Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Establishment of an intragastric surgical model using C57BL/6 mice to study the vaccine efficacy of OMV-based immunogens against Helicobacter pylori.

Chronic gastritis is one of the major symptoms of gastro-duodenal disorders typically induced by Helicobacter pylori (H. pylori). To date, no suitable model is available to study pathophysiology and therapeutic measures accurately. Here, we have presented a successful surgical infection model of H. pylori-induced gastritis in C57BL/6 mice that resembles features similar to human infection. The proposed model does not require any preparatory treatment other than surgical intervention. C57BL/6 mice were injected with wild-type SS1 (Sydney strain 1, reference strain) directly into the stomach. Seven days post infection, infected animals showed alterations in cytokine responses along with inflammatory cell infiltration in the lamina propria, depicting a prominent inflammatory response due to infection. To understand the immunogenicity and protective efficacy, the mice were immunized with outer membrane vesicles (OMVs) isolated from an indigenous strain with putative virulence factors of H. pylori [A61C (1), cag+/vacA s1m1]. In contrast to the non-immunized cohort, the OMV-immunized cohort showed a gradual increase in serum immunoglobulin(s) levels on the 35th day after the first immunization. This conferred protective immunity against subsequent challenge with the reference strain (SS1). Direct inoculation of H. pylori into the stomach influenced infection in a short time and, more importantly, in a dose-dependent manner, indicating the usefulness of the developed model for pathophysiology, therapeutic and prophylactic studies.

Emerging resistome diversity in clinical Vibrio cholerae strains revealing their role as potential reservoirs of antimicrobial resistance.

BACKGROUND: This is a unique and novel study delineating the genotyping and subsequent prediction of AMR determinants of Vibrio cholerae revealing the potential of contemporary strains to serve as precursors of severe AMR crisis in cholera. METHODS AND RESULTS: Genotyping of representative strains, VC1 and VC2 was undertaken to characterize antimicrobial resistance genes (ARGs) against chloramphenicol, SXT, nalidixic acid and streptomycin against which they were found to be resistant by antibiogram analysis in our previous investigation. strAB, sxt, sul2, qace∆1-sul1 were detected by PCR. Genome annotation and identification of ARGs with WGS helped to detect the presence of almG, varG, strA (APH(3'')-Ib), strB (APH(6)-Id), sul2, catB9, floR, CRP, dfrA1 genes. Signatures of resistance determinants and protein domains involved in antimicrobial resistance, primarily, efflux of antibiotics were identified on the basis of 30-100% homology to reference proteins. These domains were predicted to be involved in other metabolic functions on the basis of 100% identity with 100% coverage with reference protein and nucleotide sequences and were predicted to be of a diverse taxonomic origin accentuating the influence of the microbiota on AMR acquisition. Sequence analysis of QRDR (quinolone resistance-determining region) revealed SNPs. Cytoscape v3.8.2 was employed to analyse protein-protein interaction of MDR proteins, MdtA and EmrD-2, with nodes of vital AMR pathways. Vital nodes involved in efflux of different classes of antibiotics were found to be absent in VC1 and VC2 justifying the sensitivity of these strains to most antibiotics. CONCLUSIONS: The study helped to examine the resistome of VC isolated from recent outbreaks to understand the underlying reason of sensitivity to most antibiotics and also to characterize the ARGs in their genome. It revealed that VC is a reservoir of signatures of resistance determinants and serving as precursors for severe AMR crisis in cholera. This is the first study, to our knowledge, which has scrutinized and presented systematically, information on prospective domains which bear the potential of serving as AMR determinants in VC with the help of bioinformatic tools. This pioneering approach may help in the prediction of AMR landfalls and benefit epidemiological surveillance and early warning systems.

Prevalence and changing antimicrobial resistance profiles of Shigella spp. isolated from diarrheal patients in Kolkata during 2011-2019.

BACKGROUND: The primary aim of this study was to investigate the occurrence, characteristics, and antimicrobial resistance patterns of various Shigella serogroups isolated from patients with acute diarrhea of the Infectious Diseases Hospital in Kolkata from 2011-2019. PRINCIPAL FINDINGS: During the study period, Shigella isolates were tested for their serogroups, antibiotic resistance pattern and virulence gene profiles. A total of 5.8% of Shigella spp. were isolated, among which S. flexneri (76.1%) was the highest, followed by S. sonnei (18.7%), S. boydii (3.4%), and S. dysenteriae (1.8%). Antimicrobial resistance against nalidixic acid was higher in almost all the Shigella isolates, while the resistance to ß-lactamases, fluoroquinolones, tetracycline, and chloramphenicol diverged. The occurrence of multidrug resistance was found to be linked with various genes encoding drug-resistance, multiple mutations in the topoisomerase genes, and mobile genetic elements. All the isolates were positive for the invasion plasmid antigen H gene (ipaH). Dendrogram analysis of the plasmid and pulsed-field electrophoresis (PFGE) profiles revealed 70-80% clonal similarity among each Shigella serotype. CONCLUSION: This comprehensive long-term surveillance report highlights the clonal diversity of clinical Shigella strains circulating in Kolkata, India, and shows alarming resistance trends towards recommended antibiotics. The elucidation of this study's outcome is helpful not only in identifying emerging antimicrobial resistance patterns of Shigella spp. but also in developing treatment guidelines appropriate for this region.

Foodborne Outbreak by Salmonella enterica Serovar Weltevreden in West Bengal, India.

Foodborne gastroenteritis outbreaks owing to Salmonella enterica serovar Weltevreden (Salmonella Weltevreden) represent a significant global public health problem. In the past two decades, Salmonella Weltevreden has emerged as a dominant foodborne pathogen, especially in South-East Asian countries. This report describes a community foodborne outbreak of gastroenteritis caused by Salmonella Weltevreden in August 2022 following consumption of panipuri from a street vendor in the Polba block in Hooghly district, West Bengal, India. This food item was consumed by 185 people, of whom 129 had acute watery diarrhea with other clinical symptoms and 65 of them were admitted to different District hospitals for treatment. Stool specimens collected from hospitalized cases were positive for S. enterica, and further serotyped as Salmonella Weltevreden. All the Salmonella Weltevreden strains possessed the Salmonella pathogenicity islands associated genes (invA/E, orgA, ttrc, ssaQ, mgtC, misL, spi4D), the enterotoxin (stn), and hyperinvasive locus gene (hilA). Except erythromycin, all the strains were susceptible for commonly used antimicrobials in the treatment of diarrhea. The XbaI-based pulsed-field gel electrophoresis analysis indicated that all the isolates responsible for the recent outbreak were similar, but diverged from other Salmonella Weltevreden that were previously reported in West Bengal. This report indicates that foodborne infection is a major public health concern in India and demands to strengthen capacity-building measures at the local health care levels for linking causative agents of outbreaks.

Complete genomic sequence of Vibrio fluvialis strain IDH5335 isolated from a patient with diarrhea in Kolkata, India.

We isolated a Vibrio fluvialis strain (IDH5335) from a stool sample collected from a patient with diarrhea. In this announcement, we report the complete genomic sequence of this organism, which was obtained by combining Illumina and Oxford Nanopore sequencing data.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected 12% more Shigella and 13% more ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Pentavalent outer membrane vesicles immunized mice sera confers passive protection against five prevalent pathotypes of diarrhoeagenic Escherichia coli in neonatal mice.

Diarrhoeagenic Escherichia coli (DEC) pathotypes are one of the major causative agents of diarrhoea induced childhood morbidity and mortality in developing countries. Licensed vaccines providing broad spectrum protection against DEC mediated infections are not available. Outer membrane vesicles (OMVs) are microvesicles released by gram-negative bacteria during the growth phase and contain multiple immunogenic proteins. Based on prevalence of infections, we have formulated a pentavalent outer-membrane vesicles (POMVs) based immunogen targeting five main pathotypes of DEC responsible for diarrhoeal diseases. Following isolation, OMVs from five DEC pathotypes were mixed in equal proportions to formulate POMVs and 10 µg of the immunogen was intraperitoneally administered to adult BALB/c mice. Three doses of POMVs induced significant humoral immune response against whole cell lysates (WCLs), outer membrane proteins (OMPs) and lipopolysaccharides (LPS) isolated from DEC pathotypes along with significant induction of cellular immune response in adult mice. Passive transfer of POMVs immunized adult mice sera protected neonatal mice significantly against DEC infections. Overall, this study finds POMVs to be immunogenic in conferring broad-spectrum passive protection to neonatal mice against five main DEC pathotypes. Altogether, these findings suggest that POMVs can be used as a potent vaccine candidate to ameliorate the DEC-mediated health burden.

The gut microbiome of the healthy population in Kolkata, India, is a reservoir of antimicrobial resistance genes emphasizing the need of enforcing antimicrobial stewardship.

Antimicrobial resistance (AMR) alleviation warrants antimicrobial stewardship (AS) entailing the indispensability of epidemiological surveillance. We undertook a small-scale surveillance in Kolkata to detect the presence of antimicrobial resistance genes (ARGs) in the healthy gut microbiome. We found that it was a reservoir of ARGs against common antibiotics. A targeted Polymerase Chain Reaction (PCR) and sequencing-based ARGs detection against tetracyclines, macrolides, trimethoprim, sulfamethoxazole, aminoglycosides, amphenicol, and mobile genetic element (MGE) markers were deployed in 25 fecal samples. Relative abundance and frequency of ARGs were calculated. We detected markers against all these classes of antibiotics. 100% samples carried aminoglycoside resistance marker and int1U. A comparison with our previously published diarrheal resistome from the same spatial and temporal frame revealed that a higher diversity of ARGs were detected in the community and a higher rate of isolation of tetC, msrA, tmp, and sul-2 was found. The presence of common markers in the two cohorts proves that the gut microbiome has been contaminated with ARGs and which are being disseminated among different ecosystems. This is an issue of discerning concern for public health. The study raises an alarming picture of the AMR crisis in low-middle and emergent economies. It emphasizes the strict enforcement of AS in the community.

Helicobacter pylori attachment-blocking antibodies protect against duodenal ulcer disease.

The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabA's binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.

Glycyrrhizin, an inhibitor of HMGB1 induces autolysosomal degradation function and inhibits Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori is a key agent for causing gastric complications linked with gastric disorders. In response to infection, host cells stimulate autophagy to maintain cellular homeostasis. However, H. pylori have evolved the ability to usurp the host's autophagic machinery. High mobility group box1 (HMGB1), an alarmin molecule is a regulator of autophagy and its expression is augmented during infection and gastric cancer. Therefore, this study aims to explore the role of glycyrrhizin (a known inhibitor of HMGB1) in autophagy during H. pylori infection. MAIN METHODS: Human gastric cancer (AGS) cells were infected with the H. pylori SS1 strain and further treatment was done with glycyrrhizin. Western blot was used to examine the expression of autophagy proteins. Autophagy and lysosomal activity were monitored by fluorescence assays. A knockdown of HMGB1 was performed to verify the effect of glycyrrhizin. H. pylori infection in in vivo mice model was established and the effect of glycyrrhizin treatment was studied. RESULTS: The autophagy-lysosomal pathway was impaired due to an increase in lysosomal membrane permeabilization during H. pylori infection in AGS cells. Subsequently, glycyrrhizin treatment restored the lysosomal membrane integrity. The recovered lysosomal function enhanced autolysosome formation and concomitantly attenuated the intracellular H. pylori growth by eliminating the pathogenic niche. Additionally, glycyrrhizin treatment inhibited inflammation and improved gastric tissue damage in mice. CONCLUSION: This study showed that inhibiting HMGB1 restored lysosomal activity to ameliorate H. pylori infection. It also demonstrated the potential of glycyrrhizin as an antibacterial agent to address the problem of antimicrobial resistance.

Development and evaluation of a simple PCR assay and nested PCR for rapid detection of clarithromycin-resistant Helicobacter pylori from culture and directly from the biopsy samples in India.

BACKGROUND: Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. RESULTS: Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based results. Additionally, the sequencing study also identified the A to G mutation at 2143 position in 23S rRNA gene of the resistant strains only. Consequently, the newly developed Nested-ASP-PCR dealing directly with 50 biopsy specimens demonstrated 100% sensitivity and specificity with the findings of agar dilution method taken as Gold standard. Bioinformatics based analysis such as accessibility analysis and dot plot clearly stated that the base pairing probability has increased due to mutation. Computational studies revealed that the point mutation confers more stability in secondary structure due to conversion of loop to stem. Furthermore, interaction studies showed binding affinity of the CLR to the mutant type is weaker than that to the wild type. CONCLUSION: This assay outlines a rapid, sensitive and simple approach to identify point mutation that confers clarithromycin resistance as well as clarithromycin sensitive strains, providing rapid initiation of effective antibiotic treatment. Additionally, it is simple to adopt for hospital based diagnostic laboratories to evaluate the degree of regional clarithromycin resistance from biopsy specimens itself. Furthermore, in silico studies provide evidence or a signal that the prevalence of clarithromycin resistance may rise in the near future as a result of this point mutation.

Environmental water in Kolkata is suitable for the survival of Vibrio cholerae O1.

Many patients with cholera emerge in Kolkata, India throughout the year. Such emergency indicates that cholera toxin-producing Vibrio cholerae O1 (toxigenic V. cholerae O1) are widespread in Kolkata. This suggests that the suitable conditions for replication of toxigenic V. cholerae O1 is provided in Kolkata. In previous studies, we found that the replication rate of toxigenic V. cholerae O1 is low in the low ionic aqueous solution. Then we measured the ion concentration in the environmental water of Kolkata. As a control, we measured them in Japanese environmental water. The ion concentration in the environmental water of Kolkata was significantly high. Then, we examined the survival of toxigenic V. cholerae O1 in groundwater from Kolkata and found that V. cholerae O1 survive for long time in the solution but not in the solution diluted with Milli Q water. In addition, we found that V. cholerae O1 proliferated in environmental water of Kolkata to which a small amount of nutrient was added, but did not grow in the environmental water diluted with water to which the same amount of nutrient was added. These results indicate that the environmental water from Kolkata is suitable for survival of V. cholerae O1.

Deciphering the genetic network and programmed regulation of antimicrobial resistance in bacterial pathogens.

Antimicrobial resistance (AMR) in bacteria is an important global health problem affecting humans, animals, and the environment. AMR is considered as one of the major components in the "global one health". Misuse/overuse of antibiotics in any one of the segments can impact the integrity of the others. In the presence of antibiotic selective pressure, bacteria tend to develop several defense mechanisms, which include structural changes of the bacterial outer membrane, enzymatic processes, gene upregulation, mutations, adaptive resistance, and biofilm formation. Several components of mobile genetic elements (MGEs) play an important role in the dissemination of AMR. Each one of these components has a specific function that lasts long, irrespective of any antibiotic pressure. Integrative and conjugative elements (ICEs), insertion sequence elements (ISs), and transposons carry the antimicrobial resistance genes (ARGs) on different genetic backbones. Successful transfer of ARGs depends on the class of plasmids, regulons, ISs proximity, and type of recombination systems. Additionally, phage-bacterial networks play a major role in the transmission of ARGs, especially in bacteria from the environment and foods of animal origin. Several other functional attributes of bacteria also get successfully modified to acquire ARGs. These include efflux pumps, toxin-antitoxin systems, regulatory small RNAs, guanosine pentaphosphate signaling, quorum sensing, two-component system, and clustered regularly interspaced short palindromic repeats (CRISPR) systems. The metabolic and virulence state of bacteria is also associated with a range of genetic and phenotypic resistance mechanisms. In spite of the availability of a considerable information on AMR, the network associations between selection pressures and several of the components mentioned above are poorly understood. Understanding how a pathogen resists and regulates the ARGs in response to antimicrobials can help in controlling the development of resistance. Here, we provide an overview of the importance of genetic network and regulation of AMR in bacterial pathogens.

Metagenomic analysis of diarrheal stools in Kolkata, India, indicates the possibility of subclinical infection of Vibrio cholerae O1.

We examined the stools of 23 patients in Kolkata, who were diagnosed as cholera patients because Vibrio cholerae O1 was detected from their stools by culturing methods, and further explored by metagenomic sequencing analysis. Subsequently, the presence of the gene encoding A subunit of cholera toxin (ctxA) and the cholera toxin (CT) level in these stool samples were examined. ctxA was examined by both metagenomic sequencing analysis and polymerase chain reaction. In these examinations, two samples did not show positive in any of these tests. The metagenomic analysis showed that the genes for Streptococcus pneumoniae and Salmonella enterica were present in the stools of these two patients, respectively. Therefore, these two patients were not considered to have diarrhea due to V. cholerae infection. From these results, we predicted that some Kolkata residents harbor a small number of V. cholerae in their intestines as a form of subclinical infection with V. cholerae. Next, we analyzed the stool samples of 22 diarrhea patients from which V. cholerae was not isolated. The results showed that 3 of the patients seemed to have subclinical infection of V. cholerae based on the amount of the genes. These results indicated that subclinical infections with V. cholerae O1 occur in Kolkata.

Biomass derived self-assembled DNA-dot hydrogels for enhanced bacterial annihilation.

Nanotization of biomass for interesting biomedical applications is still in the nascent stage with no visible market available products. While products derived from biomass DNA and protein have unquestionable biocompatibility, induction of desired properties needs careful manipulation of the biomolecules. Herein, for the first time, we report the transformation of onion derived biomass DNA into DNA-dots through its partial hydrothermal pyrolysis to induce improved mechanical and photophysical properties. The DNA-dots were further used as crosslinkers to create a hydrogel through hybridization-mediated self-assembly with untransformed genomic DNA. The DNA dot-DNA hydrogel sustainably delivers the ciprofloxacin antibiotic as well as produces on-demand reactive oxygen species (ROS) with visible light irradiation. This prompted us to explore the hydrogel as a topical formulation for combination antibiotic Antibacterial-Photodynamic Therapy (APDT) applications. Remarkable annihilation of E. coli and S. aureus, and most importantly two drug-resistant strains of E. coli, shows the success of our sustainable approach.

Protective efficacy of fish oil nanoemulsion against non-typhoidal Salmonella mediated mucosal inflammation and loss of barrier function.

Non-typhoidal Salmonella serotypes are well adapted to utilize the inflammation for colonization in the mammalian gut mucosa and cause loss of the integrity of the epithelial barrier in the mammalian intestine. The present study assessed the protective efficacy of fish oil-in-water nanoemulsion, compared to the conventional emulsion, towards the intestinal epithelial barrier against invasive infection of Salmonella enterica serovar Typhimurium strain SL1344 in an in vivo streptomycin-treated mouse model. Non-typhoidal Salmonella enterica serovar Typhimurium strain SL1344 expresses its invasiveness by creating extreme inflammatory assault in the mammalian host lumen via its repertoire of secretory or membrane-bound proteins. Prophylactic treatment of ω-3 polyunsaturated fatty acid-rich fish oil nanoemulsion not only reduced the inflammatory markers by 4-5 fold against the established infection but also retained the gut barrier efficiency as shown by FITC-dextran permeability assay. Though the conventional emulsion also showed similar trends, the efficacy was significantly better with nanoemulsion treatment but neither the nanoemulsion nor conventional emulsion caused any significant change in the microbial colonization of the murine gut mucosa. Mechanistic assessment of the nanoemulsion against inflammation and invasion across the Caco-2 cell monolayer revealed that nanoemulsion treatment protected the expression of Zona occludens-1 along the tight junction, almost by 3-fold as compared to the infected cell monolayer. Such protection was evinced by the trans-epithelial electrical resistance value and the FITC-dextran permeability analysis as well. Fish oil nanoemulsion treatment has also shown significant reduction in pro-inflammatory cytokine expression by the Salmonella strain SL1344 infected Caco-2 cell monolayer. Conventional emulsion also showed distinct protection, but the nanoemulsion offered better protection at the same dosage of fish oil, probably due to its better bioavailability. The results proved that fish oil-loaded nanoemulsion can be efficacious towards maintaining the barrier function and protecting against systemic bacteremia during invasive intestinal infection.

Sequence Polymorphisms in Vibrio cholerae HapR Affect Biofilm Formation under Aerobic and Anaerobic Conditions.

We investigated the influence of hapR sequence mutations on the biofilm formation of Vibrio cholerae. In this study, hapR sequences from 85 V. cholerae strains belonging to both pandemic and nonpandemic serogroup were investigated through phylogenetic and sequence analyses. Biofilm formation assays under aerobic and anaerobic conditions were also performed. Sequence variations include single point mutations and insertions/deletions (indels) leading to either truncated or frameshifted HapR. Population structure analysis revealed two major hapR haplogroups, hapR1 and hapR2. Phylogenetic reconstruction displayed a hypothetical ancestral hapR sequence located within the hapR1 haplogroup. Higher numbers of single nucleotide polymorphisms and genetic diversity indices were observed in hapR1, while indels occurred dominantly in hapR2. Aerobic conditions supported more robust biofilms compared to anaerobic conditions. Strains with frameshifted HapR produced the largest amount of biofilm under both oxygen conditions. Quantitative real-time PCR assay confirmed that strains with truncated and frameshifted HapR resulted in a nonfunctional regulator as exhibited by the significantly low hapA gene expression. The present study shows that HapR mutations had a strong influence on biofilm formation and that sequence polymorphisms leading to the disruption of DNA-binding sites or dimerization of the HapR will result in more-robust V. cholerae biofilms. IMPORTANCE Our study revealed an ancestral hapR sequence from a phylogenetic reconstruction that displayed the evolutionary lineage of the nonpandemic to the pandemic strains. Here, we established hapR1 and hapR2 as major hapR haplogroups. The association of the O1 and O139 serogroups with the hapR2 haplogroup demonstrated the distinction of hapR2 in causing cholera infection. Moreover, mutations in this regulator that could lead to the disruption of transcription factor-binding sites or dimerization of the HapR can significantly affect the biofilm formation of V. cholerae. These observations on the relationship of the hapR polymorphism and V. cholerae biofilm formation will provide additional considerations for future biofilm studies and insights into the epidemiology of the pathogen that could ultimately help in the surveillance and mitigation of future cholera disease outbreaks.

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

AIMS: 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). METHODS AND RESULTS: A total of 87 V. 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Vibrio cholerae O139 genomes provide a clue to why it may have failed to usher in the eighth cholera pandemic.

Cholera is a life-threatening infectious disease that remains an important public health issue in several low and middle-income countries. In 1992, a newly identified O139 Vibrio cholerae temporarily displaced the O1 serogroup. No study has been able to answer why the potential eighth cholera pandemic (8CP) causing V. cholerae O139 emerged so successfully and then died out. We conducted a genomic study, including 330 O139 isolates, covering emergence of the serogroup in 1992 through to 2015. We noted two key genomic evolutionary changes that may have been responsible for the disappearance of genetically distinct but temporally overlapping waves (A-C) of O139. Firstly, as the waves progressed, a switch from a homogenous toxin genotype in wave-A to heterogeneous genotypes. Secondly, a gradual loss of antimicrobial resistance (AMR) with the progression of waves. We hypothesize that these two changes contributed to the eventual epidemiological decline of O139.