non-O1 Isolates Research Articles

A comparative study among clinical and environmental isolates of Vibrio cholerae at their antibiotic

in this study, the comparison of the antibiogram and molecular level among different serotype of clinical and environmental isolates of V. cholerae was done. Antibiogram of five clinical and three environmental isolates showed high susceptibility to all tested antibiotics, but non-O1 serotype had Ampicilin resistant. Plasmid profile screening revealed that V. cholerae O1 isolates and non-O1 Dial131 had no small size plasmid while non-O1 ab, 1J, 1R harbored three different small size plasmids with different appearance profile, two mega plasmids detected in the O1 isolate and only one mega plasmid resolute in non-O1 clinical and environmental isolates.

Occurrence of Vibrio cholerae non-O1 in three wastewater treatment plants in Agadir (Morocco)

A total of 21 isolates of Vibriocholerae non-O1 strains were isolated from three wastewater treatment plants in Agadir, Morocco. The isolates were analyzed by biochemical analysis, antibiogram, pulsed-field gel electrophoresis and the MALDI-TOF patterns of their protein masses were compared. Over 67% of isolates were susceptible to antimicrobial agents tested and 14% proved resistant to both trimethoprim-sulfamethoxazole and nalidixic acid. Typing by pulsed-field gel electrophoresis with NotI digestion revealed that the V. cholerae non-O1 strains from Agadir (Morocco) have a lower level of genetic homogeneity, the restriction patterns of whole-chromosomal DNA grouped the V. cholerae O1 and V. alginolyticus strains into a separate cluster from V. metschnikovii and V. cholerae non-O1 isolates. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used dendrogram based on MALDI-TOF spectral patterns generated by the BioTyper 1.1™ software. All m/z signatures of all strains tested indicate that the mass spectral data contained sufficient information to distinguish between strains of V. cholerae.

Environmental monitoring of Vibrio cholerae using chironomids in India

Environmental Vibrio cholerae strains belonging to the non-O1/non-O139 serogroups are natural inhabitants of freshwater including estuarine environments. Recent findings indicated that chironomids (Diptera: Chironomidae), the most widely distributed insects in freshwater, serve as a natural reservoir of these bacteria. Here we study the role of chironomids, particularly exuviae as carriers and as a monitoring tool for the distribution of V. cholerae in the environment. During a survey conducted in India (June 2006), 326 V. cholerae non-O1/non-O139 isolates were isolated from chironomid egg masses, larvae and exuviae. In addition, a heat-stable enterotoxin (nag-st) positive strain was isolated from exuviae during the local cholera outbreak. We identified 62 different strains in a subset of 102 isolates by analysis of variable number of tandem repeats (VNTR), demonstrating a high variation of V. cholerae on hosting chironomids. Our results show that chironomids can both maintain and distribute this overwhelming diversity of environmental V. cholerae strains, including toxigenic ones. Exuviae proved to be an efficient tool for the monitoring of environmental V. cholerae, offering simple, direct and practical access for on-shore collection. Finally, finding toxigenic V. cholerae on chironomids in endemic areas, together with molecular typing, may potentially improve monitoring of cholera in the future.

Virulence profile and clonal relationship among the Vibrio cholerae isolates from ground and surface water in a cholera endemic area during rainy season

All the V. cholerae non-O1, non-O139 isolates from ground and surface water samples collected during the rainy season (rainfall contributes significantly in the spread of cholera) contained ompW and a regulatory toxR gene, while many others possessed accessory cholera toxin (ace), hemolysin (hlyA) and outer membrane protein (ompU) genes. All the isolates lacked ctxAB, tcp, zot, rfbO1 and rfbO139 genes. The strains could be grouped into two main clusters colligating the isolates from ground water and surface water samples. The results suggest that surface water harbors various virulent V. cholerae strains that contaminate the ground water due to rain or poor hygienic practices, and result in the emergence of new toxigenic strains for cholera.

Characterization of potentially virulent non-O1/non-O139 Vibrio cholerae strains isolated from human patients

Traditional methods of typing Vibrio cholerae define virulent strains according to their recognition by sera directed against the known epidemic serogroups O1 and O139, overlooking potentially virulent non-O1/non-O139 strains. Here, we have undertaken the characterization of eight clinical isolates of non-O1/non-O139 V. cholerae, collected during cholera outbreaks in Brazil. Seven of these were typed as O26 and one, 17155, was defined as non-typable. A PCR-based approach has previously detected in these strains several virulence genes derived from the CTXφ prophage and generally associated with pathogenic strains. Here, the presence of the O1-specific wbeN gene was investigated through PCR and found to be restricted to strain 17155, as well as one of the O26 strains, 4756, although neither strain was recognized by O1-specific antisera. The same two isolates were the only strains able to express the cholera toxin in culture, assayed by western blotting. They also possessed four repeats of the heptanucleotide TTTTGAT upstream of the ctxAB genes encoding the cholera toxin. The remaining strains possessed only two intact repeats, whereas pathogenic O1 possessed four to six repeats. To define their evolutionary relationships, selected 16S–23S intergenic rRNA spacer regions were sequenced from the various strains and the resulting sequences used to build phylogenetic trees. Strains 4756 and 17155 always clustered with control O1 strains, whereas the remaining O26 strains clustered separately. These results confirm that, despite their serological phenotype, these two strains are genotypically related to O1 strains and potentially able to produce epidemic cholera.

A molecular survey on virulence associated genotypes of non-O1 non-O139 Vibrio cholerae in aquatic environment of Tehran, Iran

Thirty-seven Vibrio cholerae strains were isolated from surface water sources at 5 different locations in Tehran, Iran during 2006 and were identified as non-O1 and non-O139 isolates. PCR for SXT element and class 1 integron was positive for 19% and 5.4% of isolates, respectively. PCR for virulence associated-genes within the vibrio pathogenicity island (VPI) gene cluster showed the presence of LJ, int and RJ in 8, 59 and 30% of the isolates, respectively. None of the V. cholerae isolates contained the toxin encoding genes ( ace, zot, ctx) in the CTX genetic element. Biochemical fingerprinting using PhPlate system (PhP-RV) was able to type all strains and resulted in 8 common types (containing 78% of the isolates) and 8 single types (22%). Out of 37 isolates, only 26 isolates were typeable with pulsed-field gel electrophoresis (PFGE) producing banding patterns. The results presented in this study showed no genotyping correlation between the V. cholerae isolated from surface water and the clinical setting which had been reported previously by this laboratory. Furthermore, combination of PFGE and PhP-RV methods was proved beneficial for non-typeable V. cholerae isolates.

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (>/=100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.

Construction and Characterization of Avian Pathogenic Escherichia coli Mutants with iro and/or tsh Gene Mutation

Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive hybridization (SSH) and selective capture of transcribed sequences (SCOTS) in avian pathogenic Escherichia coli (APEC) isolates. To get more insights in the distribution and the occurrence of the iroC and tsh genes, 243 avian E. coli isolates were examined for the presences of these genes. Amongst 243 avian E. coli isolates, iroC gene was present in 84.4% strains (205/243). Of the 205 iroC-positive isolates, iroC gene was found in 184 (89.8%), 18 (8.8%) and 3 (1.5%) isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 (87.4%), 21 (12.6%) and 0 (0%) isolates with high, intermediate and low pathogenicity, respectively. Amongst tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene ( P<0.01). Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragment of iroB and 603-bp fragment of the iroN were amplified by PCR, respectively. Both these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector combined with the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm, and a resultant suicide vector combined with the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037 (Δiro), E037 (Δtsh) and E037 (ΔiroΔtsh). The 50% lethal dose (LD50) of E037, E037 (Δiro), E037 (Δtsh) and E037 (ΔiroΔtsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined as 10 5.6, 10 8.4, 10 9.0 and 10 9.5CFU, respectively. In the chicken challenge model, the mutants were evaluated on the individual role of these genes for virulence and persistence in chickens. The result indicated that Iro system and Tsh were important in the pathogenicity of APEC.

Occurrence and Expression of Luminescence in Vibrio cholerae

Several species of the genus Vibrio, including Vibrio cholerae, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of V. cholerae strains are luminescent. Analysis of 224 isolates of non-O1/non-O139 V. cholerae collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the luxA gene. In contrast, 334 non-O1/non-O139 V. cholerae strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%) luxA+ isolates. An additional 270 clinical and environmental isolates of V. cholerae serogroups O1 and O139 were tested, and none were luminescent or harbored luxA. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139 V. cholerae strains that frequently occur in certain environments. Luminescence expression patterns of V. cholerae were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the lux operon, including natural K variants, were identified.

Biofilm Acts as a Microenvironment for Plankton‐Associated Vibrio cholerae in the Aquatic Environment of Bangladesh

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.

Genetic and Phenotypic Diversity of Quorum-Sensing Systems in Clinical and Environmental Isolates of Vibrio cholerae

Vibrio cholerae is the causative agent of cholera, a severe and devastating diarrheal disease. V. cholerae lives naturally in various aquatic habitats during interepidemic periods. Recent studies reveal that quorum-sensing systems, which exist in many bacteria and help them monitor their population densities and regulate various cellular functions, control V. cholerae pathogenesis, biofilm formation, and protease production. In this study we surveyed quorum-sensing systems in 16 geographically diverse V. cholerae strains from epidemic-causing O1 and O139 strains as well as non-O1/non-O139 and environmental isolates and discovered an unexpectedly high rate of dysfunctional components. We also found that a functional quorum-sensing system conferred a survival advantage on bacteria in biofilms when the bacteria were exposed to seawater, though quorum sensing was less important to survival in a planktonic state under the same conditions. These findings suggest that variations in quorum-sensing systems are due to environmental selective pressures and might be beneficial to V. cholerae's fitness under certain conditions found in its natural reservoirs.

Characterization of filamentous phages of Vibrio cholerae O139 and O1.

We have analyzed our collection of Vibrio cholerae O139 strains to determine whether filamentous phages are produced in their culture supernatants, and whether any replicative form of DNA is detectable in cell lysates. Two types of filamentous phage, designated fs1 (6.4 kb) and fs2 (8.5 kb), were found in strains of Vibrio cholerae O139, fs1 was commonly produced from clinical isolates of Vibrio cholerae O1. Infectious particles (filamentous phages) were inducible by subculture, mitomycin C, and cultivation in a ligated ileal loop of a rabbit. Type 4 fimbriae of Vibrio cholerae O1 sensitive to D-glucose and D-mannose were suggested to be receptors for fs1 and fs2. The genome of fs1 was revealed to encode a potential new enterotoxin homologous to zonula occludens toxin. Clarification of the relation of type 4 fimbriae and these filamentous phages will provide a new understanding of the colonization of Vibrio-cholerae O1 and O139. Thus the presence of a new enterotoxin encoded by the genome of filamentous phage like fs1 may clarify the pathogenesis of cholera toxin negative clinical isolates of Vibrio cholerae O1 and non-O1. Our findings combined with the earlier report by Ehara et al. [Microbio. Immunol. 37 (1993) 679-688] suggest that type 4 fimbriae of Vibrio cholerae O1 are important for the development of an effective vaccine against cholera.

Molecular evolution of Vibrio pathogenicity island-2 (VPI-2): mosaic structure among Vibrio cholerae and Vibrio mimicus natural isolates

Vibrio cholerae is a Gram-negative rod that inhabits the aquatic environment and is the aetiological agent of cholera, a disease that is endemic in much of Southern Asia. The 57.3 kb Vibrio pathogenicity island-2 (VPI-2) is confined predominantly to toxigenic V. cholerae O1 and O139 serogroup isolates and encodes 52 ORFs (VC1758 to VC1809), which include homologues of an integrase (VC1758), a restriction modification system, a sialic acid metabolism gene cluster (VC1773-VC1783), a neuraminidase (VC1784) and a gene cluster that shows homology to Mu phage. In this study, a 14.1 kb region of VPI-2 comprising ORFs VC1773 to VC1787 was identified by PCR and Southern blot analyses in all 17 Vibrio mimicus isolates examined. The VPI-2 region in V. mimicus was inserted adjacent to a serine tRNA similar to VPI-2 in V. cholerae. In 11 of the 17 V. mimicus isolates examined, an additional 5.3 kb region encoding VC1758 and VC1804 to VC1809 was present adjacent to VC1787. The evolutionary history of VPI-2 was reconstructed by comparative analysis of the nanH (VC1784) gene tree with the species gene tree, deduced from the housekeeping gene malate dehydrogenase (mdh), among V. cholerae and V. mimicus isolates. Both gene trees showed an overall congruence; on both gene trees V. cholerae O1 and O139 serogroup isolates clustered together, whereas non-O1/non-O139 serogroup isolates formed separate divergent branches with similar clustering of strains within the branches. One exception was noted: on the mdh gene tree, V. mimicus sequences formed a distinct divergent lineage from V. cholerae sequences; however, on the nanH gene tree, V. mimicus clustered with V. cholerae non-O1/non-O139 isolates, suggesting horizontal transfer of this region between these species.

Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis

To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.

Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles.

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.

Prevalence of virulence-associated genes in clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 1999

Genes located on the CTX element and the Vibrio cholerae pathogenicity island (VPI) were investigated in 297 clinical V. cholerae O1 and 76 environmental O1 and non-O1 isolates from Brazil between 1991 and 1999. RAPD analysis suggested that serogroup O1 strains regardless of clinical or environmental source were clonal while non-O1 strains showed greater diversity. PCR analysis showed that 71% of O1 clinical isolates had a complete set of CTX element target genes ( ctxA, ctxB, zot and ace) and 68% a complete set of the VPI genes studied ( orf1, aldA, tagA, tcpA, toxT and int genes). The results also showed that 72.4% of environmental O1 isolates possessed ctxA, ctxB, zot and ace genes while environmental non-O1 strains rarely possessed virulence genes. Our data are consistent with the hypothesis that the CTX element and the VPI can have a mosaic structure in some V. cholerae strains, genotype diversity is due to the circulation of virulence genes which are more commonly found in O1 strains in Brazil. This study also shows that the aquatic environment is a potential source for virulence genes and toxigenic V. cholerae during epidemic periods.

Prevalence of virulence-associated genes in clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 1999.

Genes located on the CTX element and the Vibrio cholerae pathogenicity island (VPI) were investigated in 297 clinical V. cholerae O1 and 76 environmental O1 and non-O1 isolates from Brazil between 1991 and 1999. RAPD analysis suggested that serogroup O1 strains regardless of clinical or environmental source were clonal while non-O1 strains showed greater diversity. PCR analysis showed that 71% of O1 clinical isolates had a complete set of CTX element target genes (ctxA, ctxB, zot and ace) and 68% a complete set of the VPI genes studied (orf1, aldA, tagA, tcpA, toxT and int genes). The results also showed that 72.4% of environmental O1 isolates possessed ctxA, ctxB, zot and ace genes while environmental non-O1 strains rarely possessed virulence genes. Our data are consistent with the hypothesis that the CTX element and the VPI can have a mosaic structure in some V. cholerae strains, genotype diversity is due to the circulation of virulence genes which are more commonly found in O1 strains in Brazil. This study also shows that the aquatic environment is a potential source for virulence genes and toxigenic V. cholerae during epidemic periods.

Evidence for the emergence of non-O1 and non-O139 Vibrio cholerae strains with pathogenic potential by exchange of O-antigen biosynthesis regions.

The novel epidemic strain Vibrio cholerae O139 Bengal originated from a seventh-pandemic O1 El Tor strain by antigenic shift resulting from homologous recombination-mediated exchange of O-antigen biosynthesis (wb*) clusters. Conservation of the genetic organization of wb* regions seen in other serogroups raised the possibility of the existence of pathogenic non-O1 and non-O139 V. cholerae strains that emerged by similar events. To test this hypothesis, 300 V. cholerae isolates of non-O1 and non-O139 serogroups were screened for the presence of virulence genes and an epidemic genetic background by DNA dot blotting, IS1004 fingerprinting, and restriction fragment length polymorphism (RFLP) analysis. We found four non-O1 strains (serogroups O27, O37, O53, and O65) with an O1 genetic backbone suggesting exchange of wb* clusters. DNA sequence analysis of the O37 wb* region revealed that a novel approximately 23.4-kb gene cluster had replaced all but the approximately 4.2-kb right junction of the 22-kb O1 wbe region. In sharp contrast to the backbones, the virulence regions of the four strains were quite heterogeneous; the O53 and O65 strains had the El Tor vibrio pathogenicity island (VPI) cluster, the O37 strain had the classical VPI cluster, and the O27 strain had a novel VPI cluster. Two of the four strains carried CTXphi; the O27 strain possessed a CTXphi with a recently reported immune specificity (rstR-4** allele) and a novel ctxB allele, and the O37 strain had an El Tor CTXphi (rstR(ET) allele) and novel ctxAB alleles. Although the O53 and O65 strains lacked the ctxAB genes, they carried a pre-CTXphi (i.e., rstR(cla)). Identification of non-O1 and non-O139 serogroups with pathogenic potential in epidemic genetic backgrounds means that attention should be paid to possible future epidemics caused by these serogroups and to the need for new, rapid vaccine development strategies.

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.

Genetic diversity of clinical and environmental isolates of Vibrio cholerae determined by amplified fragment length polymorphism fingerprinting.

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.