Laboratory Culturing Techniques and Maintenance of Vibrio cholerae.

Cholera is a severe diarrheal disease caused by the consumption of food or water contaminated with the aquatic gram-negative bacterium Vibrio cholerae. Infected hosts will experience vomiting and severe watery diarrhea and if not treated properly will ultimately succumb to death by dehydration. Due to the global prevalence and severity of cholera, V. cholerae has been extensively studied in both environmental and laboratory settings. Herein, we describe proper V. cholerae maintenance, in addition to classical and El Tor biotype culturing in a laboratory setting.

Genotypic and Phenotypic Assays to Distinguish Vibrio cholerae Biotype.

Vibrio cholerae is a motile gram-negative bacterium found in brackish water and the etiological agent of the fecal-oral disease cholera. Classical and El Tor are two main biotypes that make up the V. cholerae O1 serogroup, which each display unique genotypic and phenotypic characteristics that allow for reliable biotype characterization. While treatment for cholera is much the same despite the causative strain's biotype, such classification can be imperative for laboratory experiments and may have broader impacts in the biomedical field. In the early 2000s, clinical isolates were identified that contained genotypic and phenotypic traits from both biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to be more complicated than previous single-assay identification. Herein, we describe a series of PCR-based genetic screens (tcpA and ctxB) and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and Voges-Proskauer). Together, these assays are used for reliable biotype characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000's clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate.

Vibrio cholerae is the etiological agent of the infectious disease cholera, which is characterized by vomiting and severe watery diarrhea. Recently, V. cholerae clinical isolates have demonstrated increased virulence capabilities, causing more severe symptoms with a much higher rate of disease progression than previously observed. We have identified single nucleotide polymorphisms (SNPs) in four virulence-regulatory genes (hapR, hns, luxO, and vieA) of a hypervirulent V. cholerae clinical isolate, MQ1795. Herein, all SNPs and SNP combinations of interest were introduced into the prototypical El Tor reference strain N16961, and the effects on the production of numerous virulence-related factors, including cholera toxin (CT), the toxin-coregulated pilus (TCP), and ToxT, were analyzed. Our data show that triple-SNP (hapR hns luxO and hns luxO vieA) and quadruple-SNP combinations produced the greatest increases in CT, TCP, and ToxT production. The hns and hns luxO SNP combinations were sufficient for increased TCP and ToxT production. Notably, the hns luxO vieA triple-SNP combination strain produced TCP and ToxT levels similar to those of MQ1795. Certain SNP combinations (hapR and hapR vieA) had the opposite effect on CT, TCP, and ToxT expression. Interestingly, the hns vieA double-SNP combination strain increased TCP production while decreasing CT production. Our findings suggest that SNPs identified in the four regulatory genes, in various combinations, are associated with increased virulence capabilities observed in V. cholerae clinical isolates. These studies provide insight into the evolution of highly virulent strains. IMPORTANCE Cholera, an infectious disease of the small intestine caused by the aquatic bacterium Vibrio cholerae, often results in vomiting and acute watery diarrhea. If left untreated or if the response is too slow, the symptoms can quickly lead to extreme dehydration and ultimately death of the patient. Recent anecdotal evidence of cholera patients suffering from increasingly severe symptoms and of disease progression at a much higher rate than previously observed has emerged. As recent cholera outbreaks caused by increasingly virulent strains have resulted in higher mortality rates, the need to investigate the mechanism(s) allowing this observed increased virulence is apparent. The significance of our research is in identifying the mechanism for increased virulence capabilities, which will allow the development of a model that will greatly enhance our understanding of cholera disease and V. cholerae pathogenesis, leading to broader biomedical impacts, as cholera serves as a model for other enteric diarrheal diseases.