Vibrio cholerae O1 associated with recent endemic cholera shows temporal changes in serotype, genotype, and drug-resistance patterns in Bangladesh.

BACKGROUND: Despite the advancement in our understanding of cholera and its etiological agent, Vibrio 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 CTX-prophage, 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 six 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 ß-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.

Genomic attributes of Vibrio cholerae O1 responsible for 2022 massive cholera outbreak in Bangladesh.

In 2022, one of its worst cholera outbreaks began in Bangladesh and the icddr,b Dhaka hospital treated more than 1300 patients and ca. 42,000 diarrheal cases from March-1 to April-10, 20221. Here, we present genomic attributes of V. cholerae O1 responsible for the 2022 Dhaka outbreak and 960 7th pandemic El Tor (7PET) strains from 88 countries. Results show strains isolated during the Dhaka outbreak cluster with 7PET wave-3 global clade strains, but comprise subclade BD-1.2, for which the most recent common ancestor appears to be that responsible for recent endemic cholera in India. BD-1.2 strains are present in Bangladesh since 2016, but not establishing dominance over BD-2 lineage strains2 until 2018 and predominantly associated with endemic cholera. In conclusion, the recent shift in lineage and genetic attributes, including serotype switching of BD-1.2 from Ogawa to Inaba, may explain the increasing number of cholera cases in Bangladesh.

Vibrio tarriae sp. nov., a novel member of the Cholerae clade.

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.

Vibrio cholerae O1 El Tor strains linked to global cholera show region-specific patterns by pulsed-field gel electrophoresis.

Vibrio cholerae O1 El Tor, causative agent of the ongoing seventh cholera pandemic, is native to the aquatic environment of the Ganges Delta, Bay of Bengal (GDBB). Recent studies traced pandemic strains to the GDBB and proposed global spread of cholera had occurred via intercontinental transmission. In the research presented here, NotI-digested genomic DNA extracted from V. cholerae O1 clinical and environmental strains isolated in Bangladesh during 20042014 was analyzed by pulsed-field gel electrophoresis (PFGE). Results of cluster analysis showed 94.67% of the V. cholerae strains belonged to clade A and included the majority of clinical strains of spatio-temporal origin and representing different cholera endemic foci. The rest of the strains were estuarine, all environmental strains from Mathbaria, Bangladesh, and occurred as singletons, clustered in clades B and C, or in the small clades D and E. Cluster analysis of the Bangladeshi strains and including 157 El Tor strains from thirteen countries in Asia, Africa, and the Americas revealed 85% of the total set of strains belonged to clade A, indicating all were related, yet did not form an homogeneous cluster. Overall, 15% of the global strains comprised multiple small clades or segregated as singletons. Three sub-clades could be discerned within the major clade A, reflecting distinct lineages of V. cholerae O1 El Tor associated with cholera in Asia, Africa, and the Americas. The presence in Asia and the Americas of non-pandemic V. cholerae O1 El Tor populations differing by PFGE and from strains associated with cholera globally suggests different ecotypes are resident in distant geographies.

Dynamic Subspecies Population Structure of Vibrio cholerae in Dhaka, Bangladesh.

Cholera has been endemic to the Ganges Delta for centuries. Although the causative agent, Vibrio cholerae, is autochthonous to coastal and brackish water, cholera occurs continually in Dhaka, the inland capital city of Bangladesh which is surrounded by fresh water. Despite the persistence of this problem, little is known about the environmental abundance and distribution of lineages of V. cholerae, the most important being the pandemic generating (PG) lineage consisting mostly of serogroup O1 strains. To understand spatial and temporal dynamics of PG lineage and other lineages belonging to the V. cholerae species in surface water in and around Dhaka City, we used qPCR and high-throughput amplicon sequencing. Seven different freshwater sites across Dhaka were investigated for six consecutive months, and physiochemical parameters were measured in situ. Total abundance of V. cholerae was found to be relatively stable throughout the 6-month sampling period, with 2 × 105 to 4 × 105 genome copies/L at six sites and around 5 × 105 genome copies/L at the site located in the most densely populated part of Dhaka City. PG O1 V. cholerae was present in high abundance during the entire sampling period and composed between 24 and 92% of the total V. cholerae population, only showing occasional but sudden reductions in abundance. In instances where PG O1 lost its dominance, other lineages underwent a rapid expansion while the size of the total V. cholerae population remained almost unchanged. Intraspecies richness of V. cholerae was positively correlated with salinity, conductivity, and total dissolved solids (TDS), while it was negatively correlated with dissolved oxygen (DO) concentration in water. Interestingly, negative correlation was observed specifically between PG O1 and salinity, even though the changes in this variable were minor (0-0.8 ppt). Observations in this study suggest that at the subspecies level, population composition of naturally occurring V. cholerae can be influenced by fluctuations in environmental factors, which can lead to altered competition dynamics among the lineages.

Computational screening of 645 antiviral peptides against the receptor-binding domain of the spike protein in SARS-CoV-2.

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.

Population Analysis of Vibrio cholerae in Aquatic Reservoirs Reveals a Novel Sister Species (Vibrio paracholerae sp. nov.) with a History of Association with Humans.

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.

Simultaneous Quantification of Vibrio metoecus and Vibrio cholerae with Its O1 Serogroup and Toxigenic Subpopulations in Environmental Reservoirs.

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.

A Vibrio cholerae Core Genome Multilocus Sequence Typing Scheme To Facilitate the Epidemiological Study of Cholera.

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 for Vibrio cholerae, a bacterium abundant in marine and freshwater environments and the etiologic agent of cholera. A set of 2,443 core genes ubiquitous in V. cholerae were used to analyze a comprehensive data set 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 that 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 for V. cholerae research and epidemiological surveillance in the future. This cgMLST scheme is publicly available on PubMLST (https://pubmlst.org/vcholerae/).IMPORTANCE Toxigenic Vibrio cholerae isolates of the O1 and O139 serogroups are the causative agents of cholera, an acute diarrheal disease that plagued the world for centuries, if not millennia. Here, we introduce a core genome multilocus sequence typing scheme for V. cholerae Using this scheme, we have standardized the definition for subspecies-level classification, facilitating global collaboration in the surveillance of V. cholerae In addition, this typing scheme allows for quick identification of outbreak-related isolates that can guide subsequent analyses, serving as an important first step in epidemiological research. This scheme is also easily scalable to analyze thousands of isolates at various levels of resolution, making it an invaluable tool for large-scale ecological and evolutionary analyses.

Draft Genome Sequences of Eight Vibrio sp. Clinical Isolates from across the United States That Form a Basal Sister Clade to 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.

Draft Genome Sequences of Nine Vibrio sp. Isolates from across the United States Closely Related to Vibrio cholerae.

We are reporting whole-genome sequences of nine Vibrio sp. isolates closely related to the waterborne human pathogen Vibrio cholerae. These isolates were recovered from sources, including human samples, from different regions of the United States. Genome analysis suggests that this group of isolates represents a highly divergent basal V. cholerae lineage or a closely related novel species.