Vibrio cholerae O139 persists in Dhaka, Bangladesh since 1993.

BACKGROUND: After a multi-country Asian outbreak of cholera due to Vibrio cholerae serogroup O139 which started in 1992, it is rarely detected from any country in Asia and has not been detected from patients in Africa. METHODOLOGY/PRINCIPAL FINDINGS: We extracted surveillance data from the Dhaka and Matlab Hospitals of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to review trends in isolation of Vibrio cholerae O139 in Bangladesh. Data from the Dhaka Hospital is a 2% sample of > 100,000 diarrhoeal patients treated annually. Data from the Matlab Hospital includes all diarrhoeal patients who hail from the villages included in the Matlab Health and Demographic Surveillance System. Vibrio cholerae O139 was first isolated in Dhaka in 1993 and had been isolated every year since then except for a gap between 2005 and 2008. An average of thirteen isolates was detected annually from the Dhaka Hospital during the last ten years, yielding an estimated 650 cases annually at this hospital. During the last ten years, cases due to serogroup O139 represented 0.47% of all cholera cases; the others being due to serogroup O1. No cases with serogroup O139 were identified at Matlab since 2006. Clinical signs and symptoms of cholera due to serogroup O139 were similar to cases due to serogroup O1 though more of the O139 cases were not dehydrated. Most isolates of O139 remained sensitive to tetracycline, ciprofloxacin, and azithromycin, but they became resistant to erythromycin starting in 2009. CONCLUSIONS/SIGNIFICANCE: Cholera due to Vibrio cholerae serogroup O139 continues to cause typical cholera in Dhaka, Bangladesh.

Generation and In Vivo Characterization of Tn5-Induced Biofilm Mutants of Vibrio cholerae O139.

The Gram-negative bacterium Vibrio cholerae is a unique pathogen with an ability to colonize human intestine as well as outside environments. The biofilm, an organized polymeric structure produced by this bacterium known to be a significant factor for the survival and persistence in hostile conditions. However, the direct role of biofilm formation by this bacterium in environmental persistence, in vivo colonization, and pathogenesis remains unexplored. In this study, we have generated biofilm-altered Tn5 mutants of V. cholerae O139 and evaluated their in vivo colonization ability on mouse model. These Tn5 mutants were found to harbor an independent, single Tn5 insertion in their genome. The DNA sequence analysis revealed that genomic region wherein Tn5 insertion occurred is identified to be involved in functions like LPS biosynthesis, efflux transporters, motility, purine metabolism, stringent response, VPS synthesis, and a hypothetical protein of unknown function. In single-strain infection with the planktonic culture, the biofilm-altered as well as the biofilm intermediate mutants were found to be more or less similar in their intestinal colonization ability, however infection with their biofilm form, a marked difference was observed between the biofilm deficient and other biofilm forming strains. Further, in the competition experiments, biofilm deficient and proficient mutants were found reduced in their colonization ability and outcompeted by their parent strain. In conclusion, biofilm formation in V. cholerae O139 is a genetically complex process and the controlled and regulated production of biofilm appeared to be necessary for its efficient colonization of mouse intestine.

Increased antibiotic resistance exhibited by the biofilm of Vibrio cholerae O139.

Background: Vibrio cholerae, the aetiological agent of the deadly diarrhoeal disease cholera, is known to form biofilm. The antibiotic susceptibility status of biofilm of V. cholerae O139, an important epidemic strain in India and other countries, has not previously been studied in detail. Methods: Antibiotic susceptibility status of planktonic and biofilm cultures of V. cholerae O139 was evaluated by determining MIC, MBC and minimum biofilm eradication concentration (MBEC) values of five different classes of antibiotics using established methods. Effects of antibiotic treatment on planktonic and biofilm cultures were analysed by scanning electron microscopy. The virulence of the antibiotic-surviving population (ASP) was evaluated using an infant mouse model. The frequency of spontaneous mutants and inheritability of antibiotic resistance were determined with standard methods. Results: The antibiotic resistance exhibited by biofilm of V. cholerae O139 was found to be significantly higher (P < 0.05) than its planktonic counterpart. The biofilm-associated antibiotic resistance was found to be transient and exclusive to the biofilm culture. The frequency of ASP clones among antibiotic-treated biofilm cultures occurred at a rate of 0.012%-0.95% and these clones were found to retain the virulence and antibiotic resistance of their parent strains. Conclusions: The biofilm of V. cholerae O139 was found to be resistant to different types of antibiotics tested. This unconventional biofilm resistance highlights the hidden danger of antimicrobial escape by V. cholerae, increased risk of cholera transmission and its continued persistence in the environment.

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.

Vibrio cholerae NspS, a homologue of ABC-type periplasmic solute binding proteins, facilitates transduction of polyamine signals independent of their transport.

The polyamines norspermidine and spermidine are among the environmental signals that regulate Vibrio cholerae biofilm formation. The effects of these polyamines are mediated by NspS, a member of the bacterial periplasmic solute binding protein superfamily. Almost all members of this superfamily characterized to date are components of ATP-binding cassette-type transporters involved in nutrient uptake. Consequently, in the current annotation of the V. cholerae genome, NspS has been assigned a function in transport. The objective of this study was to further characterize NspS and investigate its potential role in transport. Our results support a role for NspS in signal transduction in response to norspermidine and spermidine, but not their transport. In addition, we provide evidence that these polyamine signals are processed by c-di-GMP signalling networks in the cell. Furthermore, we present comparative genomics analyses which reveal the presence of NspS-like proteins in a variety of bacteria, suggesting that periplasmic ligand binding proteins may be widely utilized for sensory transduction.

Phenotypic and genotypic characterization Vibrio cholerae O139 of clinical and aquatic isolates in China.

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.

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 O139 requires neither capsule nor LPS O side chain to grow inside Acanthamoeba castellanii.

Vibrio cholerae, the causative agent of cholera, has the ability to grow and survive in the aquatic free-living amoeba Acanthamoeba castellanii. The aim of the present study was to examine the ability of the clinical isolate V. cholerae O139 MO10 to grow in A. castellanii and to determine the effect of the bacterial capsule and LPS O side chain on intracellular growth. Results from co-cultivation, viable counts, a gentamicin assay, electron microscopy and statistical analysis showed that the association of V. cholerae O139 MO10 with A. castellanii did not inhibit growth of the amoeba, and enhanced growth and survival of V. cholerae O139 MO10 occurred. The wild-type V. cholerae O139 MO10 and a capsule mutant or capsule/LPS double mutant grew inside A. castellanii. Neither the capsule nor the LPS O side chain of V. cholerae O139 was found to play an important role in the interaction with A. castellanii, disclosing the ability of V. cholerae to multiply and survive inside A. castellanii, as well as the role of A. castellanii as an environmental host for V. cholerae.

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.

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.

[Transition of Vibrio cholerae into the non-culturable state under the conditions of low temperature, different mineralization and illumination of artificial culture medium]. / Perekhod cholernykh vibrionov v nekul'tiviruemoe sostoianie v usloviiakh nizkoi temperatury, razlichnoi mineralizatsii i osveshchennosti iskusstvennoi sredy.

The importance of the combined influence of temperature, mineralization and illumination of the medium on the time of the transition of V. cholerae into the uncultivable state has been shown. The reversion of 5- to 60-day variants of uncultivable forms after the elevation of temperature to 20-22 degrees C has been obtained.

Cold shock response and major cold shock proteins of Vibrio cholerae.

When exponentially growing Vibrio cholerae cells were shifted from 37 degrees C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15 degrees C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspA(VC)) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspA(VC) and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspA(VC) and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37 degrees C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5' untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.

The Vibrio cholerae O139 O-antigen polysaccharide is essential for Ca2+-dependent biofilm development in sea water.

Vibrio cholerae is both an inhabitant of estuarine environments and the etiologic agent of the diarrheal disease cholera. Previous work has demonstrated that V. cholerae forms both an exopolysaccharide-dependent biofilm and a Ca2+-dependent biofilm. In this work, we demonstrate a role for the O-antigen polysaccharide of V. cholerae in Ca2+-dependent biofilm development in model and true sea water. Interestingly, V. cholerae biofilms, as well as the biofilms of several other Vibrio species, disintegrate when Ca2+ is removed from the bathing medium, suggesting that Ca2+ is interacting directly with the O-antigen polysaccharide. In the Bay of Bengal, cholera incidence has been correlated with increased sea surface height. Because of the low altitude of this region, increases in sea surface height are likely to lead to transport of sea water, marine particulates, and marine biofilms into fresh water environments. Because fresh water is Ca2+-poor, our results suggest that one potential outcome of an increase is sea surface height is the dispersal of marine biofilms with an attendant increase in planktonic marine bacteria such as V. cholerae. Such a phenomenon may contribute to the correlation of increased sea surface height with cholera.