RTX toxin actin cross-linking activity in clinical and environmental isolates of Vibrio cholerae

Vibrio cholerae strains from diverse O-antigen groups were evaluated for RTX toxin actin cross-linking activity. This study demonstrates that the actin cross-linking domain sequence is present within rtxA in the majority of clinical and environmental isolates tested, and the RTX toxin produced by these strains catalyzes the covalent cross-linking of cellular actin.     

In vivo evaluation of pathogenicity of clinical and environmental isolates of Vibrio cholerae.

Thirty-three minimally passaged clinical and environmental isolates of Vibrio cholerae were examined for ability to survive and multiply in the upper bowel of infant mice and to elicit diarrhea. All of 21 smooth O-1 V. cholerae isolates from stool were able to multiply and elicit diarrhea. Three rough strains isolated from stool were unable to multiply or to elicit diarrhea. Two smooth O-1 isolates associated with cholera cases (from a sewer and a septic tank) also were able to cause disease. However, four O-1 strains and one non-O-1 strain from sources not associated with cholera cases did not cause mouse disease. A human gall bladder isolate was also avirulent, whereas a Louisiana shrimp isolated showed low mouse virulence. We conclude that smooth human diarrheal isolates of V. cholerae of serogroup O-1 are virulent for infant mice. Examination of sequential isolates from single patients showed that some strains isolated later in infection had a reduced ability to induce diarrhea. Comparison of epidemiologically related strains showed that an isolate from crab had a low ability to induce disease in infant mice, whereas the isolates from patients showed the expected ability to multiply and elicit diarrhea in mice.     

Genetic diversity among clinical and environmental isolates of Aspergillus fumigatus.

To determine if cases of invasive aspergillosis (IA) were caused by strains of Aspergillus fumigatus with unique characteristics, strains from immunosuppressed patients with IA were compared to strains obtained from sputa of patients with cystic fibrosis and to strains from the environment. An extremely high genomic diversity was observed among the 879 strains typed by Southern blotting with a retrotransposon-like element from A. fumigatus (C. Neuvéglise, J. Sarfati, J. P. Latgé, and S. Paris, Nucleic Acids Res. 24:1428-1434, 1996). Analysis of Southern blot hybridization patterns showed the absence of clustering between environmental isolates and clinical isolates from patients with IA or cystic fibrosis. In addition, strains could not be clustered depending on their geographical location. This study implies that practically any strain of A. fumigatus is potentially pathogenic and can provoke a case of IA when it encounters a favorable environment in an immunosuppressed host.     

Temporal quorum-sensing induction regulates Vibrio cholerae biofilm architecture

Vibrio cholerae, the pathogen that causes cholera, also survives in aqueous reservoirs, probably in the form of biofilms. Quorum sensing negatively regulates V. cholerae biofilm formation through HapR, whose expression is induced at a high cell density. In this study, we show that the concentration of the quorum-sensing signal molecule CAI-1 is higher in biofilms than in planktonic cultures. By measuring hapR expression and activity, we found that the induction of quorum sensing in biofilm-associated cells occurs earlier. We further demonstrate that the timing of hapR expression is crucial for biofilm thickness, biofilm detachment rates, and intestinal colonization efficiency. These results suggest that V. cholerae is able to regulate its biofilm architecture by temporal induction of quorum-sensing systems.     

Colonization of the rabbit small intestine by clinical and environmental isolates of non-O1 Vibrio cholerae and Vibrio mimicus.

We examined the capability of 12 isolates of non-cholera toxin-producing O1 and non-O1 Vibrio cholerae to colonize the small intestine of adult rabbits and cause diarrhea. Using the removable intestinal tie-adult rabbit diarrhea model, we found that eight environmental isolates that showed no or marginal biological activity in other diarrhea models (rabbit ileal loop, infant rabbit, and suckling mouse) appeared to be incapable of attaching to and colonizing, even transiently, the small intestinal mucosa of animals with normal clearance mechanisms. In contrast, three clinical isolates attached, proliferated rapidly, and colonized mucosal surfaces of the entire small intestine within 8 h of challenge. This led to diarrhea with strikingly high rates of mortality compared with that of rabbits given similar challenges doses with strains of O1 V. cholerae that produce cholera toxin and Vibrio mimicus, which produces a toxin similar to cholera toxin. We have further demonstrated that multiple exposures to enteric infection by these strains elicited local and serum antibodies that reacted strongly with cell surface antigens of the homologous strain and showed a high degree of cross-reactivity against the cell surface antigens of the two heterologous strains. The enteric infections appeared to engender protection against subsequent infection as well, as evidenced by reduced incidence of diarrhea and duration of fecal shedding of the challenge organism upon subsequent challenges.     

A molecular and phenotypic study of Vibrio cholerae in Iran

Vibrio cholerae is again the subject of attention on account of the current increase in the world-wide incidence of cholera. In this study, 200 clinical isolates of V. cholerae serotypes O1 and non-O1, non-O139, were collected from different provinces in Iran. The isolates were subjected to biochemical analysis, antibiogram, PCR of toxin genes, plasmid profile, ribotyping and pulsed-field gel electrophoresis (PFGE). The analysis of plasmid content showed that 33-96% of V. cholerae isolated from different provinces carry a large plasmid. PCR analysis of V. cholerae O1 showed that the genes encoding cholera toxin (ctx), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin (zot) were present in 55-97% of isolates in different provinces. Restriction fragment length polymorphism (RFLP) of BglI-digested DNA probed with five oligonucleotides revealed three different ribotype patterns in isolates of V. cholerae O1. The ribotype pattern B21 of V. cholerae O1 El Tor was found to be the predominant pattern in the isolates studied. V. cholerae non-O1, non-O139 isolates showed a single ribotype pattern. PFGE analysis also showed 10 different patterns amongst the isolates, 9 of which were in V. cholerae O1. Overall, the analysis of polymorphism of ribotypes and PFGE patterns of the isolates showed that the provinces in Iran were affected by a limited number of clones of V. cholerae O1 and non-O1, non-O139 strains.     

Plasmids and factors associated with virulence in environmental isolates of Vibrio cholerae non-O1 in Bangladesh

Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-O1 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7-115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxic strains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-O1 strains. Conjugal transfer assays demonstrated that the 115-Mda plasmid harboured by some of the toxigenic V. cholerae non-O1 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mol.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-O1 strains. Regardless of their pathogenic potential, V. cholerae non-O1 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced.     

New variant of Vibrio cholerae O1 from clinical isolates in Amazonia.

A survey of pathogenic Vibrio cholerae O1 strains from the north of Brazil by using arbitrarily primed PCR fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current El Tor epidemic strain. These strains have been analyzed by in vivo and in vitro techniques and the group has been denominated the Amazonian variant of V. cholerae O1.     

Genetic diversity of Vibrio cholerae O1 in Argentina and emergence of a new variant

The genetic diversity of Vibrio cholerae O1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpA appeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucumán showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. cholerae O1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose "Tucumán variant" as the designation for this new cluster of cholera toxin-negative V. cholerae O1 strains.     

Genetic diversity of toxigenic Vibrio cholerae O1 from Sabah,Malaysia 2015

BackgroundCholera is an important health problem in Sabah, a Malaysian state in northern Borneo; however, Vibrio cholerae in Sabah have never been characterized. Since 2002, serogroup O1 strains having the traits of both classical and El Tor biotype, designated as atypical El Tor biotype, have been increasingly reported as the cause of cholera worldwide. These variants are believed to produce clinically more severe disease like classical strains.PurposeThe purpose of this study is to investigate the genetic diversity of V. cholerae in Sabah and whether V. cholerae in Sabah belong to atypical El Tor biotype.MethodsERIC-PCR, a DNA fingerprinting method for bacterial pathogens based on the enterobacterial repetitive intergenic consensus sequence, was used to study the genetic diversity of 65 clinical V. cholerae O1 isolates from 3 districts (Kudat, Beluran, Sandakan) in Sabah and one environmental isolate from coastal sea water in Kudat district. In addition, we studied the biotype-specific genetic traits in these isolates to establish their biotype.ResultsDifferent fingerprint patterns were seen in isolates from these three districts but one of the patterns was seen in more than one district. Clinical isolates and environmental isolate have different patterns. In addition, Sabah isolates harbor genetic traits specific to both classical biotype (ctxB-1, rstR^Cla) and El Tor biotype (rstR^ET, rstC, tcpA^ET, rtxC, VC2346).ConclusionThis study revealed that V. cholerae in Sabah were genetically diverse and were atypical El Tor strains. Fingerprint patterns of these isolates will be useful in tracing the origin of this pathogen in the future.     

Genetic Diversity and Population Structure of Vibrio cholerae

Multilocus enzyme electrophoresis (MLEE) of 397 Vibrio cholerae isolates, including 143 serogroup reference strains and 244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II). Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic of division I but not in subdivision Ia, which includes 76% of the ETs. Despite this evidence of relatively frequent recombination, clonal lineages may persist for periods of time measured in at least decades. In addition to the pandemic clones of serogroups O1 and O139, which form a tight cluster of four ETs in subdivision Ia, MLEE analysis identified numerous apparent clonal lineages of non-O1 strains with intercontinental distributions. A clone of serogroup O37 that demonstrated epidemic potential in the 1960s is closely related to the pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference strain is not. A strain of serogroup O22, which has been identified as the most likely donor of exogenous rfb region DNA to the O1 progenitor of the O139 clone, is distantly related to the O1/O139 clones. The close evolutionary relationships of the O1, O139, and O37 epidemic clones indicates that new cholera clones are likely to arise by the modification of a lineage that is already epidemic or is closely related to such a clone.     

Isolation and phenotypic characterization of virulence-deficient mutants of Vibrio cholerae.

Mutants of Vibrio cholerae was isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulence-deficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.     

Genetic and phenotypic analysis of Vibrio cholerae non-O1, non-O139 isolated from German and Austrian patients

Vibrio cholerae belonging to the non-O1, non-O139 serogroups are present in the coastal waters of Germany and in some German and Austrian lakes. These bacteria can cause gastroenteritis and extraintestinal infections, and are transmitted through contaminated food and water. However, non-O1, non-O139 V. cholerae infections are rare in Germany. We studied 18 strains from German and Austrian patients with diarrhea or local infections for their virulence-associated genotype and phenotype to assess their potential for infectivity in anticipation of possible climatic changes that could enhance the transmission of these pathogens. The strains were examined for the presence of genes encoding cholera toxin and toxin-coregulated pilus (TCP), as well as other virulence-associated factors or markers, including hemolysins, repeats-in-toxin (RTX) toxins, Vibrio seventh pandemic islands VSP-1 and VSP-2, and the type III secretion system (TTSS). Phenotypic assays for hemolysin activity, serum resistance, and biofilm formation were also performed. A dendrogram generated by incorporating the results of these analyses revealed genetic differences of the strains correlating with their clinical origin. Non-O1, non-O139 strains from diarrheal patients possessed the TTSS and/or the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which were not found in the strains from ear or wound infections. Routine matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS) analysis of all strains provided reliable identification of the species but failed to differentiate between strains or clusters. The results of this study indicate the need for continued surveillance of V. cholerae non-O1, non-O139 in Germany, in view of the predicted increase in the prevalence of Vibrio spp. due to the rise in surface water temperatures.     

A rapid and reliable species-specific identification of clinical and environmental isolates of Vibrio cholerae using a three-test procedure and recA polymerase chain reaction

Purpose: Vibrio cholerae, the cause of cholera, is one of the leading causes of morbidity and mortality in many developing countries. Most laboratories initially rely on biochemical tests for a presumptive identification of these strains, followed by a polymerase chain reaction (PCR)-based method to confirm their identification. The aim of this study is to establish a rapid and reliable identification scheme for V. cholerae using a minimal, but highly specific number of biochemical tests and a PCR assay. Materials and Methods: We developed a species-specific PCR to identify V. cholerae, using a housekeeping gene recA, and used that to evaluate the sensitivity and specificity of 12 biochemical tests commonly used for screening and / or presumptive identification of V. cholerae in the clinical and environmental samples. Results: Here we introduced a combination of three biochemical tests, namely, sucrose fermentation, oxidase test, and growth in trypton broth containing 0% NaCl, as also the PCR of the recA gene, for rapid identification of V. cholerae isolates, with 100% sensitivity and specificity. The established method accurately identified a collection of 47 V. cholerae strains isolated from the clinical cases (n = 26) and surface waters (n = 21), while none of the 32 control strains belonging to different species were positive in this assay. Conclusion: The triple-test procedure introduced here is a simple and useful assay which can be adopted in cholera surveillance programs for efficient monitoring of V. cholerae in surface water and fecal samples.     

Genetic and phenotypic diversity among ampicillin-resistant, non-beta-lactamase-producing, nontypeable Haemophilus influenzae isolates.

Levels of genotypic and phenotypic diversity among 23 ampicillin-resistant, non-beta-lactamase-producing (Ampr NBLP) isolates of serologically nontypeable Haemophilus influenzae recovered from the respiratory tract were determined by multilocus enzyme electrophoresis, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Twenty distinctive multilocus enzyme genotypes were identified, among which the average level of genetic diversity per locus was equivalent to that in the species as a whole. Hence, a single, recent origin for Ampr NBLP strains is excluded. Of the growth factors tested, a requirement for methionine was significantly associated with the Ampr NBLP phenotype. In contrast to the relative homogeneity of the PBP profiles of the ampicillin-susceptible strains tested (8 PBPs detected), the PBP profiles of the Ampr NBLP strains exhibited marked heterogeneity (5 to 10 PBPs detected). Care should be taken in interpreting changes in PBP profiles and in associating these profiles with resistance for species such as H. influenzae that demonstrate variability.     

Genetic diversity among clinical Coccidioides spp. isolates in Arizona

Increasing coccidioidomycosis rates in Arizona may indicate the development of a hypervirulent strain. One hundred and twenty-one clinical Coccidioides spp. isolates were collected over 16 months from Maricopa, Graham, Yuma, and Pima counties in Arizona. The patient age distribution ranged from 9 to 91 years, with a median age of 58 years; 36% were female, and 64% male. All isolates were analyzed by measuring length polymorphisms in nine distinct microsatellite regions. The three microsatellites found to have the greatest discriminatory power for Coccidioides posadasii were: K03 (0.87), GA37 (0.83), and K01 (0.78). The majority of isolates (n=119) were C. posadasii. Duplicate isolates (n=28) from 13 patients showed single strain infections. Phylogenetic analysis of the microsatellite data showed no dominant microsatellite pattern. We conclude that the increase in reported cases of coccidioidomycosis in Arizona is not linked to a dominant, hypervirulent strain of Coccidioides posadasii.     

Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus.

Twenty clinical isolates of Vibrio vulnificus were compared with 10 environmental strains by using electron microscopy and agglutination assays with human erythrocytes, guinea pig erythrocytes, and Saccharomyces cerevisiae. In addition, the isolates were tested for ability to adhere to the human epithelial cell lines HEp-2 and A549. When examined by electron microscopy, 16 (80%) of the 20 clinical isolates demonstrated the presence of piluslike structures; the composition of the bacterial populations ranged from 0 to 68% piliated cells. In contrast, only 3 (30%) of the 10 environmental isolates were piliated, with a range from 0 to 16% piliated cells. A significant association between the presence of piliated cells and the isolate source was found (P less than 0.05). None of the 30 strains agglutinated erythrocytes or yeast cells. V. vulnificus adherence results obtained with HEp-2 cells showed 10 (50%) of 20 clinical isolates and 0 (0%) of 10 environmental isolates with averages of greater than 10 adherent bacteria per cell, demonstrating a correlation between attachment and the isolate source (P less than 0.05). Selected strains were tested to determine whether methyl alpha-D-mannopyranoside, fructose, or alpha-L-(-)-fucose would inhibit bacterial adherence to HEp-2 cells. Multiple patterns of adherence inhibition were observed. Adherence to A549 cells showed 8 (40%) of 20 clinical isolates and 0 (0%) of 10 environmental strains with averages of greater than 10 adherent bacteria per cell. A statistical association between attachment and the isolate source was demonstrated (P less than 0.05). These data suggest that the presence of piluslike structures and the ability to adhere to human epithelial cell lines may be more closely associated with V. vulnificus isolates from clinical specimens than with environmental strains.     

Genomic diversity of clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 2001 as revealed by fluorescent amplified fragment length polymorphism analysis

Vibrio cholerae is a ubiquitous and abundant organism in aquatic environments, particularly in coastal areas, estuaries, and rivers. This organism was the cause of a considerable number of deaths in Brazil during the last decade. In this study we applied the genomic fingerprinting technique fluorescent amplified fragment length polymorphism (FAFLP) to analyze 106 V. cholerae O1 and non-O1 and non-O139 strains isolated from clinical specimens and the environment between 1991 and 2001. Numerical analysis of the FAFLP patterns disclosed seven main groups of genomes, all of them originated from a variety of different places in different years, suggesting that V. cholerae is a very diverse species. O1 and non-O1 and non-O139 strains were distinguishable by FAFLP, although clinical and environmental strains clustered together in a few cases. The persistence of some strains of highly related genomes during several years and in completely different geographical regions suggests that these strains are highly successful in adapting to changing environmental conditions.     

Pathogenic potential and genetic diversity of environmental and clinical isolates of Pseudomonas aeruginosa

The aim of this study was to investigate the occurrence of virulence genes among clinical and environmental isolates of Pseudomonas aeruginosa and to establish their genetic relationships by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC‐PCR). A total of 60 P. aeruginosa isolates from environmental and clinical sources were studied. Of these, 20 bacterial isolates were from soil, 20 from water, and 20 from patients with cystic fibrosis. Analysis of ERIC‐PCR demonstrated that the isolates of P. aeruginosa showed a considerable genetic variability, regardless of their habitat. Numerous virulence genes were detected in both clinical and environmental isolates, reinforcing the possible pathogenic potential of soil and water isolates. The results showed that the environmental P. aeruginosa has all the apparatus needed to cause disease in humans and animals.     

Molecular characterization of environmental and nontoxigenic strains of Vibrio cholerae.

Environmental and nontoxigenic strains of Vibrio cholerae 0-1 were examined for genes homologous to genes encoding Escherichia coli heat-labile enterotoxin (LT). Restriction fragments encoding LT A and B subunits were isolated from the recombinant plasmid EWD299 and labeled in vitro with 32P. These probes were then hybridized to deoxyribonucleic acid extracted from strains of V. cholerae and visualized by autoradiography. None of the nontoxigenic strains of V. cholerae 0-1 from Louisiana, Alabama, Maryland, Guam, Brazil, Bangladesh, or Great Britain hybridized with the LT probes, whereas all toxigenic strains exhibited homology. In addition, strains of V. cholerae non-0-1, "group F" vibrios, V. vulnificus, and Aeromonas hydrophila were tested, and all were negative except two strains of V. cholerae non-0-1. The presence of plasmids did not correlate with toxigenicity or nontoxigenicity in any of the species examined. Thus, it appears that these strains are not simple nontoxigenic mutants, but rather do not possess any genetic material encoding cholera toxin. Such strains therefore cannot revert and serve as a reservoir of cholera.