Vibrio Cholerae O1 Strains Research Articles

Environmental Vibrio cholerae Strains Harboring Cholera Toxin and Vibrio Pathogenicity Island 1, Nigeria, 2008-2015.

Analysis of clinical and environmental Vibrio cholerae O1 strains obtained during 2008-2015 in Nigeria showed that lineages Afr9 and Afr12 carrying cholera toxin and Vibrio pathogenicity island 1 can be isolated from water. Our findings raise concerns about the role of the environment in maintenance and emergence of cholera outbreaks in Nigeria.

Epidemiological Situation on Cholera in the Russian Federation in 2023 and Forecast for 2024

The paper presents an analysis and assessment of risks of complication of epidemiological situation regarding cholera in the Russian Federation and a forecast for 2024. It briefly characterizes the dynamics of the epidemic process around the world during the last decade (2013–2022). The forecast of cholera importation into the Russian Federation, due to activity of the epidemic process in endemic countries, has been confirmed. In 2023, two cases of cholera importation from India were reported. It was established that the toxigenic strains isolated from patients belonged to the currently dominant in the world “post-Haitian” group, including in India and Bangladesh, with the genotype ctxB7 tcpACIRS rtxA4a VSP-IIΔ0495-0512. Furthermore, non-toxigenic Vibrio cholerae O1 strains were isolated from samples of biological material from persons arriving from India, containing preCTX prophage and distinct from the strain (ctxAB– tcpAEТ–) isolated from a patient with acute intestinal infection (Zaporozhye Region, Melitopol). Nontoxigenic NAG vibrios were isolated from 13 patients with acute intestinal infections, which is 1.9 times more than in 2022. Regardless of importations, a strain of V. cholerae O1 (ctxB1+tcpAEТ) of the “pre-Haitian” group was isolated from a water sample of a surface reservoir. 52 non-toxigenic V. сholerae O1 strains were found on the territory of 11 constituent entities of the Russian Federation, which is 18.9 % more than in 2022. In 2024, epidemiological risks of cholera importation from endemic countries to any administrative territory of the Russian Federation remain. The risks of epidemic manifestations of cholera are especially high in the territories of the constituent entities of the Russian Federation which are under ongoing emergency, due to migration processes, and the impact of other risk-forming factors that contribute to an increase in the degree of contact of the population with water bodies contaminated with cholera vibrios. If epidemic foci of cholera are to emerge in a neighboring country, there will be a high probability of spread of this infection in the stated constituent entities of the Russian Federation and in other territories of southern Russia. In the absence of epidemic manifestations of cholera, the possibility of occasional detection of toxigenic O1 serogroup strains in water samples due to unidentified introductions from cholera-affected areas cannot be ruled out.

The VxrAB two-component system is important for the polymyxin B-dependent activation of the type VI secretion system in Vibrio cholerae O1 strain A1552.

The type VI secretion system (T6SS) is used by bacteria for virulence, resistance to grazing, and competition with other bacteria. We previously demonstrated that the role of the T6SS in interbacterial competition and in resistance to grazing is enhanced in Vibrio cholerae in the presence of subinhibitory concentrations of polymyxin B. Here, we performed a global quantitative proteomic analysis and a targeted transcriptomic analysis of the T6SS-known regulators in V. cholerae grown with and without polymyxin B. The proteome of V. cholerae is greatly modified by polymyxin B with more than 39% of the identified cellular proteins displaying a difference in their abundance, including T6SS-related proteins. We identified a regulator whose abundance and expression are increased in the presence of polymyxin B, vxrB, the response regulator of the two-component system VxrAB (VCA0565-66). In vxrAB, vxrA and vxrB deficient mutants, the expression of both hcp copies (VC1415 and VCA0017), although globally reduced, was not modified by polymyxin B. These hcp genes encode an identical protein Hcp, which is the major component of the T6SS syringe. Thus, the upregulation of the T6SS in the presence of polymyxin B appears to be, at least in part, due to the two-component system VxrAB.

Genomic and Evolutionary Insights into Australian Toxigenic Vibrio cholerae O1 Strains.

Vibrio cholerae O1 is the causative agent of cholera, a severe diarrheal disease which can cause death if left untreated. In this study, a collection of clinical and environmental V. cholerae serogroup O1 isolates from Australia (1977 to 1987) (from local cases and cases acquired through international travel) and publicly available international isolates were characterized for genotypic features (virulence genes, mobile genetic elements [MGEs], and antimicrobial resistance gene profiles). Whole-genome sequencing (WGS) was used to investigate and compare the genetic relatedness between the 44 Australian and nine travel-associated isolates and the 60 publicly available international V. cholerae sequences representing pre-seventh-pandemic (pre-7PET) isolates and different waves of 7PET isolates. In this study, 36 (81%) Australian clinical and aquatic isolates harbored the cholera toxin-producing genes located in the CTX bacteriophage region. All the Australian environmental and clinical isolates lacked the seventh-pandemic virulence-associated genomic islands (VSP-I and -II). In silico multilocus sequence typing (MLST) classified all nine internationally acquired isolates as sequence type 69 (ST69), 36 clinical and aquatic isolates as ST70, and eight isolates from Australia as ST71. Most of the nontoxigenic clinical and aquatic isolates of ST71 had diverse genetic variations compared to ST70 Australian strains. The antimicrobial resistance-associated genes gyrA, parC, and parE had no mutations in all the environmental and clinical isolates from Australia. The SXT genetic element and class 1 integron gene sequences were not detected in Australian strains. Moreover, in this study, a Bayesian evolutionary study suggests that two distinct lineages of ST71 (new set of strains) and ST70 strains were prevalent around similar times in Australia, in ~1973 and 1969. IMPORTANCE Australia has its own indigenous V. cholerae strains, both toxigenic and nontoxigenic, that are associated with disease. Exotic strains are also detected in Australian patients returning from overseas travel. The clinical and aquatic V. cholerae O1 toxin gene-positive isolates from Australia responsible for cases in 1977 to 1987 were linked to acquisition from Queensland waterways but until now had not been characterized genetically. It is important to determine the genetic relatedness of Australian strains to international strains to assist in understanding their origin. This is the first extensive study to provide sequences and genomic analysis focused on toxigenic O1 V. cholerae clinical and environmental strains from Australia and its possible evolutionary relationship with other publicly available pre-7PET and 7PET V. cholerae strains. It is important to understand the population genetics of Australian V. cholerae from a public health perspective to assist in devising control measures and management plans for reducing V. cholerae exposure in Australia, given previous Australian disease clusters.

Emergence and Evolutionary Response of Vibrio cholerae to Novel Bacteriophage, Democratic Republic of the Congo1.

Cholera causes substantial illness and death in Africa. We analyzed 24 toxigenic Vibrio cholerae O1 strains isolated in 2015-2017 from patients in the Great Lakes region of the Democratic Republic of the Congo. Strains originating in southern Asia appeared to be part of the T10 introduction event in eastern Africa. We identified 2 main strain lineages, most recently a lineage corresponding to sequence type 515, a V. cholerae cluster previously reported in the Lake Kivu region. In 41% of fecal samples from cholera patients, we also identified a novel ICP1 (Bangladesh cholera phage 1) bacteriophage, genetically distinct from ICP1 isolates previously detected in Asia. Bacteriophage resistance occurred in distinct clades along both internal and external branches of the cholera phylogeny. This bacteriophage appears to have served as a major driver for cholera evolution and spread, and its appearance highlights the complex evolutionary dynamic that occurs between predatory phage and bacterial host.

Altered molecular attributes and antimicrobial resistance patterns of Vibrio cholerae O1 El Tor strains isolated from the cholera endemic regions of India.

The present study aimed to document the comparative analysis of differential hypervirulent features of Vibrio cholerae O1 strains isolated during 2018 from cholera endemic regions in Gujarat and Maharashtra (Western India) and West Bengal (Eastern India). A total of 87V. cholerae O1 clinical strains from Western India and 48 from Eastern India were analysed for a number of biotypic and genotypic features followed by antimicrobial resistance (AMR) profile. A novel polymerase chain reaction was designed to detect a large fragment deletion in the Vibrio seventh pandemic island II (VSP-II) genomic region, which is a significant genetic feature of the V. cholerae strains that have caused Yemen cholera outbreak. All the strains from Western India belong to the Ogawa serotype, polymyxin B-sensitive, hemolytic, had a deletion in VSP-II (VSP-IIC) region and carried Haitian genetic alleles of ctxB, tcpA and rtxA. Conversely, 14.6% (7/48) of the strains from Eastern India belonged to the Inaba serotype, polymyxin B-resistant, nonhemolytic, harboured VSP-II other than VSP-IIC type, classical ctxB, Haitian tcpA and El Tor rtxA alleles. Resistance to tetracycline and chloramphenicol has been observed in strains from both regions. This study showed hypervirulent, polymyxin B-sensitive epidemic causing strains in India along with the strains with polymyxin B-resistant and nonhemolytic traits that may spread and cause serious disease outcomes in future. The outcomes of this study can help to improve the understanding of the hyperpathogenic property of recently circulating pandemic Vibrio cholerae strains in India. Special attention is also needed for the monitoring of AMR surveillance because V. cholerae strains are losing susceptibility to many antibiotics used as a second line of defence in the treatment of cholera.

Etiological and epidemiological characteristics of Vibrio cholerae in Beijing, 2015-2021

Objective: To analyze the etiological and epidemiological characteristics of Vibrio cholerae in Beijing during 2015-2021 and provide evidence for the prevention and control of cholera. Methods: The V. cholerae strains isolated in Beijing during 2015-2021 were analyzed by serotyping and virulence genes detection. Pulsed field gel electrophoresis (PFGE) was performed for the molecular typing of the strains. Based on the collected epidemiological and clinical data of cholera cases,the epidemiological characteristics of cholera were analyzed by descriptive epidemiology method. Results: A total of 76 Vibrio cholerae O1 strains were isolated in Beijing during 2015-2021, including 61 strains from human, 10 strains from environment and 5 strains from seafood. The 76 strains consisted of 68 Ogawa strains and 8 Inaba strains. Six Ogawa strains isolated from sporadic cases carried ctxAB. After NotⅠ digestion, 76 strains were divided into 33 PFGE patterns. From 2015 to 2021, a total of 38 cholera epidemics were reported in Beijing, most of them were sporadic ones, accounting for 92.11% (35/38). A total of 45 cases were reported, and the cases occurred during June-September accounted for 97.78% (44/45). Cholera cases occurred in 9 districts of Beijing, and the cases reported in Chaoyang district accounted for 42.22% (19/45) and in Changping district accounted for 31.11% (14/45). The age of the cholera cases ranged from 19 to 63 years. Except for one case with unknown clinical symptoms, 44 cases had diarrhea symptoms with 84.09% (37/44) of the cases reporting diarrhea (3-9 times/day), followed by yellow watery stool (95.45%, 42/44), abdominal pain (68.18%, 30/44), nausea and vomiting (40.91%, 18/44) and fever (36.36%, 16/44). Conclusion: Vibrio cholerae strains isolated in Beijing during 2015-2021 were mainly O1 serotype Ogawa,most of which were non-toxigenic. The PFGE of the strains varied. Cholera epidemics occurred in 9 districts of Beijing, but most were sporadic ones with incidence peak during June-September.

Origin and Dissemination of Altered El Tor Vibrio cholerae O1 Causing Cholera in Odisha, India: Two and Half Decade's View.

The origin, spread and molecular epidemiology of altered El Tor Vibrio cholerae O1 strains isolated from cholera outbreaks/surveillance studies between 1995 and 2019 from different district of Odisha were analyzed. The stock cultures of V. cholerae O1 strains from 1995 to 2019 were analyzed through molecular analysis using different PCR assays and pulse field gel electrophoresis (PFGE) analysis. The spread map (month, year and place) was constructed to locate the dissemination of altered El Tor variants of V. cholerae O1 in this region. A total of 13 cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor carrying ctxB1 and ctxB7 genotypes. The ctxB1 alleles of V. cholerae O1 mostly confined to the coastal areas, whereas the ctxB7 genotypes, though originating in the coastal region of Odisha, concentrated more in the tribal areas. The positive correlation between virulence-associated genes (VAGs) was found through Pearson’s correlation model, indicative of a stronger association between the VAGs. The clonal relationship through PFGE between ctxB1 and ctxB7 genotypes of V. cholerae O1 strains exhibited 80% similarity indicating single- or multi-clonal evolution. It is evident from this study that the spread of multidrug-resistant V. cholerae O1-altered El Tor was dominant over the prototype El Tor strains in this region. The origin of altered El Tor variants of V. cholerae O1 occurred in the East Coast of Odisha established that the origin of cholera happened in the Gangetic belts of Bay of Bengal where all new variants of V. cholerae O1 might have originated from the Asian countries.

Variants of ctxB alleles of Vibrio cholerae O1 caused sequential cholera outbreaks in the tribal areas of Odisha, India.

Cholera localized outbreaks/epidemics accounting for high morbidity and mortality have been reported in different years both from the coastal and tribal districts of Odisha. In the present study, the emergence and spread of two sequential cholera outbreaks reported in July to October 2012 from Rayagada and Kalahandi districts of Odisha was investigated. Environmental water samples from different sources and rectal swabs from diarrhoea patients were analysed for identification, antibiogram profiles and molecular studies using DMAMA-PCR assays. The pulsed field gel electrophoresis (PFGE) was done on some selected Vibrio cholerae O1 strains isolated from these cholera outbreak areas. Results showed 42% of rectal swabs and 2.3% of water samples collected from both the districts were positive for Vibrio cholerae O1 Ogawa biotype El Tor carrying both ctxB1 and ctxB7 genotypes. The common resistance profile of V. cholerae O1 strains was ampicillin, nalidixic acid, furazolidone and co-trimoxazole. The PFGE analysis on selected V. cholerae O1 strains of ctxB1 and ctxB7 genotypes showed three pulsotypes with 96% similarity matrix exhibiting the relationship with their respective water sources. Hence, continuous surveillance is highly essential to monitor the antibiogram profile and changing pattern of ctxB genotypes of V. cholerae O1 in this region.

Comparative Analysis of Molecular-Genetic Properties in Non-Toxigenic Vibrio cholerae O1 Strains Biovar El Tor, Isolated in Russia and on Cholera Endemic Territories

Objective of the study was to perform a comparative analysis of molecular-genetic properties in non-toxigenic Vibrio cholerae O1 strains biovar El Tor, isolated in the Republic of Kalmykia and on cholera endemic territories and to reveal their phylogenetic relations to toxigenic isolates.Materials and methods. We have carried out bio-information analysis of whole genome sequences of 60 cholera vibrio strains from endemic as regards cholera regions and from Kalmykia. The presence of pathogenicity and endemicity islands in their genomes has been determined. Specifed have been the sequence-types of the examined strains and whole genome SNP-analysis conducted.Results and discussion. Non-toxigenic El Tor vibrios circulating in Kalmykia are clustered into two major genotypes: ctxA–tcpA+VPI-2+VSP– and ctxA–tcpA–VPI-2Δ+VSP–, where VPI-2 Δ+ signifes the presence of deletions of varying length in the genome of this pathogenicity island. Only the latter one is found in regions endemic for cholera. In addition, ctxA– tcpA+VPI-2+VSP+ populations circulate in cholera endemic foci, not found in Kalmykia. 17 sequence-types were identifed among the studied strains (by seven housekeeping gene loci). Phylogenetic analysis performed using SNP-typing demonstrated the absence of close genetic relation between the ctxA–tcpA+VPI-2+VSP– vibrios from Kalmykia and both toxigenic and non-toxigenic vibrios with different composition of pathogenicity and pandemicity islands in the genome. At the same time, genetic proximity of ctxA–tcpA–VPI-2Δ+VSP– cholera vibrios from endemic cholera foci with those isolated in Kalmykia was detected, which may indicate the possibility of their recurrent importation into the territory of Russia. Non-toxigenic V. cholerae strains found in the territory of Kalmykia are characterized by a high genetic diversity. Circulation of the strains with unique sequence-types suggests their potential for long-term persistence on this territory. At the same time, phylogenetic closeness and identity of certain strains with strains from endemic territories can be an evidence of repeated importation.

Genomic diversities of ctxB, tcpA and rstR alleles of Vibrio cholerae O139 strains isolated from Odisha, India.

The genome of Vibrio cholerae O139 strains has undergone cryptic changes since its first emergence in 1992 in South India. This study aimed to determine the presence of genotypic changes marked in ctxB, tcpA and rstR genes located within the CTX prophages among the strains of V. cholerae O139 isolated from 1999 to 2017 in Odisha. Antibiotic susceptibility test was conducted on 59 V. cholerae O139 strains. A conventional PCR assay was done for ctxB gene typing followed by sequencing along with identification of rstR and tcpA gene. Pulsed-field gel electrophoresis (PFGE) was carried out to reveal clonal variations among the V. cholerae O139 strains. Among V. cholerae O139 isolates more than 60% showed resistance to ampicillin, co-trimoxazole, furazolidone, streptomycin, neomycin and nalidixic acid. The ctxB sequencing and rstR allele-specific PCR assay revealed the presence of three genotypes 1, 3 and 4 with at least one copy of CTX Calc φ in addition to CTX ET and CTX Cl prophages in V. cholerae O139 isolates. PFGE analysis revealed 13 pulsotypes with two clades having 60% similarity among V. cholerae O139 strains. The circulating V. cholerae O139 strains in Odisha showed variation in genotypes with multiple clonal expansions over the years.

Haitian variant Vibrio cholerae O1 Ogawa caused cholera outbreaks in Odisha

PurposeDiarrheal disorders particularly cholera cause a significant threat resulting in high morbidity and mortality in the coastal and tribal areas of Odisha. Two sequential diarrheal outbreaks reported in 2016 from Balasore and Rayagada districts of Odisha were investigated to find out the causative organisms, antibiogram profile and molecular analysis of the isolated pathogens. MethodBacteriological analysis and antibiogram profiles of the pathogens were carried out as per the standard procedure followed. The double mismatch amplification mutation (DMAMA) PCR for ctxB gene, sequencing and pulse-field gel electrophoresis (PFGE) were carried out on Vibrio cholerae O1 strains. ResultsThe rectal swabs and water samples from these districts were positive for V. cholerae O1 Ogawa biotype El Tor. The V. cholerae O1 strains isolated from Balasore district were multidrug resistant to many antibiotics which differed from the isolates of Rayagada district. The DMAMA PCR assay on all clinical and water isolates from these areas and some strains from other districts exhibited ctxB7 allele of V. cholerae O1 which correlates with the sequencing results having different pulsotypes. The Haitian variant of V. cholerae O1 strains which were compared with the V. cholerae O1 strains of 1999 and 2000 exhibited different pulsotypes. ConclusionThe present study reports cholera outbreaks due to multidrug resistant ctxB7 allele of V. cholerae O1 from both coastal (Balasore) and tribal (Rayagada) areas of Odisha.

Dissemination of Vibrio cholerae O1 isolated from Odisha, India.

The present study reported the antimicrobial susceptibility trends, virulence genes, and drug resistance genes of Vibrio cholerae O1 strains isolated from outbreaks and epidemics over two and half decades (1995-2019) from Odisha, India. Antimicrobial susceptibility testing was performed by disc diffusion method. Virulence and drug resistance genes were detected by multiplex PCR assays. All V. cholerae O1 strains were sensitive to gentamicin, chloramphenicol, norfloxacin and ciprofloxacin while resistant to one or more antibiotics used. About 90% of the isolates of V. cholerae O1 carried antibiotic drug resistant genes (SulII, dfrA1 and strB) and SXT elements and the results correlated with the phenotypic antibiotic data obtained through disc diffusion assay. The tcpA Haitian variant V. cholerae O1 first appeared in 1999, gradually showing its increasing number upto 2019. TcpA El Tor strains only prevailed from 1995 to 2006; whereas the tcpA classical strains of V.choleraeO1 were found in less number from 1995 to 2016. Two multiplex PCR assays confirmed the presence of various toxigenic and virulence genes (toxR, ompU, ace, rtxC, ctxA, tcpA, rfbO1 and ompW) in all isolate of V. cholerae O1 strains. The present findings demonstrated the origin and spread of Haitian variants tcpA in V. cholerae O1 strains over two and half decades.

Molecular epidemiology of newly emerged V. cholerae O139 in Taiwan

A Vibrio cholerae O139 strain was isolated from a cholera patient in August 1997 in Kaohsiung County, Taiwan. This was the first case of V. cholerae O139 emerging in Taiwan. An epidemiological study showed that the infectious source was turtle eggs. One clinical isolate and fourteen isolates from environmental specimens of the turtle farm were collected. From all isolates, the genes encoded for cholera toxin (ctxA and ctxB) and for toxin-coregulated pili genes (tcpA and tcpl) were specifically amplified by polymerase chain reactions. In pulsed-field gel electrophoresis (PFGE) studies, these isolates were categorized to five subtypes using SfiI restriction digestion: whereas three distinct subtypes were identified when Notl was used for digestion. The banding pattern of the clinical isolate in PFGE only differed by 1-3 bands from those of the environmental isolates regardless of the restriction enzyme used. The Dice coefficients were found to be in the range of 0.88-1.0 and 0.87-1.0, respectively, by using SfiI and NotI for the subtyping. When these isolates were analyzed by plasmid profile analysis, the profile pattern of the clinical strain was identical to 12 of the 14 environmental strains. Based on these results, it was concluded that the fifteen strains studied were descended from the same origin, with the clinical isolate originated from the environmental isolates. The data suggests that the molecular subtyping is a powerful tool for tracing and verifying the infectious sources of V. cholerae cases.

Structural and Functional Changes in the Genome of Avirulent El Tor Biovar Vibrio cholerae ctxA + tcpA + Strains

High genomic variability of cholera pathogen, which is capable of persisting in the human organism and aquatic environments, underlies the diversity of virulent properties of its strains. However, the mechanism of emergence of the El Tor biovar Vibrio cholerae O1 strains with altered virulence remains poorly understood. A total of 21 El Tor biovar V. cholerae O1 strains were studied. Protein electrophoresis, enzyme-linked immunosorbent assay (GM1-ELISA), whole-genome sequencing, genomic analysis, and SNP genotyping were employed to study their properties. The virulence of the strains was assessed via intestinal infection of model animals. This paper reports that the genome of avirulent V. cholerae O1 strains of the El Tor biovar isolated from the aquatic environment contains the CTXφ prophage and VPI-1 pathogenicity island, which contain the ctxAB and tcpA-F genes, respectively, encoding the key pathogenicity factors, namely, the cholera toxin and toxin-coregulated pili. Comparative analysis of the nucleotide sequences of these mobile elements' genomes from two avirulent strains (89 and 147) of the ctxA+tcpA+ genotype and five clinical virulent isolates of the same genotype has not revealed any differences between them. At the same time, a change in the nucleotide sequence of the toxR global regulator gene located in the core region of pathogen’s genome has for the first time been detected in avirulent strains. This was a single nucleotide deletion (T in position 357), which resulted in the formation of a TGA stop codon. The consequence of such mutation is the production of defective transmembrane DNA-binding protein ToxR that positively regulates the toxT main virulence regulator gene expression due to the loss of 176 amino acids. It has been demonstrated that the loss of toxR protein function resulted in a significant decrease of the mRNA levels of the key regulatory (toxR and toxT) and structural (ctxA, ctxB, and tcpA) virulence genes in the studied strains. As a result, the expression of the cholera toxin genes in the mutant strains 89 and 147 was more than 20 times lower compared to the virulent strains. Whole-genome SNP analysis of the strains 89 and 147, as well as of 19 virulent strains including different genetic variants of the pathogen was carried out to infer their phylogenetic relationships. A new mechanism for the virulence change in cholera vibrio strains has been identified. A change in the nucleotide sequence of the toxR global regulator gene has been demonstrated in the avirulent strains of the ctxA+tcpA+ genotype. This mutation resulted in the formation of a defective transmembrane DNA-binding protein ToxR, which caused a dramatic decrease in the production of the toxin required for the development of the cholera disease.

Evaluation of Antibiotics Effectiveness Against Vibrio Cholerae under the Conditions of Complex Biofilm Formation

The effectiveness of 9 antibiotics against mono- and polymicrobial biofilms formed by Vibrio cholerae O1 strains was studied in vitro on fragments of chitinous exoskeleton of broad- fingered crayfish Astacus astacus in monoculture and together with oppor­tunistic bacteria Klebsiella spp. and non-O1/non-O139 V.cholerae. An increase in the antibiotic resistance of monomicrobial biofilms compared with planktonic bacteria was observed. Resistance of the polymicrobial biofilm bacteria to most antibacteri­al drugs corresponded to the resistance of the most stable strain in the monomicrobial biofilm. An increase in resistance to two and three drugs was observed in biofilms formed by classical toxigenic strains together with non-O1/non-O139 V.cholerae, while in biofilms formed together with Klebsiella spp. — to one antibacterial drug. Changes in antibiotic sensitivity during the interaction between bacteria in polymicrobial biofilm should be taken into account when developing tactics for prevention and treatment of infections.

Molecular Analysis of Selected Resistance Determinants in Diarrheal Fecal Samples Collected From Kolkata, India Reveals an Abundance of Resistance Genes and the Potential Role of the Microbiota in Its Dissemination.

Twenty-five diarrheal fecal samples from Kolkata were examined to determine the relative abundance of antimicrobial resistance genes (ARGs) against eight common classes of antibiotics with polymerase chain reaction (PCR) and Sanger sequencing. Relative abundance of an ARG was calculated as the percentage of fecal samples showing the presence of that particular ARG. The frequency of occurrence of resistance marker against each class of antibiotic was calculated as the percentage of fecal samples carrying at least one resistance marker for that particular class of antimicrobials. Antibiogram of Vibrio cholerae (V. cholerae) O1 strains isolated from four of these samples was obtained by disc diffusion method and was compared with the ARG profile of corresponding fecal samples from which the strains were isolated. A 464 bp amplicon of the V3-V4 region of bacterial 16S rDNA was obtained by PCR from 9 of these 25 samples using the primer pair S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 and sequenced to determine the major operational taxonomic unit (OTU). These 9 samples represented diarrhea due to diverse etiology and also unresolved etiology as determined by culture method. We conclude that the diarrheal intestinal microbiome has a common gene pool of ARGs against the major classes of antibiotics and may be serving as a reservoir of ARG dissemination. ARG profile of cholera stool showed that ARGs present in the gut of cholera patients may be transferred to the V. cholerae genome and pose a serious threat to the treatment of cholera by triggering resistance against potential drugs to which contemporary strains of V. cholerae were found to be sensitive in the present study. Fecal samples which were culture negative for diarrheal pathogens we tested also carried ARGs and OTU. Abundance of resistance markers against macrolides, tetracyclines, and aminoglycosides was the highest. Phylum Proteobacteria was the most abundant OTU suggesting proteobacterial blooms characteristic of disturbed gut microflora. Our study is the first comparative study of ARG profile of diarrheal samples with varying etiologic agent revealing the presence of ARGs against the most important classes of antibiotics in the gut of diarrheal patients by common, robust molecular methods, which are easily accessible by molecular epidemiological laboratories worldwide.

Usage of nutrient Medium Based on Dry Hydrolysate of Casein in Manufacturing Bivalent Chemical Cholera Vaccine

Objective of the study was to select the standardized substrate containing dry hydrolysate of casein for preparation of nutrient medium utilized for manufacturing bivalent chemical cholera vaccine under submerged cultivation of cholera vibrio strains in fermenters. Materials and methods . We used Vibrio cholerae O1 strains of classical biovar: strain 569B Inaba and strain M-41 Ogawa. Examined were two dry substrates of the medium: enzymatic hydrolysate of casein, Type I Himedia (India) and pancreatic hydrolysate of casein, produced by the State Scientific Center of Applied Microbiology and Biotechnology (Russian Federation). Produced under laboratory conditions at the premises of the RusRAPI “Microbe” medium was used as a control. Submerged cultivation was conducted in bioreactors during (9±1) h with aeration and automatic feeding of glucose and ammonia. Production of protective antigens was measured applying immunochemical and biological methods. Results and discussion . It is demonstrated that submerged cultivation of cholera vibrio production strains on nutrient media under study provides for synthesis of protective antigens the parameters of which comply with the requirements of normative documentation. More standardized and higher indicator values of the target product are ensured by cultivation of producer strains on nutrient medium with a substrate from dry enzymatic hydrolysate of casein, containing (1.5±0.1) g/l of amino nitrogen for the strain V. cholerae M-41 and (2±0.1) g/l – for V. cholerae 569 B. Transition to the use of standardized dry protein components of cultivation media does not lower the quality of the chemical cholera vaccine, but allows for the reduction of cost price and duration of technological process.

Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation.

Toxigenic Vibrio cholerae strains, including strains in serogroups O1 and O139 associated with the clinical disease cholera, are ubiquitous in aquatic reservoirs, including fresh, estuarine, and marine environments. Humans acquire cholera by consuming water and/or food contaminated with the microorganism. The genome of toxigenic V. cholerae harbors a cholera-toxin producing prophage (CT-prophage) encoding genes that promote expression of cholera toxin. The CT-prophage in V. cholerae is flanked by two satellite prophages, RS1 and TLC. Using cell surface appendages (TCP and/or MSHA pili), V. cholerae can sequentially acquire TLC, RS1, and CTX phages by transduction; the genome of each of these phages ultimately integrates into V. cholerae’s genome in a site-specific manner. Here, we showed that a non-toxigenic V. cholerae O1 biotype El Tor strain, lacking the entire RS1-CTX-TLC prophage complex (designated as RCT: R for RS1, C for CTX and T for TLC prophage, respectively), was able to acquire RCT from donor genomic DNA (gDNA) of a wild-type V. cholerae strain (E7946) via chitin-induced transformation. Moreover, we demonstrated that a chitin-induced transformant (designated as AAS111) harboring RCT was capable of producing cholera toxin. We also showed that recA, rather than xerC and xerD recombinases, promoted the acquisition of RCT from donor gDNA by the recipient non-toxigenic V. cholerae strain. Our data document the existence of an alternative pathway by which a non-toxigenic V. cholerae O1 strain can transform to a toxigenic strain by using chitin induction. As chitin is an abundant natural carbon source in aquatic reservoirs where V. cholerae is present, chitin-induced transformation may be an important driver in the emergence of new toxigenic V. cholerae strains.

Comparison and Evaluation of the Molecular Typing Methods for Toxigenic Vibrio cholerae in Southwest China

Vibrio cholerae O1 strains taken from the repository of Yunnan province, southwest China, were abundant and special. We selected 70 typical toxigenic V. cholerae (69 O1 and one O139 serogroup strains) isolated from Yunnan province, performed the pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and MLST of virulence gene (V-MLST) methods, and evaluated the resolution abilities for typing methods. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. Seventy V. cholerae obtained 50 PFGE patterns, with a high resolution. The strains could be divided into three groups with predominance of strains isolated during 1980s, 1990s, and 2000s, respectively, showing a good consistency with the epidemiological investigation. We also evaluated two MLST method for V. cholerae, one was used seven housekeeping genes (adk, gyrB, metE, pntA, mdh, purM, and pyrC), and all the isolates belonged to ST69; another was used nine housekeeping genes (cat, chi, dnaE, gyrB, lap, pgm, recA, rstA, and gmd). A total of seven sequence types (STs) were found by using this method for all the strains; among them, rstA gene had five alleles, recA and gmd have two alleles, and others had only one allele. The virulence gene sequence typing method (ctxAB, tcpA, and toxR) showed that 70 strains were divided into nine STs; among them, tcpA gene had six alleles, toxR had five alleles, while ctxAB was identical for all the strains. The latter two sequences based typing methods also had consistency with epidemiology of the strains. PFGE had a higher resolution ability compared with the sequence based typing method, and MLST used seven housekeeping genes showed the lower resolution power than nine housekeeping genes and virulence genes methods. These two sequence typing methods could distinguish some epidemiological special strains in local area.