i NKT cell expansion. C20:2 also exhibited less stringent requirements for loading onto CD1d than KRN7000, suggesting a mechanism for the immunomodulatory properties of this lipid. The differential cellular response elicited by this class of Valpha14i NKT cell agonists may prove to be useful in immunotherapeutic applications.

Zengler K, Walcher M, Clark G, Haller I, Toledo G, Holland T, Mathur EJ, Woodnutt G, Short JM, Keller M (2005) High-throughput cultivation of microorganisms using microcapsules. Methods Enzymol 397 :124-130


This chapter describes a universal and novel method that provides access to the immense reservoir of untapped microbial diversity by cultivation. This technique uses microcapsules to encapsulate single cells combined with parallel microbial cultivation under low nutrient flux conditions. Under these conditions, single encapsulated cells grow and form microcolonies within the microcapsules. Flow cytometry is used as a sensitive tool to detect growth within the microcapsules. Microcapsules that contain microcolonies (originated from a single encapsulated cell) are sorted individually into microtiter dishes containing organic-rich medium. This high-throughput cultivation can provide more than 10,000 bacterial and fungal isolates per environmental sample.