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      Characterization of Vibrio cholerae isolates from freshwater sources in northwest Ohio

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          Abstract

          Vibrio cholerae is a natural inhabitant of aquatic ecosystems worldwide, typically residing in coastal or brackish water. While more than 200 serogroups have been identified, only serogroups O1 and O139 have been associated with epidemic cholera. However, infections other than cholera can be caused by nonepidemic, non-O1/non-O139 V. cholerae strains, including gastroenteritis and extraintestinal infections. While V. cholerae can also survive in freshwater, that is typically only observed in regions of the world where cholera is endemic. We recently isolated V. cholerae from several locations in lakes and rivers in northwest Ohio. These isolates were all found to be non-O1/non-O139 V. cholerae strains, that would not cause cholera. However, these isolates contained a variety of virulence genes, including ctxA, rtxA, rtxC, hlyA, and ompU. Therefore, it is possible that some of these isolates have the potential to cause gastroenteritis or other infections in humans. We also investigated the relative motility of the isolates and their ability to form biofilms as this is important for V. cholerae survival in the environment. We identified one isolate that forms very robust biofilms, up to 4x that of our laboratory strains. Finally, we investigated the susceptibility of these isolates to a panel of antibiotics. We found that many of the isolates showed decreased susceptibility to some of the antibiotics tested, which could be of concern. While we do not know if these isolates are pathogenic to humans, increased surveillance to better understand the public health risk to the local community should be considered.

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          Most cited references32

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          Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae.

          Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. The disease is characterized by a devastating watery diarrhea which leads to rapid dehydration, and death occurs in 50 to 70% of untreated patients. Cholera is a waterborne disease, and the importance of water ecology is suggested by the close association of V. cholerae with surface water and the population interacting with the water. Cholera toxin (CT), which is responsible for the profuse diarrhea, is encoded by a lysogenic bacteriophage designated CTXPhi. Although the mechanism by which CT causes diarrhea is known, it is not clear why V. cholerae should infect and elaborate the lethal toxin in the host. Molecular epidemiological surveillance has revealed clonal diversity among toxigenic V. cholerae strains and a continual emergence of new epidemic clones. In view of lysogenic conversion by CTXPhi as a possible mechanism of origination of new toxigenic clones of V. cholerae, it appears that the continual emergence of new toxigenic strains and their selective enrichment during cholera outbreaks constitute an essential component of the natural ecosystem for the evolution of epidemic V. cholerae strains and genetic elements that mediate the transfer of virulence genes. The ecosystem comprising V. cholerae, CTXPhi, the aquatic environment, and the mammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen.
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            Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans

            Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.
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              Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW.

              The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 ( approximately 98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.
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                Author and article information

                Contributors
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 September 2020
                2020
                : 15
                : 9
                : e0238438
                Affiliations
                [001]Department of Medical Microbiology and Immunology, University of Toledo, Toledo, Ohio, United States of America
                Western Michigan University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤a]

                Current address: Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America

                [¤b]

                Current address: Division of Integrated Biomedical Sciences, University of Detroit Mercy School of Dentistry, Detroit, Michigan, United States of America

                [¤c]

                Current address: Department of Biological Sciences, University of Toledo, Toledo, Ohio, United States of America

                Author information
                http://orcid.org/0000-0002-7160-4771
                Article
                PONE-D-20-15102
                10.1371/journal.pone.0238438
                7470319
                32881972
                5c2d6b02-7cb9-4197-a8bf-1ecadf60ac09
                © 2020 Daboul et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 May 2020
                : 17 August 2020
                Page count
                Figures: 3, Tables: 2, Pages: 12
                Funding
                This study was supported by startup funds from the University of Toledo (JSM). JD and LW were supported by funds from the UT Medical Student Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Vibrio Cholerae
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Vibrio Cholerae
                Biology and Life Sciences
                Organisms
                Bacteria
                Vibrio
                Vibrio Cholerae
                Biology and Life Sciences
                Microbiology
                Bacteriology
                Bacterial Biofilms
                Biology and Life Sciences
                Microbiology
                Biofilms
                Bacterial Biofilms
                Biology and Life Sciences
                Toxicology
                Toxic Agents
                Toxins
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Toxicology
                Toxic Agents
                Toxins
                Medicine and Health Sciences
                Medical Conditions
                Infectious Diseases
                Bacterial Diseases
                Cholera
                Medicine and Health Sciences
                Medical Conditions
                Tropical Diseases
                Neglected Tropical Diseases
                Cholera
                Ecology and Environmental Sciences
                Aquatic Environments
                Freshwater Environments
                Fresh Water
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Freshwater Environments
                Fresh Water
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Virulence Factors
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Antibiotic Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Antibiotic Resistance
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Virulence Factors
                Pathogen Motility
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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