Laboratory Techniques Used to Maintain and Differentiate Biotypes of <em>Vibrio cholerae</em> Clinical and Environmental Isolates

Abstract

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.

Feasibility of avian antibodies as prophylaxis against enterotoxigenic escherichia coli colonization

“…To determine the impact of anti-adhesin-tip MEFA IgY on bacterial viability, growth inhibition assays were conducted by OD method as previously described ( 23 ), with minor variations. Three biological replicates of ETEC isolates encoding CFA/II, CFA/III, CFA/IV, or CS3 ( Table 1 ) and a commensal E. coli strain that does not encode any of the adhesins included on MEFA (BL21; Genotype: F - , omp T, hsd S B (r B –, m B –), dcm , gal , λ(DE3), pLysS, Cm r ; Promega, Madison, WI, USA) were evaluated.…”

Occurrence, antimicrobial resistance, virulence, and biofilm formation capacity of Vibrio spp. and Aeromonas spp. isolated from raw seafood marketed in Bangkok, Thailand

“…The hemolytic activity of the bacterial isolates secreting hemolytic enzymes that lyse red blood cells was investigated using blood agar as previously described [32]. All plates were incubated at 37°C for 24 h. The degree of hemolysis was visualized on blood agar plates.…”

A Review of the Environmental Trigger and Transmission Components for Prediction of Cholera

“…Historically, detection of V. cholerae was achieved by determining its presence clinically during cholera outbreaks [24]. However, before the advent of epifluorescent microscopy [25,26] and molecular markers [27][28][29][30][31], detection of the presence of V. cholerae in the environment was accomplished by employing culture-based techniques [32]. Such investigations significantly underestimated V. cholerae populations in the environment, namely because the bacterium can enter a viable but non-culturable (VBNC) state [33].…”