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Background Despite the advancement in our understanding of cholera and its etiological agent, V. cholerae, the prevention and treatment of the disease are often hindered due to rapid changes in drug response pattern, serotype, and the major genomic islands namely, the cholera toxin phage, and related genetic characteristics. In the present study, V. cholerae (n = 172) associated with endemic cholera in Dhaka during the years 2015–2021 were analyzed for major phenotypic and genetic characteristics, including drug resistance patterns. Results Results revealed that the V. cholerae strains belonged to serogroup O1 biotype El Tor carrying El Tor -specific genes rtxC, tcpA El Tor, and hlyA El Tor, but possessed classical-biotype cholera toxin. Serotypes of V. cholerae strains differed temporally in predominance with Inaba during 2015–2017, and again in 2020–2021, while Ogawa was the predominant serotype in 2018–2019. Also, ctxB1 was predominant in V. cholerae associated with cholera during 2015–2017, while ctxB7 was predominant in 2018, and in the subsequent years, as observed until 2021. V. cholerae strains differed in their antibiotic resistance pattern with a majority (97%) being multi-drug resistant (MDR) and belonging to eight sub-groups. Notably, one of these MDR strains was resistant to eleven of the eighteen antibiotics tested, with resistance to fourth-generation cephalosporin (cefepime), and aztreonam. This extreme drug resistant (XDR) strain carried resistance-related genes namely, extended-spectrum β-lactamases (ESBL), blaOXA-1 and blaPER-3. Conclusion The observed temporal switching of serotypes, as well as the ctxB genotype, and the emergence of MDR/XDR V. cholerae and their association with endemic cholera in Dhaka underscore the need for routine monitoring of the pathogen for proper patient management.

Bangladesh is currently experiencing one of the worst cholera outbreaks in its history. The icddr,b hospital has treated a record number of patients, more than 1400 per day and ca. 40,000 diarrheal cases from the end of March through April 20221. A recent genomic study showed temporal progression of two lineages, BD-1 and BD-2, with the former linked to the 7th pandemic wave-3 global clade and the latter predominant in endemic cholera in Dhaka during 2013 and 20172. Here, we present genomic attributes of V. cholerae O1 responsible for the 2022 Dhaka cholera epidemic and genome phylogeny of 960 7th pandemic El Tor strains from 88 countries. Results show the Dhaka cholera etiological agent clustered with the 7th pandemic El Tor wave-3 global clade, but comprises a new subclade, BD-1.2, for which the most recent common ancestor appears to be of the globally distributed sublineage predominantly associated with recent endemic cholera in India. Results also suggest BD-1.2 was present in Bangladesh since 2016. However, it was not until 2018 that strains of the subclade successfully established dominance over the BD-2 during an expansion of the wave-3 global clade. It is concluded that the recent shift in predominant lineage and the observed genetic changes including serotype switch in BD-1.2 from Ogawa to Inaba may explain the increasing number of infections and massive outbreak of cholera during 2022 in Bangladesh.

A number of bacteria with close resemblance to Vibrio cholerae have been isolated over the years by the Centres for Disease Control and Prevention (CDC), which could not be assigned a proper taxonomic designation on the basis of the results from preliminary identification methods. Nine such isolates have been found to share 16S rRNA gene identity exceeding 99 % with V. cholerae, yet DNA–DNA hybridization (60.4–62.1 %) and average nucleotide identity values (94.4–95.1 %) were below the species cut-off, indicating a potentially novel species. Phylogenetic analysis of core genomes places this group of isolates in a monophyletic clade, within the ‘Cholerae clade’, but distinct from any other species. Extensive phenotypic characterization reveals unique biochemical properties that distinguish this novel species from V. cholerae . Comparative genomic analysis reveals a unique set of siderophore genes, indicating that iron acquisition strategies could be vital for the divergence of the novel species from a common ancestor with V. cholerae . On the basis of the genetic, phylogenetic and phenotypic differences observed, we propose that these isolates represent a novel species of the genus Vibrio , for which the name Vibrio tarriae sp. nov. is proposed. Strain 2521-89 T (= DSM 112461=CCUG 75318), isolated from lake water, is the type strain.

Successful treatment against infectious agents depends on rapid and accurate detection of the causative organisms. Misdiagnosis can hamper such success while leading to improper advising of antibiotics. In Bangladesh, the majority of the diagnostic centers detect and identify pathogens through culture and biochemical test-based methods and suggest antibiotics based solely on the results of disk-diffusion methods. This pilot study tried to validate the identity of the isolates characterized by diagnostic facilities near Dhaka. One hundred and twenty pre-characterized clinical isolates were analyzed biochemically and genotypically. Random Amplification of Polymorphic DNA-PCR, rcsA, and phoA genes-based PCR, and Loop-Mediated Isothermal Amplification (LAMP)-based identification of Klebsiella pneumoniae and Escherichia coli, respectively, followed by 16S rRNA sequencing confirmed misidentification of some clinical pathogens of other genera as Klebsiella spp. and E. coli. According to the antibiotic susceptibility testing guidelines, antibiotic choice, sensitivity pattern, and breakpoint measurement are different for each group of organisms. The lack of adherence to proper standards results in misdiagnosis and may facilitate the development of antibiotic resistance. The pilot study observers misidentification of clinical pathogens identified by the diagnostic centers. Well-characterized rapid molecular techniques like LAMP are suggested in clinical diagnosis to avoid misdiagnosis and subsequently circumvent antibiotic resistance development.

Most efforts to understand the biology of Vibrio cholerae have focused on a single group, the pandemic-generating lineage harboring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in the V. cholerae populations inhabiting regions with a history of cholera cases and those lacking such a history, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka, Bangladesh (where cholera is endemic), located in the Ganges Delta, and those in Falmouth, MA (no known history of cholera), a small coastal town on the United States east coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka, which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny for the V. cholerae species and were sufficiently differentiated genetically and phenotypically to form a novel species. A retrospective search revealed that strains from this species have been anecdotally found from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhea. In 1935, Gardner and Venkatraman unofficially referred to a member of this group as Vibrio paracholerae. In recognition of this earlier designation, we propose the name Vibrio paracholerae sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species. IMPORTANCE Cholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution, and environmental adaptation of the causative agent (Vibrio cholerae) and tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics of Vibrio cholerae in an inland locality, which is known as endemic for cholera, and compared them with those of a cholera-free coastal location. We found the consistent presence of the pandemic-generating lineage of V. cholerae in Dhaka, where cholera is endemic, and an exclusive presence of a lineage phylogenetically distinct from other V. cholerae lineages. Our study suggests that this lineage represents a novel species that has pathogenic potential and a human link to its environmental abundance. The possible association with human populations and coexistence and interaction with toxigenic V. cholerae in the natural environment make this potential human pathogen an important subject for future studies.

The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in binding and internalization through the alpha-helix (AH) of human angiotensin-converting enzyme 2 (hACE2). Thus, it is a potential target for designing and developing antiviral agents. Inhibition of RBD activity of the S protein may be achieved by blocking RBD interaction with hACE2. In this context, inhibitors with large contact surface area are preferable as they can form a potentially stable complex with RBD of S protein and would not allow RBD to come in contact with hACE2. Peptides represent excellent features as potential anti-RBD agents due to better efficacy, safety, and tolerability in humans compared to that of small molecules. The present study has selected 645 antiviral peptides known to inhibit various viruses and computationally screened them against the RBD of SARS-CoV-2 S protein. In primary screening, 27 out of 645 peptides exhibited higher affinity for the RBD of S protein compared to that of AH of the hACE2 receptor. Subsequently, AVP1795 appeared as the most promising candidate that could inhibit hACE2 recognition by SARS-CoV 2 as was predicted by the molecular dynamics simulation. The critical residues in RBD found for protein-peptide interactions are TYR 489, GLY 485, TYR 505, and GLU 484. Peptide-protein interactions were substantially influenced by hydrogen bonding and hydrophobic interactions. This comprehensive computational screening may provide a guideline to design the most effective peptides targeting the spike protein, which could be studied further in vitro and in vivo for assessing their anti-SARS CoV-2 activity., Graphical abstract Image 1

Most efforts to understand the biology ofVibrio choleraehave focused on a single group, the pandemic-generating lineage harbouring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in theV. choleraepopulations inhabiting in regions with varying history of cholera cases and how that might influence the abundance of pandemic strains, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka (cholera endemic) located in the Ganges delta, and of Falmouth (no known history of cholera), a small coastal town on the US East Coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny ofV. choleraespecies, sufficiently differentiated genetically and phenotypically to form a novel species. Strains from this species have been anecdotally isolated from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhoea. In 1935 Gardner and Venkatraman unofficially referred to a member of this group asVibrio paracholerae. In recognition of this earlier designation, we propose the nameVibrio paracholerae, sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species.ImportanceCholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution and environmental adaptation of the causative agentVibrio choleraeand tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics ofVibrio choleraein an inland locality which is known as endemic for cholera and compared with that of a cholera free coastal location. We found the consistent presence of the pandemic generatingV. choleraein cholera-endemic Dhaka and an exclusive presence of a lineage phylogenetically distinct from otherV. cholerae. Our study suggests that this lineage represents a novel species having pathogenic potential and a human link to its environmental abundance. The possible association with human population, co-existence and interaction with toxigenicV. choleraein the natural environment make this potential human pathogen an important subject for future studies.

Vibrio metoecus is a recently described aquatic bacterium and opportunistic pathogen, closely related to and often coexisting with Vibrio cholerae. To study the relative abundance and population dynamics of both species in aquatic environments of cholera-endemic and cholera-free regions, we developed a multiplex qPCR assay allowing simultaneous quantification of total V. metoecus and V. cholerae (including toxigenic and O1 serogroup) cells. The presence of V. metoecus was restricted to samples from regions that are not endemic for cholera, where it was found at 20% of the abundance of V. cholerae. In this environment, non-toxigenic O1 serogroup V. cholerae represents almost one-fifth of the total V. cholerae population. In contrast, toxigenic O1 serogroup V. cholerae was also present in low abundance on the coast of cholera-endemic regions, but sustained in relatively high proportions throughout the year in inland waters. The majority of cells from both Vibrio species were recovered from particles rather than free-living, indicating a potential preference for attached versus planktonic lifestyles. This research further elucidates the population dynamics underpinning V. cholerae and its closest relative in cholera-endemic and non-endemic regions through culture-independent quantification from environmental samples.

Core genome multilocus sequence typing (cgMLST) has gained popularity in recent years in epidemiological research and subspecies level classification. cgMLST retains the intuitive nature of traditional MLST but offers much greater resolution by utilizing significantly larger portions of the genome. Here, we introduce a cgMLST scheme forVibrio cholerae, a bacterium abundant in marine and freshwater environments and the etiologic agent of cholera. A set of 2,443 core genes ubiquitous inV. choleraewere used to analyze a comprehensive dataset of 1,262 clinical and environmental strains collected from 52 countries, including 65 newly sequenced genomes in this study. We established a sublineage threshold based on 133 allelic differences that creates clusters nearly identical to traditional MLST types, providing backwards compatibility to new cgMLST classifications. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying strains from the same outbreak and closely related isolates which could give clues on outbreak origin. Using cgMLST, we confirmed the South Asian origin of modern epidemics and identified clustering affinity among sublineages of environmental isolates from the same geographic origin. Advantages of this method are highlighted by direct comparison with existing classification methods, such as MLST and single nucleotide polymorphism-based methods. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use. We anticipate this scheme will serve as a basis for a universally applicable and standardized classification system forV. choleraeresearch and epidemiological surveillance in the future. This cgMLST scheme is publicly available on PubMLST (https://pubmlst.org/vcholerae/).IMPORTANCEToxigenicVibrio choleraeof the O1 and O139 serogroups are the causative agent of cholera, an acute diarrheal disease that plagued the world for centuries, if not millennia. Here, we introduce a core genome multilocus sequence typing (cgMLST) scheme forV. cholerae. Using cgMLST, we established an outbreak threshold that can efficiently identify outbreak related strains and potential sources of introduction. We also defined a sublineage threshold that is similar to traditional MLST sequence type which will provide context to this new typing method by relating it to previous MLST results. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use, making this scheme the most suitable method forV. choleraetyping and surveillance worldwide.

Vibrio metoecusis a recently described and little studied causative agent of opportunistic infections in humans, often coexisting withV. choleraein aquatic environments. However, the relative abundance ofV. metoecuswithV. cholerae and their population dynamics in aquatic reservoirs is still unknown. We developed a multiplex qPCR assay with a limit of detection of three copies per reaction to simultaneously quantify totalV. metoecusandV. choleraeabundance, as well as the toxigenic and O1 serogroup subpopulations ofV. choleraefrom environmental samples. Four different genes were targeted as specific markers for individualVibriospecies or subpopulations;viuB, a gene encoding a vibriobactin utilization protein, was used to quantify the totalV. choleraepopulation. The cholera toxin genectxAprovided an estimation of toxigenicV. choleraeabundance, while therfbO1gene specifically detected and quantifiedV. choleraebelonging to the O1 serogroup, which includes almost all lineages of the species responsible for the majority of past and ongoing cholera pandemics. To measureV. metoecusabundance, the genemcp, encoding methyl accepting chemotaxis protein, was used. Marker specificity was confirmed by testing several isolates ofV. choleraeandV. metoecusalongside negative controls of isolates within and outside of theVibriogenus. Analysis of environmental water samples collected from four different geographic locations including cholera-endemic (Dhaka, Kuakata and Mathbaria in Bangladesh) and non-endemic (Oyster Pond in Falmouth, Massachusetts, USA) regions showed thatV. metoecuswas only present in the USA site, recurring seasonally. Within the coastal USA site, the non-toxigenic O1 serogroup represented up to ∼18% of the totalV. choleraepopulation.V. choleraetoxigenic O1 serogroup was absent or present in low abundance in coastal Bangladesh (Kuakata and Mathbaria) but constituted a relatively high proportion of the totalV. choleraepopulation sustained throughout the year in inland Bangladesh (Dhaka). A preference for host/particle attachment was observed, as the majority of cells from bothVibriospecies (>90%) were identified in the largest water size fraction sampled, composed of particles or organisms >63 μm and their attached bacteria. This is the first study to apply a culture-independent method to quantifyV. choleraeorV. metoecusdirectly in environmental reservoirs of areas endemic and non-endemic for cholera on significant temporal and spatial scales.SIGNIFICANCECholera is a life-threatening disease that requires immediate intervention; it is of prime importance to have fast, accurate and sensitive means to detectV. cholerae. Consistent environmental monitoring of the abundance ofV. choleraealong with its toxigenic and O1 serogroup subpopulations could facilitate the determination of the actual distribution of this organism in aquatic reservoirs and thus help to predict an outbreak before it strikes. The lack of substantial temporal and spatial environmental sampling, along with specific quantitative measures, has made this goal elusive so far. The same is true forV. metoecus, a close relative ofV. choleraewhich has been associated with several clinical infections and could likely pose an emerging threat, readily exchanging genetic material with its more famous relative.

We sequenced the genomes of eight isolates from various regions of the United States. These isolates form a monophyletic cluster clearly related to but distinct from Vibrio cholerae .

We sequenced the genomes of eight isolates from various regions of the United States. These isolates form a monophyletic cluster clearly related to but distinct from Vibrio cholerae., We sequenced the genomes of eight isolates from various regions of the United States. These isolates form a monophyletic cluster clearly related to but distinct from Vibrio cholerae. Phylogenetic and genomic analyses suggest that they represent a basal lineage highly divergent from Vibrio cholerae or a novel species.

Vibrio cholerae O1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, before the El Tor (ET) biotype initiated the seventh pandemic in Asia in the 1970s by completely displacing the CL biotype. Although the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. cholerae CL and ET biotypes, including a hybrid ET, were found associated with areas of cholera endemicity in Mexico between 1991 and 1997. In this study, CL biotype strains isolated from areas of cholera endemicity in Mexico between 1983 and 1997 were characterized in terms of major phenotypic and genetic traits and compared with CL biotype strains isolated in Bangladesh between 1962 and 1989. According to sero- and biotyping data, all V. cholerae strains tested had the major phenotypic and genotypic characteristics specific for the CL biotype. Antibiograms revealed the majority of the Bangladeshi strains to be resistant to trimethoprim-sulfamethoxazole, furazolidone, ampicillin, and gentamicin, while the Mexican strains were sensitive to all of these drugs, as well as to ciprofloxacin, erythromycin, and tetracycline. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA revealed characteristic banding patterns for all of the CL biotype strains although the Mexican strains differed from the Bangladeshi strains in 1 to 2 DNA bands. The difference was subtle but consistent, as confirmed by the subclustering patterns in the PFGE-based dendrogram, and can serve as a regional signature, suggesting the pre-1991 existence and evolution of the CL biotype strains in the Americas, independent from Asia.