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PubMed2002

Characterization of a self-transferable plasmid from Salmonella enterica serotype typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes.

An unusual self-transferable virulence-resistance plasmid (pUO-StVR2) was found in nine multidrug-resistant (ACSSuT phenotype) Salmonella enterica serotype Typhimurium clinical isolates that were assigned to four different phage types and a single and distinctive XbaI pulsed-field gel electrophoresis profile. pUO-StVR2 is an IncFII plasmid of about 140 kb in length carrying the spvA, spvB, and spvC (Salmonella plasmid virulence) and rck (resistance to complement killing) genes. It also carries the oxa1/aadA1a (ampicillin resistance and streptomycin-spectinomycin resistance) gene cassette configuration located within a class 1 integron with qacEDelta1/sul1 (ammonium antiseptics resistance and sulfadiazine resistance); the transposon genes merA, tnpA, and tnpR (mercury resistance, transposase, and resolvase of Tn21, respectively); and the catA1 (chloramphenicol resistance) and tet(B) (tetracycline resistance) genes. The insertion of resistance genes into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and presents a serious public health problem.

Bacteriophage Typing↗
PubMed2006

Associations among Listeria monocytogenes genotypes and distinct clinical manifestations of listeriosis in cattle.

OBJECTIVE: To determine whether specific strains of Listeria monocytogenes, as determined by genetic characteristics and virulence phenotypes, were associated with distinct clinical manifestations of listeriosis in cattle and thus may potentially have tissue specificity. ANIMALS: 32 cattle. PROCEDURE: DNA sequence data for the virulence genes actAand inlAwere used to infer the phylogeny of L. monocytogenes and to test for positive selection. Isolates were screened for the presence or absence of internalin genes and assigned an internalin profile. Plaquing assays were performed to determine the relative cytopathogenicity of each isolate. Categorical data analyses were performed to describe associations among L. monocytogenes genotypes, virulence phenotypes, and clinical manifestations of listeriosis. RESULTS: Results confirmed that L. monocytogenes represents 2 deeply separated evolutionary lineages. Genes actA and inlA contained amino acid sites under positive selection, and specific residues at some sites were associated with lineage and manifestation of listeriosis. Whereas lineage I was clonal and predominantly composed of isolates from cases of encephalitis, lineage II was more genetically diverse and equally represented by isolates from cases of encephalitis versus septicemia and fetal infection. Lineage I isolates also had greater cytopathogenicity in vitro, compared with lineage II isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that L. monocytogenes virulence genes underwent positive selection that is consistent with the diversification of 2 evolutionary lineages: lineage I is clonal and associated with encephalitis, and lineage II is more genetically diverse and equally likely to cause both major forms of listeriosis in cattle.

Animals↗
PubMed1994

Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis.

Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis.

Animals↗
PubMed2005

Genotypic and phenotypic characteristics of Yersinia enterocolitica isolated from the surface of chicken eggshells obtained in Argentina.

The prevalence of Yersinia enterocolitica on chicken eggshell surfaces in San Luis, Argentina, was investigated. The pathogenic potential of recovered isolates was assessed by means of phenotypic virulence tests and the presence of the 72-kb pYV plasmid. Antimicrobial susceptibility was determined by the agar diffusion method. DNA digested with XbaI was analyzed by pulsed-field gel electrophoresis (PFGE), and relationships between genomic DNA profiles were established. Eight Y. enterocolitica B2 O:9 strains were recovered after enrichment, for a prevalence of 2.27%. All strains harbored the virulence pYV plasmid, bound Congo red, grew in a low-calcium medium, and autoagglutinated at 37 degrees C. They lacked pyrazinamidase activity and did not hydrolyze esculin. These Y. enterocolitica strains were susceptible to amikacin, ciprofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole and were resistant to rifampin. According to the genomic DNA patterns obtained by PFGE, the isolates clustered into two groups, I and II. The highest similarity coefficient observed between Y. enterocolitica strains was 0.947. Microbiological controls on production stages of eggs and good culinary practices are necessary to reduce the risk of Y. enterocolitica infection for consumers.

Animals↗
PubMed2006

Prevalence and characterization of Escherichia coli O157 isolates from Minnesota dairy farms and county fairs.

Samples were collected from 26 organic and conventional farms and 12 county fairs in Minnesota during 2001 and 2002 to identify the presence of Escherichia coli O157. Immunomagnetic separation was used for isolation of E. coli O157. Isolates were further characterized by the presence of virulence marker genes (stx1, stx2, eaeA, E-hly, katP, etpD, and espP), antimicrobial susceptibility profiles, and genotypes. During 2001, E. coli O157 was isolated from 16 (5.2%) of 305 fecal samples and from 7 (36.8%) of 19 farms. During 2002, E. coli O157 was isolated from 6 (4.5%) of 132 fecal samples from weaned calves at 4 (23.5%) of 17 farms. During 2001 and 2002, cattle manure samples were collected from 12 county fairs, and E. coli O157 was isolated from 19 (11%) of 178 samples and 9 (75%) of 12 county fairs. Among 40 E. coli O157 isolates, 17 isolates (43%) had both the stx1 and stx2 genes, and 21 strains (53%) had the stx2 gene only. Thirteen percent of O157 isolates were resistant to tetracycline, and 25% were resistant to sulfadimethoxine. Heterogeneity of E. coli O157 strains was demonstrated by the presence of 22 different pulsed-field gel electrophoresis (PFGE) patterns. Four PFGE patterns matched those of isolates previously found in humans. The presence of E. coli O157 at county fairs suggests the potential for transmission to the public, who may have contact with cattle or their environment.

Animals↗
PubMed1988

Flagella, motility and invasive virulence of Pseudomonas aeruginosa.

The role of motility as a virulence factor in Pseudomonas aeruginosa burn wound sepsis was examined using mutants deficient in the Fla or Mot phenotype. Physiological profiles of parental strains and Fla- and Mot- mutants were similar with respect to antibiograms, O antigen types, growth rates, and proteolytic, exotoxin A and phospholipase activities, providing evidence for isogenicity. Lethality studies using a subcutaneous mouse burn model showed that three Fla- mutants and one Mot- mutant were much less virulent (10(2) to 10(5) times) than the parent wild-type. Topical challenges in the flame burn model showed that a Fla- mutant of strain M-2 was approximately tenfold less virulent. A reduction in virulence, although somewhat less than tenfold, was also observed in the scald burn model for M-2 Fla-, and Mot- strains. Tissue colonization experiments revealed a characteristic, rapidly systemic infection in burned mice challenged with wild-type organisms. Nonmotile mutants similarly proliferated in the burn wound, but the characteristic bacteraemia and systemic invasion were markedly absent. The infection remained localized in the skin wound and the mice survived. The pattern of infection by nonmotile mutants in the colonization studies was very similar to that obtained with Fla+ cells in burned animals passively treated with antiflagellar antibody. These results add substantial support to the concept of motility as a P. aeruginosa virulence factor in invasive infections.

Animals↗
PubMed2003

ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients.

Toxigenic Vibrio cholerae cause cholera, a severe diarrheal disease responsible for significant morbidity and mortality worldwide. Two determinants, cholera enterotoxin (CT) and toxin coregulated pilus (TCP) are critical factors responsible for this organism's virulence. The genes for these virulence determinants belong to a network of genes (the ToxR regulon) whose expression is modulated by transcriptional regulators encoded by the toxRS, tcpPH, and toxT genes. To define the ToxR regulon more fully, mutants defective in these regulatory genes were transcriptionally profiled by using V. cholerae genomic microarrays. This study identified 13 genes that were transcriptionally repressed by the toxT mutation (all involved in CT and TCP biogenesis), and 27 and 60 genes that were transcriptionally repressed by the tcpPH and toxRS mutations, respectively. During the course of this analysis, we validated the use of a genomic DNA-based reference sample as a means to standardize and normalize data obtained in different microarray experiments. This method allowed the accurate transcriptional profiling of V. cholerae cells present in stools from cholera patients and the comparison of these profiles to those of wild-type and mutant strains of V. cholerae grown under optimal conditions for CT and TCP expression. Our results suggest that vibrios present in cholera stools carry transcripts for these two virulence determinants, albeit at relatively low levels compared with optimal in vitro conditions. The transcriptional profile of vibrios present in cholera stools also suggests that the bacteria experienced conditions of anaerobiosis, iron limitation, and nutrient deprivation within the human gastrointestinal tract.

Bacterial Proteins↗
PubMedDate not supplied

[Shigellae isolated in 1997--plasmid profiles and antibiotic resistance].

INTRODUCTION: Shigella spp is one of the most frequently isolated bacteria causing acute diarrhea with us. Genetics of pathogenicity of Shigella spp. includes chromosomal and plasmid genes. Most virulence factors are coded by invasion plasmid antigen genes residing on a 180-230 MDa plasmid. There is a big problem with multiple resistance of Shigella spp. strains, which is mostly plasmid-borne. Genetic analysis of bacterial cells, that is plasmid profile analysis, is important for investigation of sources and ways of spreading of the infection. All isolates originating from the same clone have identical plasmid profiles, i.e. number and size of plasmids. The aim of the investigation was: comparing the type of resistance to antimicrobical agents found in epidemic and nonepidemic. Shigella strains isolated in 1997, analyzing plasmid profiles of these isolates and confirming their epidemic connection. MATERIAL AND METHODS: Susceptibility to antibiotics was examined by a standard disc-diffusion method. Plasmid profiles of 40 strains (20 from the outbreak and 20 from sporadic cases) were tested using a method of alkaline lysis by Birnboim and Doly followed by electrophoresis in agarose gel. RESULTS: Shigella strains were resistant to antimicrobial agents which are most commonly used. Epidemic isolates shared the same resistance type, they were resistant to cephalexin, streptomycin and co-trimoxazole. The dominant type of resistance of nonepidemic strains was to ampicillin, streptomycin and co-trimoxazole. Strains isolated during the outbreak had identical plasmid profiles (2 plasmid bands of 55 and 1.5 MDa). Non-epidemic isolates had different plasmid profiles as well as type of resistance. CONCLUSION: Strains of Shigella spp. isolated during an outbreak had the same type of resistance and the same plasmid profiles, which indicated their origin from the same clone. The plasmid profile analysis is a reliable and precise method for determination of epidemic connection of Shigella isolates.

DNA, Bacterial↗
PubMed2004

Isolation and characterisation of an Indian strain of Mycoplasma mycoides subsp. mycoides type LC from a case of caprine arthritis.

Isolation and characterisation of an Indian strain of Mycoplasma mycoides subsp. mycoides LC from a case of caprine arthritis is reported in the study. The isolate was identified based on biochemical, digitonin sensitivity and growth inhibition tests. The virulence of the organism was studied by pathogenicity test in mice and goat. The antigenic and genomic profile of the isolate was compared with that of the standard strain (Y-Goat). Using different sets of primers, polymerase chain reaction was employed for rapid detection of the strain.

Animals↗
PubMed1985

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

Bacterial Outer Membrane Proteins↗
PubMed2006

Enhanced therapeutic efficacy of G207 for the treatment of glioma through Musashi1 promoter retargeting of gamma34.5-mediated virulence.

G207 is a conditionally replicating derivative of herpes simplex virus type1 (HSV-1) engineered with deletions of both ICP34.5 loci and a lacZ insertion disabling the ICP6 gene. G207 exhibits an efficient oncolytic activity in vitro and in vivo, yet minimal toxicity in normal tissue, and is now in clinical trial for malignant glioma. According to the results of clinical trials, however, although G207 was proved to be safe, the efficacy was not so impressive. Deletion of the ICP34.5 gene coding for virulence made G207 extremely safe, but it markedly reduced the cytotoxicity mediated by HSV-1. To enhance the therapeutic efficacy of G207 without diminishing its safety, we used a defective vector containing Musashi1 promoter/ICP34.5, with G207 as helper virus. P/musashi1 was functional selectively in human glioma cell lines (U87MG, U251, T98G) in this study and dvM345 showed a much higher therapeutic efficacy both in culture and in the in vivo glioma model, than G207 alone, without diminishing its favorable toxicity profile. These results suggest that transcriptional regulation of ICP34.5 by P/musashi1 can be used to target HSV-1 virulence toward gliomas while maintaining the desirable neuroattenuated phenotype.

Animals↗
PubMed2006

Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences.

The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be used in conjunction with microarrays to determine global gene expression by a pathogen during infection. We used this method to study genes expressed by Salmonella enterica serovar Typhi, the etiological agent of typhoid fever, within human macrophages. Global expression profiles of Typhi grown in vitro and within macrophages at different time points were obtained and compared. Known virulence factors, such as the SPI-1- and SPI-2-encoded type III secretion systems, were found to be expressed as predicted during infection by Salmonella, which validated our data. Typhi inside macrophages showed increased expression of genes encoding resistance to antimicrobial peptides, used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. Genes coding for the flagellar apparatus, chemotaxis, and iron transport systems were down-regulated in vivo. Many cDNAs corresponding to genes with unknown functions were up-regulated inside human macrophages and will be important to consider for future studies to elucidate the intracellular lifestyle of this human-specific pathogen. Real-time quantitative PCR was consistent with the microarray results. The combined use of selective capture of transcribed sequences and microarrays is an effective way to determine the bacterial transcriptome in vivo and could be used to investigate transcriptional profiles of other bacterial pathogens without the need to recover many nanograms of bacterial mRNA from host and without increasing the multiplicity of infection beyond what is seen in nature.

Cell Line↗
PubMed2001

Characterisation and clonal relationships of Shiga-toxigenic Escherichia coli (STEC) isolated from Australian dairy cattle.

A total of 136 Shiga toxin-producing Escherichia coli (STEC) isolated during a longitudinal survey of three Australian dairy farms were examined to determine their virulence factors, serotype and genomic relationships. This study aimed to assess the potential of these STEC to cause disease in humans and to analyse the on-farm ecology of STEC. Virulence factors (stx, eae, ehxA) were used as determinants of potential to be enterohaemorrhagic E. coli (EHEC) and were examined using polymerase chain reaction (PCR). Among the cattle groups tested, calves, both before and during weaning, shed the most putative EHEC and were the main source of serotypes commonly associated with human disease. E. coli O157:H7 and E. coli O26:H11 represented 9.4 and 7.8% of cattle STEC isolates respectively, with other putative EHEC serotypes reported for the first time from cattle. Based on serotype and virulence factors, 20% of STEC were putative EHEC. Pulsed-field gel electrophoresis (PFGE) was used to compare the genomic profiles of STEC from dairy farms. Isolates common to cattle and the farm environment were identified. Multiple strains of STEC with high clonal turnover were detected in the faeces of cattle, and isolates appeared to be specific to individual farms. To fully assess the pre-slaughter EHEC risk factors on-farm, examination of STEC virulence is as important as determination of STEC prevalence.

Adhesins, Bacterial↗
PubMed2001

Virulence in Candida species.

Candida species other than Candida albicans now account for up to 50% of deep candidiasis cases, yet little attention has been paid to the virulence attributes of these fungi. Adherence to host tissues, response to environmental changes and the secretion of hydrolases are all thought to be important in Candida virulence. The identification of virulence attributes unique to a particular Candida species could provide powerful insights into the pathogenic process but will require the use of genome-wide approaches such as transcript profiling, signature-tagged mutagenesis and in vivo expression technology.

Animals↗
PubMed2000

Evidence for a signaling system in Helicobacter pylori: detection of a luxS-encoded autoinducer.

Helicobacter pylori possesses a homolog of the luxS gene, initially identified by its role in autoinducer production for the quorum-sensing system 2 in Vibrio harveyi. The genomes of several other species of bacteria, notably Escherichia coli, Salmonella enterica serovar Typhimurium, and Vibrio cholerae, also include luxS homologs. All of these bacteria have been shown to produce active autoinducers capable of stimulating the expression of the luciferase operon in V. harveyi. In this report, we demonstrate that H. pylori also synthesizes a functional autoinducer (AI-2) that can specifically activate signaling system 2 in V. harveyi. Maximal activity is produced during early log phase, and the activity is diminished when cells enter stationary phase. We show that AI-2 is not involved in modulating any of the known or putative virulence factors in H. pylori and that a luxS null mutant has a two-dimensional protein profile identical to that of its isogenic parent strain. We discuss the implications of having an AI-2-like quorum-sensing system in H. pylori and suggest possible roles that it may play in H. pylori infection.

Amino Acid Sequence↗
PubMed2004

Differential display RT-PCR analysis of ECV304 endothelial-like cells infected with dengue virus type 2 reveals messenger RNA expression profiles of multiple human genes involved in known and novel roles.

Differential display (DD)-RT-PCR was employed to analyze mRNAs from ECV304 human endothelial-like cells undergoing apoptosis following infection with the virulent New Guinea C strain of dengue virus type 2 in order to elucidate the cellular gene responses to dengue viral infection at the transcriptional level. We isolated, sequenced, and identified 203 differentially expressed and overlapping cDNA fragments, all of which were of human origin except 1 that was of viral origin. Out of these, 78 were individual distinct clones comprising 46 and 32 expressed sequence tags (ESTs) that exhibited upregulated and downregulated trends, respectively. Of the 78 differentially expressed mRNAs, 16 did not match any characterized genes or ESTs. The remaining 62 mRNAs modified by dengue virus infection matched known genes, including those encoding components of the cell cycle (Anillin, CDC27), cytoskeleton (epsilon-tubulin), signal transduction (OPHN1, PPP2R2A, TIRAP), protein translation and modification (EIF3S10, IF2, TMEM1), transcriptional regulation (alpha-NAC, C20orf104, EGR1, ELP2), apoptotic cell death (RICK), membrane (BPAG1), and mitochondrial-related proteins. Semiquantitative RT-PCR and real-time RT-PCR authenticated further the altered expression patterns of selected genes of interest. These data demonstrate the feasibility of mRNA DD in providing insights into the complex responses of the transcriptional machinery of permissive and apoptotic human endothelial-like cells in the pathogenesis of dengue and/or its complications induced by the virulent dengue virus type 2.

Apoptosis↗
PubMed1993

Characterization of mesophilic Aeromonas from clinical specimens by computerized analysis of SDS-PAGE protein profiles and by enzymatic activity.

Mesophilic Aeromonas (Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae) have recently been considered important aetiological agents of human diseases, mainly gastrointestinal infections. Although several findings have pointed out the significance of this group of microorganisms as enteric pathogens and suggested the presence of virulence factors, epidemiological and clinical studies are limited by the difficulty of correctly identifying mesophilic Aeromonas at the species level. SDS-PAGE of radiolabelled total protein profiles and bacterial enzymatic activities were used to type 31 clinical isolates (6 A. hydrophila, 7 A. sobria and 18 A. caviae) from patients with gastroenteritis and from healthy controls. Analysis of SDS-PAGE protein patterns, reinforced by the UPGMA-grouping system (AMBIS software) provided a good characterization of A. caviae strains as a homogeneous group of microorganisms, possessing significant differences from the other two species of mesophilic Aeromonas, in good agreement with biochemical and enzymatic tests. Data obtained in analyzing A. sobria protein profiles clearly showed two groups, with a correlation coefficient (CC) = 0.70, which in our experience is a doubtful value for assigning two strains to the same species. Strains biochemically identified as A. hydrophila showed a CC = 0.64, which is equally not acceptable for species assignment. Inter-species comparison highlighted this heterogeneity, showing two mixed subgroups, both containing strains that were assigned to A. sobria and A. hydrophila species on the basis of biochemical features.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Changing surface carbohydrate configurations during the growth of Leishmania major.

Surface carbohydrates of leishmanial promastigotes change during their growth cycle. These changes were monitored in a cloned line of Leishmania major in 2 media. A freshly isolated virulent strain was also examined. Infectivity, surface sugar moieties, and released glycoconjugates (EF) were examined and compared during the growth cycle. When promastigotes of the same clone were grown in different media, their lectin-mediated agglutination profiles were dissimilar, and both quantitative and qualitative variation was seen in the antigenic glycoconjugates released into the media. When labeled with fluorescent lectins, the virulent strain showed no loss of galactose, an increase of N-acetylglucosamine, and a midcycle decrease of N-acetylgalactosamine. Promastigotes of this strain were infective to hamsters throughout the growth cycle. The results presented indicate that culture medium components regulate carbohydrate surface configurations and, thereby, antigenic expression. Infectivity of the virulent strain was not dependent on the expression of a single surface carbohydrate.

Agglutination Tests↗