ctxB Gene Research Articles

Wastewater-based surveillance of Vibrio cholerae: Molecular insights on biofilm regulatory diguanylate cyclases, virulence factors and antibiotic resistance patterns

Vibrio cholerae is an inherent inhabitant of aquatic ecosystems. The Indian state of West Bengal, especially the Gangetic delta region is the highest cholera affected region and is considered as the hub of Asiatic cholera. V. cholerae were isolated from publicly accessible wastewater of Midnapore, West Bengal, India. Serotyping determined all isolates to be of non-O1/non-O139 serogroups. Moderate biofilm-forming abilities were noticed in most of the isolates (74.7 %) while, high biofilm formation was recorded for only 6.3 % isolates and 19 % of isolates exhibited low/non-biofilm-forming abilities. PCR-based screening of crucial diguanylate cyclases (DGCs) involved in cyclic-di-GMP-mediated biofilm signaling was performed. cdgH and cdgM were the most abundant DGCs among 93.7 % and 91.5 % of isolates, respectively. Other important DGCs, i.e., cdgK, cdgA, cdgL, and vpvC were present in 84 %, 75.5 %, 72 % and 68 % of isolates, respectively. Besides, the non-O1/non-O139 isolates were screened for the occurrence of virulence factor encoding genes. Moreover, among these non-O1/non-O139 isolates, two strains (3.17 %) harbored both ctxA and ctxB genes, which encode the cholera toxin associated with epidemic cholera. ompU was the most prevalent virulence factor, present in 24.8 % of isolates. Other virulence factors like, zot and st were found in 4.7 % and 9.5 % of isolates. Genes encoding tcp and ace were found to be PCR-negative for the isolates. Additionally, crucial virulence factor regulators, toxT, toxR and hapR were found to be PCR-positive in all the isolates. Antibiotic resistance patterns displayed further vulnerabilities with decreased sensitivity towards commonly used antibiotics with multiple antibiotic resistance index ranging between 0.37 and 0.62. The presence of cholera toxin-encoding multi-drug resistant (MDR) V. cholerae strains in environmental settings is alarming. High occurrence of DGCs are considered to encourage further investigations to use them as alternative therapeutic targets against MDR cholera pathogen due to their unique presence in bacterial systems.

Secretome of adipose derived-mesenchymal stem cells reduces the Vibrio cholerae attachment to Caco-2 cells and subsequent inflammatory responses.

Mesenchymal Stem Cells (MSCs) can repair gastrointestinal tract damage. The Secretome of MSCs has a high capacity to inhibit bacterial colonization and the subsequent inflammatory responses of Vibrio cholerae. The Caco-2 cells were treated with adipose-derived MSCs (AD-MSCs) secretome and then infected with V. cholerae. Subsequently, the bacterial attachment and invasion, cholera toxin gene expression, PGE2 and IL-6 secretion, TNF-α, IL-1β, and IL-8 expression, and apoptosis of Caco-2 cells were evaluated. The secretome of AD-MSCs significantly reduced the V. cholerae attachment and internalization on Caco-2 epithelial cells (P<0.0001). The cholera toxin (Ctx-B) gene expression (FR=4.56 ± 0.66) and PGE2 production (P=0.0007) were also significantly reduced. The production of NO and TNF-α, IL-1β, and IL-8 pro-inflammatory cytokines were significantly (P<0.05) reduced in exposure to the secretome of AD-MSCs. Secretome also improved a significant 81.33% increase in IL-6 production (128.1 ± 37.6 pg/mL) and showed a 12.36% significant decrease in epithelial cell apoptosis (P< 0.0001) after exposure to V. cholerae. The secretome of AD-MSCs can play a critical role in inhibiting bacterial colonization, and subsequent inflammatory responses, and maintaining the integrity of the epithelial barrier. The secretome may be effective in the prevention of hypovolemic shock.

Analysis of changes in the genome of Vibrio cholerae O1 El Tor genovariants during the current period of the cholera pandemic

Introduction. The genome variability of genetic variants of El Tor cholera agent has led to the emergence of strains carrying mutations in various genes associated with epidemically important pathogen properties. This situation requires an assessment of the trends in these changes in order to predict the pathogenic potential of previously unknown variants and promptly develop new tools for their diagnostics and prevention.&#x0D; The purpose of this work was to analyze the dynamic changes in pathogenicity and drug resistance genes of V. cholerae El Tor genetic variants from endemic countries and Russia.&#x0D; Materials and methods. We analyzed complete genome nucleotide sequences of 104 V. cholerae El Tor strains from the NCBI Gen Bank and European Nucleotide Archive databases, as well as those obtained by us. The nucleotide sequences were analyzed using the UGEN v. 45.1 software. The dendrogram was constructed using maximum parsimony algorithm in BioNumerics v.7.6 software package based on the multiple alignment generated using the Snippy 4.6.0 program.&#x0D; Results. Genomic sequences of 103 strain genovariants isolated on the territory of nine endemic countries of Asia and Africa, as well as in Russia in 1991-2022, have been compared. It is shown that the process of genovariant genome changing was multistage and occurred due to the continuous accumulation of point mutations in key (ctxB and tcpA) and additional (rtxA) genes of pathogenicity and core genes of antibiotic resistance (gyrA, parC and carR), as well as a deletion in SXT element. The most important was the change in the ctxB gene and the emergence of new genovariants with the ctxB7 allele, which replaced the previously prevalent strains. Analysis of altered genome regions of 83 strains from endemic regions has revealed eight genotypes, while the strains (21 isolates) imported to Russia belonged to only five of them including highly virulent strains with the ctxB7 allele and lost PolR biovar-specific feature due to carR gene mutation. The established close phylogenetic relatedness of genovariants from Russia with strains from endemic Asian countries confirms their importation from this region.&#x0D; Conclusion. The sequential occurrence and accumulation of mutations in the pathogenicity and drug resistance genes in the genome of genovariants in endemic regions have been shown, which leads to a change in their epidemically important features. The importation of new highly virulent genovariants into Russia has been established, which indicates the need for an ongoing assessment of changes in the genome of this pathogen for the timely development of adequate means of gene diagnostics and prevention.

Podophyllotoxin, Deoxypodophyllotoxin and Ursolic Acid as Potential Inhibitors of tcpA, ompW, and ctxB Genes in Vibrio cholerae: An in-Silico Study

Podophyllotoxin, Deoxypodophyllotoxin and Ursolic Acid as Potential Inhibitors of tcpA, ompW, and ctxB Genes in Vibrio cholerae: An in-Silico Study

Immunization with a bicistronic DNA vaccine modulates systemic IFN-γ and IL-10 expression against Vibrio cholerae infection.

Introduction. Cholera is an acute enteric infection caused by Vibrio cholerae, particularly in areas lacking access to clean water. Despite the global effort to improve water quality in these regions, the burden of cholera in recent years has not yet declined. Interest has therefore extended in the use of bicistronic DNA vaccine encoding ctxB and tcpA genes of V. cholerae as a potential vaccine.Hypothesis/Gap Statement. The potential of a bicistronic DNA vaccine, pVAX-ctxB-tcpA has not been determined in vitro and in vivo.Aim. The goal of present study was to evaluate in vitro expression and in vivo potential of pVAX-ctxB-tcpA vaccine against V. cholerae.Methodology. The pVAX-ctxB-tcpA was transiently transfected into mammalian COS-7 cells, and the in vitro expression was assessed using fluorescence and Western blot analyses. Next, the vaccine was encapsulated into sodium alginate using water-in-oil emulsification and evaluated for its efficiency in different pH conditions. Subsequently, oral vaccination using en(pVAX-ctxB-tcpA) was performed in vivo. The animals were challenged with V. cholerae O1 El Tor after 2 weeks of vaccination using the Removable Intestinal Tie-Adult Rabbit Diarrhoea (RITARD) model. Following the infection challenge, the rabbits were monitored for evidence of symptoms, and analysed for systemic cytokine expression level (TNF-α, IFN-γ, IL-6 and IL-10) using quantitative real-time polymerase chain reaction.Results. The in vitro expression of pVAX-ctxB-tcpA was successfully verified via fluorescence and Western blot analyses. Meanwhile, in vivo analysis demonstrated that the en(pVAX-ctxB-tcpA) was able to protect the RITARD model against V. cholerae infection due to a lack of evidence on the clinical manifestations of cholera following bacterial challenge. Furthermore, the bicistronic group showed an upregulation of systemic IFN-γ and IL-10 following 12 days of vaccination, though not significant, suggesting the possible activation of both T-helper 1 and 2 types of response. However, upon bacterial challenge, the gene expression of all cytokines did not change.Conclusion. Our findings suggest that the bicistronic plasmid DNA vaccine, pVAX-ctxB-tcpA, showed a potential role in inducing immune response against cholera through upregulation of in vitro gene and protein expression as well as in vivo cytokine gene expression, particularly IFN-γ and IL-10.

Prevalence of Different Types of Integrative Conjugative Element SXT/R391 Encoding Multiple Antibiotic Resistance Among Clinical Strains of Cholera Agent

The aim of the work was to study the prevalence of different types of SXT element with different composition of antibiotic resistance genes among clinical strains of the El Tor cholera pathogen isolated in Russia, Ukraine and cholera-endemic countries in Asia and Africa.Materials and methods. The subject of the study was 27 strains and nucleotide sequences of 77 strains of Vibrio cholerae El Tor available from the NCBI GenBank. The structure of the SXT element and its type were determined using the Mauve and BLAST v.2.9.0 programs. Phylogenetic relations of strains with different types of SXT were identified using Snippy v.4.6.0 and MrBayes v.3.2.7 software. Assessment of strain sensitivity to antibiotics was carried out in accordance with Methodological Regulations 4.2.2495-09.Results and discussion. Two types of SXT element (ICEVchInd5 and ICEVchBan9) have been identified among the studied strains from Russia and Ukraine, which have different composition of antibiotic resistance genes: floR, strAB, sul2, dfrA1 and floR, tetAR, strAB, sul2, dfrA1, respectively. At the same time, the studied strains from Asia and Africa contain five types of SXT: ICEVchInd5, ICEVchBan9, ICEVchBan5, SXTTET, ICEVchInd5ΔVRIII, which differ in size and/or composition of resistance genes. Of these, the last three have not been found in Russia and Ukraine. Due to the high level of genomic diversity of SXT in the population of V. cholerae in endemic regions, there is a risk of importation of cholera pathogen strains with altered resistance to antibiotics into Russia. Phylogenetic relations of 76 strains with different SXT types and different alleles of the ctxB gene encoding the B subunit of cholera toxin have been assessed based on SNP analysis. A close phylogenetic relation between strains with the same type of SXT isolated in Russia and Asian countries has been demonstrated, which confirms the importation of the causative agent of cholera with multiple resistance to antibiotics from this region and the need for constant monitoring of the sensitivity of V. cholerae to antimicrobial drugs.

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.

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

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.

MLST/MVLST Analysis and Antibiotic Resistance of Vibrio cholerae in Shandong Province of China.

Background:Vibrio cholerae is an important bacterium causing profuse watery diarrhea. Cholera had swept the whole Shandong province from 1975 to 2013.Methods:From epidemiological data and pulsed-field gel electrophoresis data, we selected 86 V. cholerae isolates appearing in Shandong Province in China from 1975 to 2013 and characterized them by multilocus sequence typing (MLST)/multi-virulence locus sequence typing (MVLST), antibiogram and analysis of genes related to antibiotic resistance.Results:Combined MLST/MVLST data revealed 33 sequence types and a major group. Within the group, 3 subgroups (ST1, ST24 and ST29) were revealed, prevalent in the strains isolated during the 1980s, 1990s and 21st century, respectively. All the O1 isolates after 1990 were found to be El Tor variants harboring the classical ctxB gene. The tcpA gene of O139 strains had a mutation at amino acid position 62 (N→D). Antibiotic resistance of V. cholerae increased over time. Most El Tor variants between 1998 and 1999 were resistant to trimethoprim/sulfamethoxazole. The O139 strain, since its appearance in 1997, had significantly broader spectrum of antibiotic resistance than O1 variants. The presence of the SXT element corresponds to the trend of growing drug resistance.Conclusion:The analysis of genotypic polymorphism and enhanced resistance of V. cholerae indicated continuous variation and evolution of this pathogenic agent in Shandong Province.

Characterization of V. cholerae O1 biotype El Tor serotype Ogawa possessing the ctxB gene of the classical biotype isolated from well water associated with the cholera outbreak in Kerala, South India.

We investigated 22 water samples (17 well water and five pipe water - both chlorinated) and six soil samples from the surroundings of wells of the households of suspected patients from Palakkad district, Kerala (India), from where a cholera outbreak was reported during June-July 2016. A total of 25 Vibrio cholerae isolates were collected from three well water samples during a recent cholera outbreak. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. PCR assays confirmed the occurrence of ctxB, ctxA, hlyA, tcpA El Tor,VPI, ace, zot, ompW, rfbO1 and toxR genes in all isolates. The presence of the ctxB gene of the classical biotype in all of the El Tor isolates suggests that it is a new variant of El Tor biotype. Antibiogram profile of all V. cholerae O1 isolates revealed resistance towards five classes of antibiotics island and indicates that they were multidrug resistant. ERIC-PCR and PFGE finger prints showed the clonal relationship among the V. cholerae O1 isolates. The results of this study revealed the emergence of a new variant of El Tor biotype in the water samples from Palakkad district, from where a cholera outbreak was reported.

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.

Genetic Diversity of ctxB Gene Among Classical O1 and El Tor Strains of Vibrio cholerae using High-Resolution Melting Curve Analysis.

Background & Objective: Vibrio cholerae is a natural inhabitant of the environment and causes severe diarrhea ailments (cholera) that affects thousands of people each year worldwide. The most important virulence factors of this pathogen are cholera toxin (cholera toxin CT) and Type IV pili (toxin co-regulated pili TCP), which are encoded within the genome of the filamentous bacteriophage CTXφ. In the present study, according to researchers’ report on genotypic variations of cholera toxin, we evaluated the sequence of ctxB subunit obtained from 100 strains of patients infected with cholera in Iran.Methods:The evaluation of genotype variations of cholera toxin was made by high-resolution melting curve analysis illustrating a single nucleotide change. Then, ctxB gene sequencing was performed. Through this analysis and the sequencing process, two standard samples were studied.Results: Using serologic tests, all the strains analyzed in this study were identified to be in O1 serotype. However, there have been differences in sequences of ctxB as some were similar to V. cholerae O1 biovar El Tor str. N16961 while others were similar to the genotype of V. cholerae ATCC 14035. We did not observe any particular pattern within the process of mutation.Conclusion:The analysis of the new samples of ctxB showed that they were potentially different. It seems that these complicated species were affected by a new genetic exchange of El Tor and classic genotypes.

Spread of Haitian Variant Vibrio cholerae O1 Causing Cholera Outbreaks in Odisha, India.

Cholera posed a significant threat causing outbreaks/epidemics with high morbidity and mortality in Odisha. This study envisages the characterisation of isolated pathogen from two cholera outbreaks reported in 2018 and 2019 from Bargarh and Rayagada districts of Odisha respectively. Vibrio cholerae O1 were isolated following standard techniques. The different virulent and drug resistant genes were detected by multiplex PCR assays; whereas the ctxB genotypes were characterised through double mismatch amplification mutation (DMAMA) PCR assay. The ctxB genes were further sequenced and pulse-field gel electrophoresis (PFGE) was done on some selected strains. The clinical and water isolates of Haitian variant (HCT) V. cholerae O1 Ogawa biotype El Tor with multi drug resistant strains were isolated from both the places. All the V. cholerae O1 strains were positive for virulence genes. The antibiotic resistant genes like dfrA1 (100%), strB (76.9%), intSXT (61.5%) were detected. The PFGE results on V. cholerae O1 strains exhibited two different pulsotypes. These cholera outbreaks were due to multidrug resistant HCT variant V. cholerae O1 strains which were circulating and caused the cholera outbreaks in Odisha. So continuous surveillance on diarrheal disorders is highly essential to prevent the future diarrheal outbreaks in this region.

A molecular and epidemiological study of Vibrio cholerae isolates from cholera outbreaks in southern Ghana.

Cholera remains a major global public health threat and continuous emergence of new Vibrio cholerae strains is of major concern. We conducted a molecular epidemiological study to detect virulence markers and antimicrobial resistance patterns of V. cholerae isolates obtained from the 2012–2015 cholera outbreaks in Ghana. Archived clinical isolates obtained from the 2012, 2014 and 2015 cholera outbreaks in Ghana were revived by culture and subjected to microscopy, biochemical identification, serotyping, antibiotic susceptibility testing, molecular detection of distinct virulence factors and Multi-Locus Variable-Number of Tandem-Repeat Analysis (MLVA). Of 277 isolates analysed, 168 (60.6%) were confirmed to be V. cholerae and 109 (39.4%) isolates constituted other bacteria (Escherichia coli, Aeromonas sobria, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococci faecalis). Serotyping the V. cholerae isolates identified 151 (89.9%) as Ogawa, 3 (1.8%) as Inaba and 14 (8.3%) as non-O1/O139 serogroup. The O1 serogroup isolates (154/168, 91.7%) carried the cholera toxin ctxB gene as detected by PCR. Additional virulence genes detected include zot, tcpA, ace, rtxC, toxR, rtxA, tcpP, hlyA and tagA. The most common and rare virulence factors detected among the isolates were rtxC (165 isolates) and tcpP (50 isolates) respectively. All isolates from 2014 and 2015 were multidrug resistant against the selected antibiotics. MLVA differentiated the isolates into 2 large unique clones A and B, with each predominating in a particular year. Spatial analysis showed clustering of most isolates at Ablekuma sub-district. Identification of several virulence genes among the two different genotypes of V. cholerae isolates and resistance to first- and second-line antibiotics, calls for scaleup of preventive strategies to reduce transmission, and strengthening of public health laboratories for rapid antimicrobial susceptibility testing to guide accurate treatment. Our findings support the current WHO licensed cholera vaccines which include both O1 Inaba and Ogawa serotypes.

Deciphering the possible role of ctxB7 allele on higher production of cholera toxin by Haitian variant Vibrio cholerae O1.

Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside GM1. In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.

Incidence of Bacterial Enteropathogens among Diarrhea Patients from Tribal Areas of Odisha.

Infectious diarrheal diseases remain a leading cause of morbidity and mortality in developing and underdeveloped countries. The present study documented the etiology of bacterial enteropathogens in three tribal districts of Odisha from July 2010 to September 2013. A total of 1427 rectal swabs were collected and bacteriologically analyzed by following standard procedure. Among the 930 (65.2%) culture positive samples, Escherichia coli (E. coli) constituted 636 (44.6%); Vibrio cholerae (V. cholerae) O1, 146 (10.2%); Salmonella species (spp.), 10 (0.7%); Shigella spp., 79 (5.5%); and Aeromonas spp., 59 (4.1%). Of the 729 environmental water samples taken from river, open well, Nala (a small stream), and Chua (a shallow pit on a river bed), 14 (1.9%) contained non-O1/non-O139 V. cholerae and 13 (1.8%) had V. cholerae O1 strains. An analysis of the demographics showed that people in the 14 to 40-year age group were highly susceptible to diarrhea caused by V. cholerae which occurred mainly during the rainy and post-rainy seasons. All enteropathogens were multidrug-resistant and found throughout the study period. The V. cholerae strains isolated were El Tor variants carrying the classical, El Tor, and Haitian cholera toxin subunit B (ctxB) genes. The classical ctxB was the dominant allele, and the prevalence of the Haitian ctxB allele increased during the test period. These findings indicate that active surveillance is needed to monitor the changing antibiotic resistance patterns of V. cholerae serogroups and biotypes present in this region.

Genome Dynamics of Vibrio cholerae Isolates Linked to Seasonal Outbreaks of Cholera in Dhaka, Bangladesh.

The temporal switching of serotypes from serotype Ogawa to Inaba and back to Ogawa was identified in Vibrio cholerae O1, which was responsible for seasonal outbreaks of cholera in Dhaka during the period 2015 to 2018. In order to delineate the factors responsible for this serotype transition, we performed whole-genome sequencing (WGS) of V. cholerae O1 multidrug-resistant strains belonging to both the serotypes that were isolated during this interval where the emergence and subsequent reduction of the Inaba serotype occurred. The whole-genome-based phylogenetic analysis revealed clonal expansion of the Inaba isolates mainly responsible for the peaks of infection during 2016 to 2017 and that they might have evolved from the prevailing Ogawa strains in 2015 which coclustered with them. Furthermore, the wbeT gene in these Inaba serotype isolates was inactivated due to insertion of a transposable element at the same position signifying the clonal expansion. Also, V. cholerae isolates in the Inaba serotype dominant clade mainly contained classical ctxB allele and revealed differences in the genetic composition of Vibrioseventh pandemic island II (VSP-II) and the SXT integrative and conjugative element (SXT-ICE) compared to those of Ogawa serotype strains which remerged in 2018. The variable presence of phage-inducible chromosomal island-like element 1 (PLE1) was also noted in the isolates of the Inaba serotype dominant clade. The detailed genomic characterization of the sequenced isolates has shed light on the forces which could be responsible for the periodic changes in serotypes of V. cholerae and has also highlighted the need to analyze the mobilome in greater detail to obtain insights into the mechanisms behind serotype switching.IMPORTANCE The switching of serotype from Ogawa to Inaba and back to Ogawa has been observed temporally in Vibrio cholerae O1, which is responsible for endemic cholera in Bangladesh. The serospecificity is key for effective intervention and for preventing cholera, a deadly disease that continues to cause significant morbidity and mortality worldwide. In the present study, WGS of V. cholerae allowed us to better understand the factors associated with the serotype switching events observed during 2015 to 2018. Genomic data analysis of strains isolated during this interval highlighted variations in the genes ctxB, tcpA, and rtxA and also identified significant differences in the genetic content of the mobilome, which included key elements such as SXT ICE, VSP-II, and PLE. Our results indicate that selective forces such as antibiotic resistance and phage resistance might contribute to the clonal expansion and predominance of a particular V. cholerae serotype responsible for an outbreak.

Haitian-like genetic traits with creeping MIC of Azithromycin in Vibrio cholerae O1 isolates from Puducherry, India.

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity.Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates.Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method.Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB, tcpA and rtxA gene, 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex, att-prfC, setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml-1 to 2 µg ml-1.Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.

Simple and Sensitive Quantification of Toxigenic Vibrio cholerae DNA by Real-Time Loop-Mediated Isothermal Amplification Based on ctxB Gene

Background: Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and specific method for the detection of bacterial pathogens within a sample. Methods: We designed and evaluated a new quantitative LAMP primer set specific to the ctxB gene for the rapid measurement of the load of toxigenic V. cholerae DNA in the reaction. Unlike the previously reported LAMP assays for V. cholerae detection, our LAMP primer set was able to detect and quantify toxigenic V. cholerae DNA with a detection limit of 2.8 pg of the ctxB gene equivalent to 8.3 copies of genomic DNA per reaction. Results: Our LAMP assay was found to be highly specific with no amplification detected in non-toxigenic V. cholerae bacterial strains. Thus, the ctxB-based LAMP assay developed in this study can serve as a simple, sensitive, specific, and quantitative molecular tool for the monitoring and epidemiological study of cholera. Conclusions: Overall, the low cost and simplicity of the LAMP assay can make it a preferable molecular method in the detection of pathogenic organisms.

The global burden of cholera outbreaks in Niger: an analysis of the national surveillance data, 2003-2015.

Niger has experienced multiple cholera outbreaks since the occurrence of cholera in West Africa in 1970-1971. Understanding the origins of these geographic variations at different scales in the region and in health districts is an essential step in the rational and operational fight against this disease. We conducted a retrospective review of all suspected cholera cases in Niger from 2003 to 2015. Data from 2010 to 2015 were reviewed to determine the number and percentage of stool specimens tested, with distribution by serotype. We also determined the time between sampling and sending to a laboratory. From 2003 through 2015 a total of 16 328 cases of cholera, including 578 deaths, were reported to the National Surveillance Database (overall case fatality rate 3.5%). The number of cases was greater among males (63.29%). Patients >15 y of age accounted for 69.80% of cases. All regions recorded cholera cases throughout the period 2003 to 2015, except in 2009, when no outbreak was reported. All epidemics recorded were due to the subtype serogroup O1 serotype Ogawa and all strains confirmed the presence of the ctxB, rstR, tcpA, zot, ace and ctxA genes encoding the toxin of this serotype. The global burden of cholera remains high in Niger. To successfully prevent and control outbreaks, adapted guidelines and strategies should be reviewed to provide more focused information and assign clear roles and responsibilities.