Genotypic and Phenotypic Assays to Distinguish Vibrio cholerae Biotype

Abstract

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.

Aerobic Metabolism in Vibrio cholerae Is Required for Population Expansion during Infection

“…Classical V. cholerae predominated among epidemic isolates prior to 1961, when it was supplanted by the El Tor biotype ( 21 ). The biotypes are differentiated by numerous physiological attributes that contributed to displacement of the classical biotype by the El Tor biotype ( 22 – 25 ). Some of these are encoded on genomic islands unique to the El Tor biotype that contribute to phage resistance or acquisition of substrates ( 26 , 27 ).…”

Advances in cholera research: from molecular biology to public health initiatives

“…Several diagnostic methods have been developed to distinguish between Classical and El Tor biotypes in recent years. PCR-based genotypic assays typically screen for specific sequence variations in virulence genes including tcpA , ctxA , ctxB , and toxR , while another genome-based method targets unique small RNA genes ( Crumfield et al, 2018 ; Greig et al, 2018 ; Ahmed et al, 2019 ). Biotype can be distinguished phenotypically by evaluating antibiotic and phage susceptibility, capability for hemolysis and proteolysis, and variations in metabolism of citrate and glucose ( Crumfield et al, 2018 ; Lee et al, 2020 ).…”

“…PCR-based genotypic assays typically screen for specific sequence variations in virulence genes including tcpA , ctxA , ctxB , and toxR , while another genome-based method targets unique small RNA genes ( Crumfield et al, 2018 ; Greig et al, 2018 ; Ahmed et al, 2019 ). Biotype can be distinguished phenotypically by evaluating antibiotic and phage susceptibility, capability for hemolysis and proteolysis, and variations in metabolism of citrate and glucose ( Crumfield et al, 2018 ; Lee et al, 2020 ). One simple diagnostic measure for distinguishing between Classical and El Tor biotype strains in clinical settings has been the susceptibility of Classical strains to polymyxin B while El Tor strains have demonstrated resistance to this antibiotic, though this may not always be a reliable metric as El Tor strains continue to evolve ( Crumfield et al, 2018 ).…”

Environmental parameters associated with incidence and transmission of pathogenic Vibrio spp.

“…The toxin‐coregulated pilus (TCP, encoded by tcpA ) and CT (encoded by ctx ) are two main virulence factors of V. cholerae and allow the bacterium to colonize and establish infection. Isolates of V. cholerae serogroup O1 are categorized into two biotypes, classical and El Tor, each of which displays unique genotypic and phenotypic characteristics (Brumfield et al ., 2018). Purified V. cholerae isolates are available for modern scientific study only from the sixth (1899–1923) and seventh (1961–present) cholera pandemics but not from any of the earlier pandemics (Hu et al ., 2016).…”