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Results for “Virulence profiling”

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PubMed1986

Characterization of outer membrane proteins of virulent phase I Shigella sonnei strains and their avirulent phase I derivatives.

Comparison of polyacrylamide gel electrophoretic protein profiles of four isogenic sets of virulent phase I Sh. sonnei strains and their avirulent phase I cells revealed no differences in outer membrane protein composition between virulent and avirulent derivatives. However, significant qualitative and quantitative differences were found in composition of major outer membrane proteins between strains of different origin. All four strains tested contained one major protein of 33K. This protein was susceptible to proteolytic enzymes and was found to be heat modifiable. Other major proteins of 35K and 37K present in three strains and 36K present in one strain were identified as peptidoglycan associated proteins.

Bacterial Outer Membrane Proteins↗

PubMed2005

Contamination of retail foods, particularly turkey, from community markets (Minnesota, 1999-2000) with antimicrobial-resistant and extraintestinal pathogenic Escherichia coli.

To assess the food supply as a possible vehicle for antimicrobial-resistant and extraintestinal pathogenic Escherichia coli (ExPEC), we defined the prevalence, density, clonal diversity, virulence characteristics, and antimicrobial resistance profiles of E. coli among diverse retail food items. A microbiological survey was undertaken of 346 food items (vegetables, produce, beef, pork, chicken, and turkey) purchased as a convenience sample from 16 retail markets within the Minneapolis-St. Paul area in 1999-2000, with selective cultures for E. coli and extensive molecular and phenotypic characterization of E. coli isolates. Meats, particularly turkey products, were often extensively contaminated with antimicrobial-resistant E. coli and ExPEC, to a much greater extent than were produce items, even those from farmer's markets. Moreover, meat-source E. coli differed substantially from produce-source E. coli with respect to phylogenetic background (more commonly from virulence-associated phylogenetic groups B2 or D), virulence genotype (more extensive), and antimicrobial resistance profile (more extensive). Molecular typing methods matched four turkey-source isolates to selected human clinical and fecal isolates representing the O7:K1:H-, O83:K1, and O73/O77:K52:H18 ("clonal group A") clonal groups of ExPEC. Meats purchased in grocery stores, particularly turkey products, are frequently contaminated with antimicrobial-resistant E. coli and ExPEC. Further study is needed regarding the origins and health consequences of these foodborne organisms, both to clarify the need for and to guide the possible development of appropriate regulatory and monitoring systems and preventive interventions.

Animals↗

PubMed1981

Increased virulence of Neisseria meningitidis after in vitro iron-limited growth at low pH.

At low pH (6.6) and under conditions of iron limitation, Neisseria meningitidis group B (strain SD1C) exhibited an atypical outer membrane protein profile and an increased relative virulence for the mouse. Cells grown in a buffered medium were effectively deprived of iron by the addition of ethylenediamine-diorthohydroxyphenylacetate. The pH of the medium selected for characteristic colonial morphologies: type M3 predominated at pH 6.6, and type M5 predominated at pH 7.7. A mixed population of M1, M3, and M5 colonies was observed at pH 7.2. Isolated outer membrane proteins were analyzed by sodium dodecyl 99 99 sulfate-polyacrylamide gel electrophoresis, and surface exposed proteins were labeled by the [125I]lactoperoxidase method and subsequently identified by autoradiography. Cells grown at pH 6.6 elaborated a major outer membrane protein (protein III; molecular weight, 69,000), which was also present in the outer membrane of iron-limited cells grown at pH 7.2. At pH 7.2 in an iron-sufficient medium, protein III was present only in small quantities in sodium dodecyl sulfate-polyacrylamide gel was present only in small quantities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A study of the relative virulence (50% lethal dose) of the meningococcus for C57/BL mice revealed that iron-limited cells grown at low pH had an increased relative virulence 1,200-fold (50% lethal dose, 4.0 CFU) greater than that of cells grown in the same medium but at pH 7.2 and with sufficient iron. These studies indicate that pH and iron can be important factors in the determination of meningococcal virulence.

Animals↗

PubMed1999

Propagation of TEM- and PSE-type beta-lactamases among amoxicillin-resistant Salmonella spp. isolated in France.

A survey conducted between 1987 and 1994 at the University Hospital of Besançon, France, demonstrated a dramatic increase (from 0 to 42. 5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 beta-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these beta-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the bla(PSE-1) gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the bla(TEM-1) gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.

Amoxicillin↗

PubMed1997

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.

Bacterial Typing Techniques↗

PubMed1990

[The molecular heterogeneity of Shigella sonnei lipopolysaccharide based on polyacrylamide gel electrophoretic data].

The molecular heterogeneity of S. sonnei lipopolysaccharide (LPS), reflecting the size of lateral O-specific polysaccharide chains, has been established by the method of electrophoresis in acrylamide gel in the presence of sodium dodecyl sulfate and urea. The dominating components fall into three types, viz. those with 0-3, 10-16 and 35-40 repeating structures, the remaining components being minor ones. The electrophoretic profile of S. sonnei LPS considerably differs from the profiles of Escherichia coli and S. flexneri LPS, but coincides with the LPS profiles of other strains with different virulence. The preparations of LPS obtained by extraction with trichloroacetic acid have the same electrophoretic profiles as LPS obtained by the method of aqueous phenol extraction. The domination of certain molecular variants reflects, seemingly, specific features of the biosynthesis of LPS, characteristic of a given strain. The mechanisms of the preferable synthesis of lateral O-specific chains of the definite size and the importance of the molecular parameters of lateral chains for the biological properties of LPS require further study.

Electrophoresis, Polyacrylamide Gel↗

PubMed2006

Global gene expression of a murein (Braun) lipoprotein mutant of Salmonella enterica serovar Typhimurium by microarray analysis.

Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.

Flagella↗

PubMed1993

Staphylococcus hyicus virulence in relation to exudative epidermitis in pigs.

Staphylococcus hyicus strains with different phage types, plasmid profiles, and antibiotic resistance patterns were isolated from piglets with exudative epidermitis. The strains could be divided into virulent strains, producing exudative epidermitis, and avirulent strains, producing no dermal changes when injected in experimental piglets. The results showed that both virulent and avirulent strains were present simultaneously on diseased piglets. This constitutes a diagnostic problem. Concentrated culture supernatants from nine virulent strains injected in the skin of healthy piglets produced a crusting reaction in all piglets. Acanthosis was observed in the histopathological examination of the crustaceous skin. Concentrated culture supernatants from nine avirulent strains produced no macroscopic or microscopic skin changes. Protein profiles from all virulent strains and seven out of nine avirulent strains showed a high degree of protein band homology. An approximately 30 kDa protein present in all concentrated culture supernatants capable of producing skin changes, could not be detected in samples that did not produce skin changes. No other protein showed a similar association. It is concluded that crusting reaction of piglet skin is a suitable indicator of virulence in S. hyicus in relation to exudative epidermitis, and that virulent strains produce a 30 kDa protein, absent in concentrated culture supernatants from avirulent strains. This 30 kDa protein might be an exfoliative toxin.

Animals↗

PubMed2000

Clonal analysis of Escherichia coli strains isolated from pigs with post-weaning diarrhea by pulsed-field gel electrophoresis.

The genetic diversity of 82 isolates of Escherichia coli recovered from pigs with post-weaning diarrhea was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with NotI and XbaI. The isolates were of serogroups O138, O139, O141, and O149 and possessed F4 and F18 fimbrial as well as LTI, STI, STII enterotoxin and shiga toxin 2e virulence markers. The restriction pattern profiles showed a high degree of polymorphism among isolates of different serotypes and a close relationship between strains belonging to the same serological group. In addition, some bacterial strains with identical virulence determinants exhibited distinct PFGE types. The results obtained showed that RFLP analysis is a powerful tool to reveal the clonal nature and genetic differences among E. coli strains responsible for diarrhea in weaned pigs.

Animals↗

PubMed2006

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

Botrytis↗

PubMed2003

Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasília, Brazil.

Escherichia coli strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid. Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%). The genotypic and phenotypic virulence markers of these strains were further analyzed. Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.05). Likewise, pic sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.005) and controls (P < 0.025). Furthermore, the association of pAA positivity (pAA(+)) and pic positivity (pic(+)) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA(+) pic(+) strains may represent a subset of pAA(+) strains associated with disease in children. Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern. The frequency of occurrence of enteropathogenic E. coli (EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups. Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea. The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea. More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea. Our results show that pAA(+) strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative E. coli virulence markers, serotypes, and extraintestinal virulence markers are considered.

Adult↗

PubMed2002

Repression of the Staphylococcus aureus accessory gene regulator in serum and in vivo.

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (agr locus) as well as multiple virulence-associated genes in Staphylococcus aureus. Gene expression was examined during growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of agr and any staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of agr and virulence factors was also observed. These results suggest that agr activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity.

Animals↗

PubMedDate not supplied

Involvement of protein tyrosine kinases and phosphatases in uptake and intracellular replication of virulent and avirulent Leishmania donovani promastigotes in mouse macrophage cells.

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.

Actins↗

PubMed2006

Identification of differentially expressed genes in virulent and nonvirulent Entamoeba species: potential implications for amebic pathogenesis.

Entamoeba histolytica is a protozoan parasite that causes colitis and liver abscesses. Several Entamoeba species and strains with differing levels of virulence have been identified. E. histolytica HM-1:IMSS is a virulent strain, E. histolytica Rahman is a nonvirulent strain, and Entamoeba dispar is a nonvirulent species. We used an E. histolytica DNA microarray consisting of 2,110 genes to assess the transcriptional differences between these species/strains with the goal of identifying genes whose expression correlated with a virulence phenotype. We found 415 genes expressed at lower levels in E. dispar and 32 genes with lower expression in E. histolytica Rahman than in E. histolytica HM-1:IMSS. Overall, 29 genes had decreased expression in both the nonvirulent species/strains than the virulent E. histolytica HM-1:IMSS. Interestingly, a number of genes with potential roles in stress response and virulence had decreased expression in either one or both nonvirulent Entamoeba species/strains. These included genes encoding Fe hydrogenase (9.m00419), peroxiredoxin (176.m00112), type A flavoprotein (6.m00467), lysozyme (6.m00454), sphingomyelinase C (29.m00231), and a hypothetical protein with homology to both a Plasmodium sporozoite threonine-asparagine-rich protein (STARP) and a streptococcal hemagglutinin (238.m00054). The function of these genes in Entamoeba and their specific roles in parasite virulence need to be determined. We also found that a number of the non-long-terminal-repeat retrotransposons (EhLINEs and EhSINEs), which have been shown to modulate gene expression and genomic evolution, had lower expression in the nonvirulent species/strains than in E. histolytica HM-1:IMSS. Our results, identifying expression profiles and patterns indicative of a virulence phenotype, may be useful in characterizing the transcriptional framework of virulence.

Animals↗

PubMed1995

Polymorphism of rRNA genes and plasmid analysis in the typing of Salmonella enterica serovar enteritidis from a Spanish health area.

A molecular epidemiological study of Salmonella enterica serovar Enteritidis (S. enteritidis) in a Spanish Health Area was carried out using two genotypic methods; polymorphism of rRNA genes (ribotyping) and plasmid analysis. The series included 100 isolates randomly selected from among those collected over the period 1984-92 (50 from sporadic episodes and 50 from 10 outbreaks). Ribotyping using HindIII, SmaI, and EcoRI showed a notable degree of homogeneity within Enteritidis serovar, it being able to discriminate only 1, 2, and 3 ribotypes, respectively. Such data suggest that there are a limited number of types or clonal lines, of which only one was widely disseminated within the health area under study (including 90% of isolates from sporadic episodes and all of those from outbreaks). Twelve plasmid profiles were differentiated. Sixty isolates carried only the virulence plasmid of 36 Md; whereas 24 isolates, grouped into 10 profiles, carried the 36 Md together with other plasmids; 7 isolates carried only other plasmids and 9 isolates were plasmid free. The combination of results from both methods revealed 15 subtypes, 9 of these including two or more isolates, one subtype being endemic and predominant (54% of the sporadic isolates and isolates from 6 outbreaks).

Bacterial Typing Techniques↗

PubMed2005

Spontaneous conversion to quinolone and fluoroquinolone resistance among wild-type Escherichia coli isolates in relation to phylogenetic background and virulence genotype.

Human clinical isolates of Escherichia coli that are resistant to quinolone or fluoroquinolone agents typically exhibit fewer extraintestinal virulence factors (VFs) than susceptible isolates, along with a different phylogenetic background. To experimentally assess the basis for this as-yet-unexplained phenomenon, 40 E. coli strains (20 E. coli Reference collection members and 20 Israeli cystitis isolates) were subjected to serial selective passaging to obtain derivatives resistant to nalidixic acid (NA) and ciprofloxacin (C). PCR-based VF profiling and phylotyping were performed on the parents and their respective resistant derivatives. All 40 susceptible parent strains yielded NA- and C-resistant derivatives after a median of 6 (range, 4 to 12) serial selective passages on agar plates containing increasing concentrations of NA and C. The numbers of passages required for resistance did not differ by collection origin, phylogenetic group, basal VF profile, source (urine versus fecal), or host group (human versus animal). With the development of C resistance, only one VF was lost in a single strain. Resistant derivatives exhibited the same phylotype as their susceptible parents. These findings suggest that the sparse VF profiles and the low-virulence phylogenetic background of NA- and C-resistant E. coli clinical isolates probably are not attributable to the loss of VFs from intrinsically high-virulence strains during conversion to resistance or to enhanced emergence of drug resistance among intrinsically low-virulence strains. A more likely explanation is the importation of resistant strains from an as-yet-undefined low-virulence external selection reservoir.

Anti-Infective Agents↗

PubMed2026

A 29-plex MOL-PCR assay for simultaneous detection of selected major, non-typing, and accessory virulence genes in Clostridium perfringens.

Clostridium perfringens is an important pathogen of humans and animals, responsible for a broad spectrum of diseases mediated by diverse toxins and virulence factors. Precise and extended toxin-gene profiling is valuable for strain characterization and molecular epidemiological surveillance. Here, we describe the development of a 29-plex Multiple Oligonucleotide Ligation PCR (MOL-PCR) assay that enables the simultaneous detection of a large and important panel of 27 C. perfringens toxin-related genes - covering major typing toxins as well as an extended panel of non-typing and accessory virulence genes - thus moving beyond the classical toxinotyping framework. The assay was evaluated in comparison with six multiplex qPCR assays. In both systems, the gene encoding the Clostridium perfringens-specific serine O-acetyltransferase (EpsC) was used as a molecular marker for species confirmation, and an internal amplification control was included to detect potentially false-negative results. Analytical specificity testing confirmed exclusive amplification in C. perfringens and sequencing confirmed the toxin-gene profiles of reference strains. Comparative analysis of 72 reference and field isolates (1,944 data points) demonstrated complete concordance for 637 positive detections, yielding 100% positive agreement and 99.7% negative agreement relative to the comparative qPCR method. The limit of detection was 100 fg/µl (approx. 3 × 101 genome equivalents; GE) for qPCR and 1  pg/µl (approx. 3 × 102 GE) for MOL-PCR. Despite its high multiplex level, MOL-PCR showed high agreement with qPCR. The developed MOL-PCR method provides a rapid, high-throughput, and cost-effective tool for expanded toxin-gene profiling of C. perfringens isolates targeting major typing toxins and selected non-typing and accessory virulence genes. Therefore, it may support advanced toxin-gene characterization, molecular epidemiology, and One Health-oriented surveillance of evolving virulence landscapes.

Clostridium perfringens↗

PubMed2003

Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica.

For intracellular pathogens such as Salmonellae, Mycobacteriae and Brucellae, infection requires adaptation to the intracellular environment of the phagocytic cell. The transition from extracellular to intravacuolar environment has been expected to involve a global modulation of bacterial gene expression, but the precise events have been difficult to determine. We now report the complete transcriptional profile of intracellular Salmonella enterica sv. Typhimurium following macrophage infection. During replication in murine macrophage-like J774-A.1 cells, 919 of 4451 S. Typhimurium genes showed significant changes in transcription. The expression profile identified alterations in numerous virulence and SOS response genes and revealed unexpected findings concerning the biology of the Salmonella-macrophage interaction. We observed that intracellular Salmonella are not starved for amino acids or iron (Fe2+), and that the intravacuolar environment is low in phosphate and magnesium but high in potassium. S. Typhimurium appears to be using the Entner-Douderoff pathway to use gluconate and related sugars as a carbon source within macrophages. Almost half the in vivo-regulated genes were of unknown function, suggesting that intracellular growth involves novel macrophage-associated functions. This is the first report that identifies the whole set of in vivo-regulated genes for any bacterial pathogen during infection of mammalian cells.

Cell Culture Techniques↗