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.

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.

[TYPING OF VIBRIO CHOLERAE NON O1/NON O139 STRAINS, ISOLATED IN ROSTOV REGION IN 2014].

AIM: Comparative study of antibiotics resistance and VNTR-typing of Vibrio cholerae non O1/ non O139 strains, isolated on the territory of Rostov region in 2014. MATERIALS AND METHODS: Antibioticogramms of strains were determined by serial dilution method in dense nutrient medium according to MG 4.2.2495-09 (2009). Pheno-, sero- and VNTR-typing was carried out by conventional-methods. RESULTS: The studied strains belonged to V. cholerae species, did not agglutinate with O1 and O139 sera, were atoxigenic hemolysis-positive, did not contain genes of cholera toxin and toxin-coregulating pili of adhesion, contained genes of hemagglutinin/protease, protease PrtV, collagenase, cytotonic factor Cef, outer membrane protein-OmpW, tol- and -vps-clusters, regulatory genes toxR and hapR. Antibioticogramms of the strains have shown the presence of cultures, resistant to ampicillin, ceftazidime-furazolidone, trimethoprim/sulfamethoxazole with intermediate resistance to streptomycin, kanamycin, gentamycin, amikacin, netilmicin, Approximately 20% of isolates had multiple drug resistance. Data of VNTR- and genotyping confirmed a possibility of water transmission route of the infection. CONCLUSION: Execution of monitoring of cultures from environmental samples is necessary for timely detection of genetic characteristics, antibiotics resistance.

IncA/C plasmids harboured in serious multidrug-resistant Vibrio cholerae serogroup O139 strains in China.

Vibrio cholerae serogroup O139 emerged in 1992 and is one of two major serogroups to have caused cholera epidemics. After 1998, serious multidrug-resistant (MDR) O139 strains quickly became common in China, showing a multidrug resistance profile to eight antibiotics. It is a great threat to public health, and elucidation of its mechanisms of resistance will provide a helpful guide for the clinical treatment and prevention of cholera. In this study, mega-plasmids from MDR V. cholerae O139 strains were identified by pulsed-field gel electrophoresis (PFGE) without enzyme digestion. One plasmid was isolated and sequenced, belonging to the IncA/C family. Ten antibiotic resistance genes were found in the MDR regions, including a blaTEM-20 gene, and these genes endowed the host with resistance to seven antibiotics. This kind of plasmid was positive in 71.2% (198/278) of toxigenic O139 strains, and the rate of plasmid positivity was consistent with the yearly change in MDR rates of these strains. This study reveals an important role of the IncA/C family plasmid in the spread of multiple antibiotic resistance of epidemic V. cholerae serogroup O139 strains, which has recombined with plasmids from different bacterial species and transferred among V. cholerae strains.

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.

Accumulation of mutations in DNA gyrase and topoisomerase IV genes contributes to fluoroquinolone resistance in Vibrio cholerae O139 strains.

High resistance rates to nalidixic acid (NAL) in Vibrio cholerae serogroup O139 strains have been found, and ciprofloxacin (CIP) resistance is also observed. In this study, mutations within the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV from NAL-resistant O139 strains were analysed. The predominant mutation profile was S83I in GyrA in combination with S85L in ParC. In addition, the combination substitutions of D87N in GyrA and D420N in ParE in combination with S83I in GyrA and S85L in ParC as well as D87N in GyrA and P439S in ParE in combination with S83I in GyrA and S85L in ParC were found in the CIP-resistant strains. A series of site-directed mutants comprising D87 in GyrA, D420 in ParE and P439 in ParE were constructed from a wild-type V. cholerae O139 strain carrying the common mutations S83I in GyrA and S85L in ParC. Introduction of the mutation D87N in GyrA increased the CIP minimum inhibitory concentration (MIC) of the mutant strain by nearly 4-fold compared with the initial strain. The second introduction of D420N in ParE further significantly increased the CIP MIC to ca. 23-fold compared with the initial strain. A second introduction of P439S in ParE also increased the CIP MIC by 17-fold. Therefore, it is concluded that the emergence of D87N in GyrA and D420N or P439S in ParE dramatically induces resistance to fluoroquinolones in V. cholerae O139, and the accumulation of multiple mutations in the QRDRs confers significant resistance to fluoroquinolones in V. cholerae.

Prevalence and molecular characterization of Vibrio cholerae O1, non-O1 and non-O139 in tropical seafood in Cochin, India.

The objective of this study was to determine the prevalence of O1, O139, and non-O1 and non-O139 Vibrio cholerae, which were associated with fresh and raw seafood samples harvested from Cochin, India waters during 2009-2011. Results from V. cholerae-specific biochemical, molecular, and serological assays identified five El Tor V. cholerae O1 Ogawa strains and 377 non-O1, non-O139 V. cholerae strains from 265 seafood samples. V. cholerae O139 strains were not isolated. Polymerase chain reaction assays confirmed the presence of V. cholerae O1 El Tor biotype in seafood. Antibiotic susceptibility analysis revealed that the V. cholerae O1 strains were pansusceptible to 20 test antibiotics, whereas 26%, 40%, 62%, and 84% of the non-O1, non-O139 V. cholerae strains were resistant to cefpodoxime, ticarcillin, augmentin, and colistin, respectively. Detection of virulence and regulatory genes in V. cholerae associated with seafood revealed the presence of virulence and regulatory genes (i.e., ctx, zot, ace, toxR genes) in V. cholerae O1 strains, nevertheless, presence of ace and toxR genes were detected in non-O1, non-O139 in 9.8 and 91% strains, respectively. In conclusion, the presence of pathogenic V. cholerae in seafood harvested from local Cochin waters warrants the introduction of a postharvest seafood monitoring program, which will lead to a greater understanding of the distribution, abundance, and virulence of diverse pathogenic Vibrio populations that inhabit these different coastal regions so that a risk management program can be established.

Multiple antibiotic resistance of Vibrio cholerae serogroup O139 in China from 1993 to 2009.

Regarded as an emerging diarrheal micropathogen, Vibrio cholerae serogroup O139 was first identified in 1992 and has become an important cause of cholera epidemics over the last two decades. O139 strains have been continually isolated since O139 cholera appeared in China in 1993, from sporadic cases and dispersed foodborne outbreaks, which are the common epidemic types of O139 cholera in China. Antibiotic resistance profiles of these epidemic strains are required for development of clinical treatments, epidemiological studies and disease control. In this study, a comprehensive investigation of the antibiotic resistance of V. cholerae O139 strains isolated in China from 1993 to 2009 was conducted. The initial O139 isolates were resistant to streptomycin, trimethoprim-sulfamethoxazole and polymyxin B only, while multidrug resistance increased suddenly and became common in strains isolated after 1998. Different resistance profiles were observed in the isolates from different years. In contrast, most V. cholerae O1 strains isolated in the same period were much less resistant to these antibiotics and no obvious multidrug resistance patterns were detected. Most of the non-toxigenic strains isolated from the environment and seafood were resistant to four antibiotics or fewer, although a few multidrug resistant strains were also identified. These toxigenic O139 strains exhibited a high prevalence of the class I integron and the SXT element, which were rare in the non-toxigenic strains. Molecular subtyping of O139 strains showed highly diverse pulsed-field gel electrophoresis patterns, which may correspond to the epidemic state of sporadic cases and small-scale outbreaks and complex resistance patterns. Severe multidrug resistance, even resistance transfers based on mobile antibiotic resistance elements, increases the probability of O139 cholera as a threat to public health. Therefore, continual epidemiological and antibiotic sensitivity surveillance should focus on the occurrence of multidrug resistance and frequent microbial population shifts in O139 strains.

Characterization of Vibrio cholerae O139 belonging to multiple ribotypes and isolated from diarrhoeal patients in Kerala, southern India.

Twenty-four Vibrio cholerae O139 strains isolated from Kerala, southern India were characterized by PCR, CTX typing and ribotyping; all of which, except three strains, carried the core of the CTX genetic element, colonization-toxin co-regulated pilus, the adherence outer membrane protein, haemolysin, central regulatory protein encoded toxR, SXT genetic element, and produced cholera toxin and biofilm. Results of RFLP analysis revealed twenty-one of the O139 strains possess two copies of CTXΦ and pre-CTXФ always preceded by tandemly arranged RS1 element; one had two copies of pre-CTXΦ and two a single copy of pre-CTXΦ. Nucleotide sequencing detected classical ctxB in CTX(ET)Φ and CTX(Calc)Ф with additional change at 28th amino acid position of CTX(Calc)Ф. Ribotype analysis revealed the presence of multiple ribotypes, including B-I and B-II, and new ribotypes designated B-VIIIa, B-VIIIb and B-IX, not reported earlier among V. cholerae O139 strains. These observations thus indicate that genetic recombination or mutations had occurred in conserved rrn operon and variations in CTXΦ may have implications on the evolution of the organism.

[In vitro induction of transmissive resistance to tetracycline, chloramphenicol and ampicillin chloramphenicol in Vibrio cholera non-O1/non-O139 serogroups isolated within 1990-2005].

Inducible character of resistance to tetracycline, chloramphenicol and ampicillin was investigated in 20 strains of Vibrio cholera non-O1/non-O139 serogroups isolated from inhabitants of Uzbekistan in 1990 (10 strains, ctx+) and in 2001 (5 strains, ctx-) and from inhabitants of Kalmykiya within 2003-2005 (5 strains, ctx-). Eight of the 20 isolates showed not only capacity for induction of the antibiotic resistance, but also its possible self transfer to Escherichia coli and reverse crosses in El Tor V. cholerae P-5879. It was shown that the effect of the antibacterial on the isolates phenotypic susceptibility could increase the resistance markers expression, when the genomes contained sites responsible for their expression, that required constant bacteriological control of the treatment efficacy and the use of the isolates antibioticograms for early replace of the inefficient drug by the efficient one. The prevalence of V. cholerae O1 and non-O1/non-O13 serogroups with multiple resistance to the antibacterial and the genetic potency for the antibiotic resistance development in the pathogen made difficult the choice of efficient drugs for prophylaxis and treatment of diseases caused by V. cholerae.

Effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains.

We examined the effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains. We found that one strain of V. cholerae O139 VO146P showed loss of the complete phage array, and other strain VO170P showed partial loss of the phage array giving rise to altered strains designated as VO146N and VO170N. Results of PCR and RFLP analysis revealed that both strains (VO146P and VO170P) possessed a single copy of pre-CTX(ET)Phi and two copies of CTXPhi comprising CTX(Class)Phi and CTX(Calc)Phi arranged in tandem, and integrated in the large chromosome. The presence of classical ctxB was detected in CTX(Calc)Phi of both V. cholerae O139 strains. Nucleotide sequencing of three housekeeping genes showed no difference between parent and altered strains of V. cholerae O139.

Establishment of an adult mouse model for direct evaluation of the efficacy of vaccines against Vibrio cholerae.

We describe here a new animal model that offers the prospect of using conventional adult mice for direct evaluation of the protective potential of new cholera vaccines. Pretreatment of adult mice with oral streptomycin allowed intestinal colonization by streptomycin-resistant Vibrio cholerae strains of either the O1 or the O139 serogroup. Bacteria were recovered in greatest numbers from the cecum and large intestine, but recoveries from all regions of the gut correlated significantly with bacterial excretion in fresh fecal pellets, which thus provides a convenient indicator of the extent and duration of gut colonization. Mice immunized mucosally or systemically with viable or inactivated V. cholerae were shown to be comparatively refractory to colonization after challenge with the immunizing strain. Several variables were examined to optimize the model, the most significant being the size of the challenge inoculum; surprisingly, a smaller challenge dose resulted in more consistent and sustained colonization. Studies with mutant strains unable to produce cholera toxin or toxin-coregulated pili revealed that neither factor contributed significantly to colonization potential. Protection against V. cholerae challenge was shown to be serogroup restricted, and significant inverse correlations were detected between serum and intestinal anti-lipopolysaccharide antibody responses and the levels of excretion of challenge organisms.

Changing characteristics of Vibrio cholerae: emergence of multidrug resistance and non-O1, non-O139 serogroups.

The serogroups and antimicrobial susceptibility patterns of V. cholerae isolated in Hubli, India during the years 2000 to 2004 were monitored. A total of 256 V. cholerae isolates were obtained during the study period, of which 129 (50.4%) belonged to serogroup O1 while the O139 and non-O1, non-O139 serogroups constituted 61 (23.8%) and 66 (25.8%) isolates, respectively. V. cholerae O1 Ogawa was the predominant isolate during the first 2 years of the study. However, this was replaced by V. cholerae non-O1, non-O139 serogroups in the following years. The V. cholerae, which was susceptible to most enteric antimicrobials in 2000, was found to be multidrug resistant in subsequent years, with the development of fluroquinolone resistance since 2002. Surveillance of the epidemiological and microbiological characteristics of V. cholerae provides useful information for managing cholera cases. The V. cholerae non-O1, non-O139 serogroups coupled with multiple antimicrobial resistance may form a group of emerging diarrheal pathogens in the tropics.

Incidence of bacterial enteropathogens among hospitalized diarrhea patients from Orissa, India.

Bacteriological analysis of 1,551 stool/rectal swabs from all age groups of diarrhea patients of different hospitals of Orissa from January 2004 to December 2006 was carried out using standard procedures. Among all enteropathogens isolated in 886 culture-positive samples, Escherichia coli constituted 75.5%, including 13.2% pathogenic E. coli; Vibrio cholerae O1 constituted 17.3%; V. cholerae O139, 1%; Shigella spp., 4.5% (Shigella flexneri type 6, 2.9%, S. dysenteriae type I, 0.7%, S. sonnei, 0.6%, and S. boydii, 0.3%); Salmonella spp., 0.7%; and Aeromonas spp., only 2.0%. The isolation of bacterial enteropathogens was highest during July, 2005, followed by September, 2006. The prevalence of shigellosis in this region was relatively low. Cholera cases were more frequent during the rainy seasons. The dominance of V. cholerae O1 Inaba over Ogawa serotypes was observed in 2005, whereas this trend was reversed in 2006. The resistance profile of V. cholerae O1 was co-trimoxazole (Co), furazolidone (Fr), and nalidixic acid (Na); for Aeromonas spp., it was ampicillin (A), Fr, ciprofloxacin (Cf), Na, norfloxacin (Nx), and Co. Pathogenic E. coli strains were resistant to A, Fr, Co, streptomycin (S), Cf, Na, Nx, and neomycin (N); Shigella spp. were resistant to Fr, Na, Co, and S; and Salmonella spp. were resistant to A and Fr. Active surveillance should be continued among diarrhea patients to look for different enteropathogens and to define the shifting antibiogram patterns in this region.

Vibrio cholerae O139 multiple-drug resistance mediated by Yersinia pestis pIP1202-like conjugative plasmids.

A conjugative plasmid, pMRV150, which mediated multiple-drug resistance (MDR) to at least six antibiotics, including ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was identified in a Vibrio cholerae O139 isolate from Hangzhou, eastern China, in 2004. According to partial pMRV150 DNA sequences covering 15 backbone regions, the plasmid is most similar to pIP1202, an IncA/C plasmid in an MDR Yersinia pestis isolate from a Madagascar bubonic plague patient, at an identity of 99.99% (22,180/22,183 nucleotides). pMRV150-like plasmids were found in only 7.69% (1/13) of the O139 isolates tested during the early period of the O139 epidemic in Hangzhou (1994, 1996, and 1997); then the frequency increased gradually from 60.00% (3/5) during 1998 and 1999 to 92.16% (47/51) during 2000 to 2006. Most (42/51) of the O139 isolates bearing pMRV150-like plasmids were resistant to five to six antibiotics, whereas the plasmid-negative isolates were resistant only to one to three antibiotics. In 12 plasmid-bearing O139 isolates tested, the pMRV150-like plasmids ranged from approximately 140 kb to 170 kb and remained at approximately 1 or 2 copies per cell. High (4.50 x 10(-2) and 3.08 x 10(-2)) and low (0.88 x 10(-8) to 3.29 x 10(-5)) plasmid transfer frequencies, as well as no plasmid transfer (under the detection limit), from these O139 isolates to the Escherichia coli recipient were observed. The emergence of pMRV150-like or pIP1202-like plasmids in many bacterial pathogens and nonpathogens occupying diverse niches with global geographical distribution indicates an increasing risk to public health worldwide. Careful tracking of these plasmids in the microbial ecosystem is warranted.

[Vibrio cholerae serogroups O1 and O139: susceptibility to antibiotics during 7th cholera pandemic].

The review presents data on circulation of antibiotic resistant and susceptible strains of Vibrio cholerae serogroups O1 and O139 isolated from cholera patients and healthy persons as well as from the environment, in Asia, Africa, Australia, and Europe (including New Independent States) during 7th cholera pandemic.

Antibacterial activity of ciprofloxacin against clinical strains of Vibrio cholerae O139 recently isolated from India.

We determined the in vitro antibacterial activity of ciprofloxacin against Vibrio cholerae O139 recently isolated from cholera patients in India. Ciprofloxacin showed excellent antibacterial activity against the O139 strains, and ciprofloxacin-resistant O139 strains were not observed. The lack of incidence of ciprofloxacin resistance in O139 strains may be because O139 strains appeared comparatively recently and have not been extensively treated with antibacterial agents including fluoroquinolones.

[Activity of 22 antibacterials against O1 and O139 serogroup Vibrio cholerae strains isolated from humans within 1927-2005 in various regions of the world].

Analysis of antibioticograms of 390 O1 and O139 serogroup Vibrio cholerae strains isolated from humans within 1927-2005 in various regions of the world showed that the strains of V. cholerae isolated within 1927-1966 were susceptible to 22 antibacterials, the strains isolated within 1938-1993 possessed 1-3 resistance markers and the strains isolated within 1994-2005 had 3-8 resistance markers including resistance to fluoroquinolones. All the strains of O139 serogroup V. cholerae isolated in 1993 and 1994 possessed 3 resistance markers. Studies on albino mice with generalized experimental cholera due to the V. cholerae eltor 1 strain (P-18826, 2005) isolated from a cholera patient, which was highly resistant to nalidixic acid, streptomycin, ampicillin and trimethoprim/sulfamethoxazole and showed cross resistance to fluoroquinolones (ciprofloxacin, ofloxacin, pefloxacin and norfloxacin) and moderate resistance to ceftriaxone and cefotaxime, revealed that the only efficient antibiotics were tetracyclines and aminoglycosides (except streptomycin). The investigation demonstrated an extension of the antibiotic resistance spectra of the epidemically significant strains of the cholera pathogen and the necessity of using antibacterial drugs in strict accordance with the antibioticograms in emergent prophylaxis and therapy of cholera and immediate replacement of the drug by a more active one.