Deletion of gene encoding the nucleoid-associated protein H-NS unmasks hidden regulatory connections in El Tor biotype Vibrio cholerae.

Hypervirulent atypical El Tor biotype Vibrio cholerae O1 isolates harbour mutations in the DNA-binding domain of the nucleoid-associated protein H-NS and the receiver domain of the response regulator VieA. Here, we provide two examples in which inactivation of H-NS in El Tor biotype vibrios unmasks hidden regulatory connections. First, deletion of the helix-turn-helix domain of VieA in an hns mutant background diminished biofilm formation and exopolysaccharide gene expression, a function that phenotypically opposes its phosphodiesterase activity. Second, deletion of vieA in an hns mutant diminished the expression of σE, a virulence determinant that mediates the envelope stress response. hns mutants were highly sensitive to envelope stressors compared to wild-type. However, deletion of vieA in the hns mutant restored or exceeded wild-type resistance. These findings suggest an evolutionary path for the emergence of hypervirulent strains starting from nucleotide sequence diversification affecting the interaction of H-NS with DNA.

Adherence to Intestinal Cells Promotes Biofilm Formation in Vibrio cholerae.

Vibrio cholerae, the etiological agent of cholera, is known to form biofilms to persist in the environment. It is demonstrated here that even during infection, biofilm genes are upregulated, and microscopic observation indicated that biofilm formation is initiated almost immediately after adherence of V. cholerae to intestinal cells. About 7-fold upregulation of the biofilm regulatory gene vpsT was observed within 30 minutes of adherence of V. cholerae to the intestinal cell line INT 407, and a massive induction of about 700-fold was observed in rabbit ileal loops. The upregulation was observed in the classical and El Tor biotype strains of serogroup O1 that is most frequently associated with epidemic cholera. vpsT upregulation was primarily dependent on the virulence master regulator AphA. Of possible clinical relevance was the observation that V. cholerae in the INT 407-associated biofilms was significantly more resistant to antibiotics than unadhered planktonic cells.

An in silico, in vitro and in vivo investigation of indole-3-carboxaldehyde identified from the seawater bacterium Marinomonas sp. as an anti-biofilm agent against Vibrio cholerae O1.

Biofilm formation is a major contributing factor in the pathogenesis of Vibrio cholerae O1 (VCO1) and therefore preventing biofilm formation could be an effective alternative strategy for controlling cholera. The present study was designed to explore seawater bacteria as a source of anti-biofilm agents against VCO1. Indole-3-carboxaldehyde (I3C) was identified as an active principle component in Marinomonas sp., which efficiently inhibited biofilm formation by VCO1 without any selection pressure. Furthermore, I3C applications also resulted in considerable collapsing of preformed pellicles. Real-time PCR studies revealed the down-regulation of virulence gene expression by modulation of the quorum-sensing pathway and enhancement of protease production, which was further confirmed by phenotypic assays. Furthermore, I3C increased the survival rate of Caenorhabditis elegans when infected with VCO1 by significantly reducing in vivo biofilm formation, which was corroborated by a survivability assay. Thus, this study revealed, for the first time, the potential of I3C as an anti-biofilm agent against VCO1.

Survival of Vibrio cholerae O1 on fomites.

It is well established that the contamination sources of cholera causing bacteria, Vibrio cholerae, are water and food, but little is known about the transmission role of the fomites (surfaces that can carry pathogens) commonly used in households. In the absence of appropriate nutrients or growth conditions on fomites, bacteria have been known to assume a viable but non-culturable (VBNC) state after a given period of time. To investigate whether and when V. cholerae O1 assumes such a state, this study investigated the survival and viable quantification on a range of fomites such as paper, wood, glass, plastic, cloth and several types of metals under laboratory conditions. The fomites were inoculated with an outbreak strain of V. cholerae and its culturability was examined by drop plate count method at 30 min intervals for up to 6 h. For molecular detection, the viable/dead stain ethidium monoazide (EMA) which inhibits amplification of DNA from dead cells was used in combination with real-time polymerase chain reaction (EMA-qPCR) for direct quantitative analyses of viable V. cholerae at 2, 4, 6, 24 h and 7 day time intervals. Results showed that V. cholerae on glass and aluminum surfaces lost culturability within one hour after inoculation but remained culturable on cloth and wood for up to four hours. VBNC V. cholerae on dry fomite surfaces was detected and quantified by EMA-qPCR even 7 days after inoculation. In conclusion, the prolonged survival of V. cholerae on various household fomites may play vital role in cholera transmission and needs to be further investigated.

Stepwise changes in viable but nonculturable Vibrio cholerae cells.

Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.

Elevated concentrations of polymyxin B elicit a biofilm-specific resistance mechanism in Vibrio cholerae.

Vibrio cholerae can form biofilms in the aquatic environment and in the human intestine, facilitating the release of hyper-infectious aggregates. Due to the increasing antibiotic resistance, alternatives need to be found. One of these alternatives is antimicrobial peptides, including polymyxin B (PmB). In this study, we first investigated the resistance of V. cholerae O1 El Tor strain A1552 to various antimicrobials under aerobic and anaerobic conditions. An increased resistance to PmB is observed in anaerobiosis, with a 3-fold increase in the dose required for 50 % growth inhibition. We then studied the impact of the PmB on the formation and the degradation of V. cholerae biofilms to PmB. Our results show that PmB affects more efficiently biofilm formation under anaerobic conditions. On the other hand, preformed biofilms are susceptible to degradation by PmB at concentrations close to the minimal inhibitory concentration. At higher concentrations, we observe an opacification of the biofilm structures within 20 min post-treatment, suggesting a densification of the structure. This densification does not seem to result from the overexpression of matrix genes but rather from DNA release through massive cell lysis, likely forming a protective shield that limits the penetration of the PmB into the biofilm.

Multi-drug resistant toxigenic Vibrio cholerae O1 is persistent in water sources in New Bell-Douala, Cameroon.

BACKGROUND: Cholera has been endemic in Douala, since 1971 when it was first recorded in Cameroon. Outbreaks have often started in slum areas of the city including New Bell. Despite the devastating nature of outbreaks, always resulting in high mortality and morbidity, a paucity of information exists on the reservoirs of the causative agent, V. cholerae, and factors maintaining its persistence. This has complicated disease prevention, resulting in frequent outbreaks of cholera. We investigated water sources in New Bell for contamination with V. cholerae O1 with pathogenic potential, to highlight their role in disease transmission. Antibiotic susceptibility pattern of isolates and the environmental factors maintaining its persistence were investigated. METHOD: Water samples from various sources (taps, dug wells, streams) were analyzed for contamination with V. cholerae O1 using standard methods. Antibiotic susceptibility was determined by disc diffusion method. Pathogenic potential of isolates was determined by analyzing for genes for cholera toxin (ctx), toxin co-regulated pilus (tcpA), and zonula occludens toxin (zot) by PCR. Physico-chemical characteristics of water (pH, temperature and salinity) were investigated using standard methods. The Spearman's Rank correlation was used to analyze the relationship between physico-chemical factors and the occurrence of V. cholerae O1. Differences were considered significant at P≤0.05. RESULTS: Twenty-five V. cholerae O1 strains were isolated from stream and well samples in both dry and rainy seasons. Twenty-three (92%) isolates were multidrug resistant. All isolates had genes for at least one virulence factor. Cholera toxin gene was detected in 7 isolates. Of the 15 isolates positive for tcpA gene, two had Classical type tcpA while 13 had tcpA El Tor. All tcpA Classical positive isolates were positive for ctx gene. Isolates were grouped into nine genotypes based on the genes analyzed. pH and salinity significantly correlated with isolation of V. cholerae O1. CONCLUSION: Multidrug resistant Vibrio cholerae O1 with pathogenic potential is present in some wells and streams in study area. pH and salinity are among the factors maintaining the persistence of the organism. Findings indicate an urgent need for potable water supply in study area and in addition, regular disinfection of water from contaminated sources to prevent outbreak of cholera.

Viability kinetics, induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio cholerae O1 in freshwater microcosm.

AIM: To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4°C) and starvation. METHODS AND RESULTS: Vibrio cholerae O1 cells were inoculated in freshwater microcosm and incubated at 4°C. The cells became coccoid, rugose and subsequently nonculturable by day 16 on tryptic soy agar (TSA) and by day 23 on TSA-SP, while 87 and 65% of the cells retained their membrane integrity, respectively. Viable cells were observed until day 30 using direct fluorescent antibody-direct viable count method. In vitro resuscitation was demonstrated by temperature upshift. Utilizing 16S rRNA as an endogenous control, the DNA pol II (27·43-fold), fliG (12·44-fold), ABC transporter (27·11-fold), relA (60·76-fold) and flaC (15·29-fold) were significantly up-regulated in VBNC cells, while the expression of fadL-3 was comparable. The expression of DNA pol II, fliG, ABC transporter, relA and flaC was 3·3, 1·1, 5·9, 5·8 and 1·2-fold, respectively, for resuscitated cells. VBNC cells were found to be virulent, as ctxA and tcpA were expressed. CONCLUSIONS: Vibrio cholerae undergoes both phenotypic alteration and genotypic modulation to protect itself from stress in freshwater. SIGNIFICANCE AND IMPACT OF THE STUDY: Induction and resuscitation of the VBNC state in freshwater is important for an understanding of the epidemiology of cholera in the freshwater environment.

Diarrheagenic pathogens in polymicrobial infections.

During systematic active surveillance of the causes of diarrhea in patients admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata, India, we looked for 26 known gastrointestinal pathogens in fecal samples from 2,748 patients. Samples from about one-third (29%) of the patients contained multiple pathogens. Polymicrobial infections frequently contained Vibrio cholerae O1 and rotavirus. When these agents were present, some co-infecting agents were found significantly less often (p = 10 (-5) to 10 (-33), some were detected significantly more often (p = 10 (-5) to 10 (-26), and others were detected equally as often as when V. cholerae O1 or rotavirus was absent. When data were stratified by patient age and season, many nonrandom associations remained statistically significant. The causes and effects of these nonrandom associations remain unknown.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

Cholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred. IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

The cyclic AMP (cAMP)-cAMP receptor protein signaling system mediates resistance of Vibrio cholerae O1 strains to multiple environmental bacteriophages.

Toxigenic Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, interacts with diverse environmental bacteriophages. These interactions promote genetic diversity or cause selective enrichment of phage-resistant bacterial clones. To identify bacterial genes involved in mediating the phage-resistant phenotype, we screened a transposon insertion library of V. cholerae O1 El Tor biotype strain C6706 to identify mutants showing altered susceptibility to a panel of phages isolated from surface waters in Bangladesh. Mutants with insertion in cyaA or crp genes encoding adenylate cyclase or cyclic AMP (cAMP) receptor protein (CRP), respectively, were susceptible to a phage designated JSF9 to which the parent strain was completely resistant. Application of the cyaA mutant as an indicator strain in environmental phage monitoring enhanced phage detection, and we identified 3 additional phages to which the parent strain was resistant. Incorporation of the cyaA or crp mutations into other V. cholerae O1 strains caused similar alterations in their phage susceptibility patterns, and the susceptibility correlated with the ability of the bacteria to adsorb these phages. Our results suggest that cAMP-CRP-mediated downregulation of phage adsorption may contribute to a mechanism for the V. cholerae O1 strains to survive predation by multiple environmental phages. Furthermore, the cyaA or crp mutant strains may be used as suitable indicators in monitoring cholera phages in the water.

Spatial dynamics and the basic reproduction number of the 1991-1997 Cholera epidemic in Peru.

After being cholera free for over 100 years, Peru experienced an unprecedented epidemic of Vibrio cholerae O1 that began in 1991 and generated multiple waves of disease over several years. We developed a mechanistic transmission model that accounts for seasonal variation in temperature to estimate spatial variability in the basic reproduction number ([Formula: see text]), the initial concentration of vibrios in the environment, and cholera reporting rates. From 1991-1997, cholera spread following a multi-wave pattern, with weekly incidence concentrated during warm seasons. The epidemic first hit the coastal departments of Peru and subsequently spread through the highlands and jungle regions. The correlation between model predictions and observations was high (range in R2: 58% to 97%). Department-level population size and elevation explained significant variation in spatial-temporal transmission patterns. The overall R0 across departments was estimated at 2.1 (95% CI: 0.8,7.3), high enough for sustained transmission. Geographic-region level [Formula: see text] varied substantially from 2.4 (95% CI: 1.1, 7.3) for the coastal region, 1.9 (0.7, 6.4) for the jungle region, and 1.5 (0.9, 2.2) for the highlands region. At the department level, mean [Formula: see text] ranged from 0.8 to 6.9. Department-level [Formula: see text] were correlated with overall observed attack rates (Spearman ρ = 0.59, P = 0.002), elevation (ρ = -0.4, P = 0.04), and longitude (ρ = -0.6, P = 0.004). We find that both [Formula: see text] and the initial concentration of vibrios were higher in coastal departments than other departments. Reporting rates were low, consistent with a substantial fraction of asymptomatic or mild cases associated with the El Tor cholera biotype. Our results suggest that cholera vibrios, autochthonous to plankton in the natural aquatic environment, may have triggered outbreaks in multiple coastal locations along the Pacific coast of Peru. Our methodology could be useful to investigate multi-wave epidemics of cholera and could be extended to conduct near real-time forecasts and investigate the impact of vaccination strategies.

Changing Susceptibility Pattern of Vibrio cholerae O1 Isolates to Commonly Used Antibiotics in the Largest Diarrheal Disease Hospital in Bangladesh during 2000-2018.

The efficacy of commonly used antibiotics for treating severe cholera has been compromised over time because of the reduced antibiotic susceptibility. This study aimed to describe the rate of detection of Vibrio cholerae O1 from fecal samples and antimicrobial susceptibility profiles of V. cholerae O1 serotypes to commonly used antibiotics. During January 2000-December 2018, V. cholerae O1 was detected in fecal samples of 7,472 patients. Vibrio cholerae O1 Inaba serotype was predominant, ranging from 60% to 86% during the period 2000-2006 except for 2003 and 2005 when the Ogawa serotype was predominant. Later on, the Ogawa serotype became predominant from 2007 to 2015, fluctuating between 52% and 100%. However, in 2016 and 2017, isolation rates declined to 2% and 1%, respectively, but surged again to 75% in 2018. Nearly 100% of V. cholerae O1 strains were sensitive to tetracycline during 2000-2004. Thereafter, a declining trend of sensitivity was observed to be continued and dropped down to < 6% during 2012-2017 and again increased to 76% in 2018. Susceptibility to azithromycin and ciprofloxacin was nearly 100%, and susceptibility to cotrimoxazole and furazolidone was 01% throughout the study period. We also found the emergence of resistance to erythromycin in 2005 and sensitivity to cotrimoxazole in 2018. Thus, the rapid decline of the sensitivity of V. cholerae O1 to tetracycline and a reversed peak after 6 years need continued monitoring and reporting.

Molecular diversification in the quorum-sensing system of Vibrio cholerae: Role of natural selection in the emergence of pandemic strains.

Two haplotypes of the Vibrio cholerae quorum-sensing system regulator hapR are described: hapR1, common among nonpandemic, non-O1, non-O139 strains, and hapR2, associated with pandemic O1 and O139 and epidemic O37 V. cholerae strains. The hapR2 has evolved under strong natural selection, implying that its fixation was influenced by conditions that led to cholera pandemics.

Vibrio cholerae O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant.

Retrospective analysis led to the detection of two Vibrio cholerae variant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml(-1)) resistance, positivity in Voges-Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to Kolkata V. cholerae O1 strains isolated on or before 1992. NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.

A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India.

A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes - ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot - in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXPhi. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.

Flagella-dependent inhibition of biofilm formation by sub-inhibitory concentration of polymyxin B in Vibrio cholerae.

Biofilm formation is a common strategy used by bacteria in order to survive and persist in the environment. In Vibrio cholerae (V. cholerae), a Gram-negative pathogen responsible for the cholera disease, biofilm-like aggregates are important for the pathogenesis and disease transmission. Biofilm formation is initiated by the attachment of the bacteria to a surface, followed by maturation stages involving the formation of a biofilm matrix. In V. cholerae, flagella are essential for the initial step of biofilm formation, allowing the bacteria to swim and to detect a surface. In this study, we explored the effect of polymyxin B (PmB), a cationic bacterial antimicrobial peptide, on biofilm formation in pathogenic V. cholerae strains belonging to the O1 and O139 serotypes. We found that sub-inhibitory concentration of PmB induces a reduction of the biofilm formation by V. cholerae O1 and O139. Experiment on preformed biofilm demonstrated that the biofilm formation inhibition occurs at the initial step of biofilm formation, where the flagella are essential. We further characterize the effect of PmB on V. cholerae flagellation. Our results demonstrate that the flagellin expression is not reduced in presence of sub-inhibitory concentration of PmB. However, a decrease of the abundance of flagellin associated with the bacterial cells together with an increase in the secretome was observed. Electron microscopy observations also suggest that the abundance of aflagellated bacteria increases upon PmB supplementation. Finally, in agreement with the effect on the flagellation, a reduction of the bacterial motility is observed. Altogether, our results suggest that the PmB affect V. cholerae flagella resulting in a decrease of the motility and a compromised ability to form biofilm.

Vibrio cholerae RND family efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse small intestine.

Vibrio cholerae is a gram-negative human intestinal pathogen that causes the diarrheal disease cholera. Humans acquire cholera by ingesting V. cholerae-contaminated food or water. Upon ingestion, V. cholerae encounters several barriers to colonization, including bile acid toxicity and antimicrobial products of the innate immune system. In many gram-negative bacteria, resistance to the antimicrobial effects of these products is mediated by RND (resistance-nodulation-division) family efflux systems. In this study we tested the hypothesis that the V. cholerae RND efflux systems are required for antimicrobial resistance and virulence. The six V. cholerae genes encoding RND efflux pumps were deleted from the genome of the O1 El Tor strain N16961, resulting in the generation of 14 independent RND deletion mutants, including one RND-null strain. Determination of the antimicrobial susceptibilities of the mutants revealed that the RND efflux systems were responsible for resistance to multiple antimicrobial compounds, including bile acids, antimicrobial peptides, and antibiotics. VexB (VC0164) was found to be the RND efflux pump primarily responsible for the resistance of V. cholerae to multiple antimicrobial compounds in vitro. In contrast, VexD (VC1757) and VexK (VC1673) encoded efflux pumps with detergent-specific substrate specificities that were redundant with VexB. A strain lacking VexB, VexD, and VexK was attenuated in the infant mouse model, and its virulence factor production was unaffected. In contrast, a V. cholerae RND-null strain produced significantly less cholera toxin and fewer toxin-coregulated pili than the wild type and was unable to colonize the infant mouse. The decreased virulence factor production in the RND-null strain was linked to reduced transcription of tcpP and toxT. Our findings show that the V. cholerae RND efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse.

Association of ompU gene in Vibrio cholerae from patients and environment with bile resistance.

The objective of this study was to determine whether Vibrio cholerae, possessing ompU isolated from patients and the environment, conferred bile resistance and whether other virulence genes were also related to bile resistance. Fifty-two V cholerae O1 and non-O1 isolates were examined by PCR for the presence of the virulence-associated and regulatory genes, ctxA, tcpA, zot, ace, ompU, toxR, hlyA and stn/sto. V. cholerae possessing ompU resistant to equal or greater than 10% sodium deoxycholate were found in 93% of isolates but only in 9% of V. cholerae isolates not possessing ompU. The effects of other virulence genes on bile resistance could not be ascertained in this study. Thus V cholerae non-O1 with ompU and possibly other virulence genes isolated from the environment have the potential of affecting public health.

Phenotypic and genotypic traits and epidemiological implication of Vibrio cholerae O1 and O139 strains in India during 2003.

During 2003, Vibrio cholerae O1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identical wbeT region among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTX(El), CTX(Cal) and truncated CTX(Cal) (devoid of ctxAB) prophages was unique among the majority of these O139 strains.