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Molecular characterization of Vibrio cholerae O1 strains circulating in Assam: a north eastern state of India.

ABSTRACT: Background and Objectives:Information on the genetic epidemiology of cholera in Assam, a northeastern state of India is lacking despite cholera being a major public health problem. The study aimed to determine the virulence genes and genes encoding antibiotic resistance in Vibrio cholerae isolates and to determine the prevalent genotypes based on the presence or absence of the virulence genes and ctxB genotype. Materials and Methods:Twenty-five V. cholerae strains were subjected to conventional biotyping and serotyping followed by multiplex PCR to detect ctxA, ctxB, zot, ace, O1rfb, tcpA, ompU, ompW, rtxC, hly and toxR and antibiotic resistance genes. Cholera toxin B (ctxB) gene was amplified followed by sequencing. Results:All the V. cholerae O1 isolates were El Tor Ogawa and showed the presence of the core toxin region representing the genome of the filamentous bacteriophage CTXø. The complete cassette of virulence genes was seen in 48% of the isolates which was the predominant genotype. All the isolates possessed amino acid sequences identical to the El Tor ctxB subunit of genotype 3. sulII gene was detected in 68% of the isolates, dfrA1 in 88%, strB in 48% and SXT gene was detected in 36% of the isolates. Conclusion:Toxigenic V. cholerae O1 El Tor Ogawa strains of ctxB genotype 3 carrying a large pool of virulence genes are prevailing in Assam. Presence of a transmissible genetic element SXT in 36% of the strains is of major concern as it indicates the emergence of multiple drug resistance among the V. cholerae isolates.

SUBMITTER: Sharma A

PROVIDER: S-EPMC8629826 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Vibrio cholerae is an autochthonous inhabitant of the aquatic environment. Several molecular methods have been used for typing V. cholerae strains, but there is no proper database for such scheme, including multilocus sequence typing (MLST) for V. cholerae O1 and O139 strains. We used 54 V. cholerae O1 and three O139 strains isolated from clinical and environmental sources and regions of India during the time period of 1975-2015 to determine the presence of virulence genes and production of biofilm. We devised a MLST scheme and developed a database for typing V. cholerae strains. Also, we performed pulsed-field gel electrophoresis to see the genomic diversity among them and compared it with MLST. We used the MEGA 7.0 software for the alignment and comparison of different nucleotide sequences. The advanced cluster analysis was performed to define complexes. All strains of V. cholerae, except five strains, showed variation in phenotypic characteristics but carried virulence-associated genes indicating they belonged to the El Tor/hybrid/O139 variants. MLST analysis showed 455 sequences types among V. cholerae strains, irrespective of sources and places of isolation. With these findings, we set up an MLST database on PubMLST.org using the BIGSdb software for V. cholerae O1 and O139 strains, which is available at https://pubmlst.org/vcholerae/ under the O1/O139 scheme. The pulsed-field gel electrophoresis (PFGE) fingerprint showed six fingerprint patterns namely E, F, G, H, I, and J clusters among 33 strains including strain N16961 carrying El Tor ctxB of which cluster J representing O139 strain was entirely different from other El Tor strains. Twenty strains carrying Haitian ctxB showed a fingerprint pattern classified as cluster A. Of the five strains, four carrying classical ctxB comprising two each of El Tor and O139 strains and one El Tor strain carrying Haitian ctxB clustered together under cluster B along with V. cholerae 569B showing pattern D. This study thus indicates that V. cholerae strains are undergoing continuous genetic changes leading to the emergence of new strains. The MLST scheme was found more appropriate compared to PFGE that can be used to determine the genomic diversity and population structure of V. cholerae.

Project description: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.

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Molecular characterization of Vibrio cholerae O1 strains circulating in Assam: a north eastern state of India.

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