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

Abstract

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 dataset 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 which 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 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.

Virulence Pattern and Genomic Diversity of Vibrio cholerae O1 and O139 Strains Isolated From Clinical and Environmental Sources in India

“…The above MLST was reproducible that can be used for typing V. cholerae O1 and O139 strains. Another method, core genome multilocus sequence typing (cg-MLST), was introduced recently, which can provide good resolution, standardization, and ease of use for V. cholerae typing ( Liang et al, 2020 ). It will require whole-genome sequencing data for generating MLST for V. cholerae.…”

Molecular epidemiology in current times

“…A set of conserved core genes is extracted from a comprehensive dataset of (hopefully high quality) genome sequences. Then, a sub‐lineage threshold of allelic differences needs to be defined that creates clusters nearly identical to traditional MLST types, providing backwards compatibility to new cgMLST classifications (Liang et al ., 2020). A ring trial demonstrated the high reproducibility of cgMLST allele profiles, only 3 of 183 927 (0.0016%) cgMLST allele calls were wrong (Mellmann et al ., 2017).…”

“…For example, within 1 month in 2020 cgMLST schemes have been published for Campylobacter jejuni (Hsu et al ., 2020), Leptospira spp. (Grillová and Picardeau, 2020), Pseudomonas aeruginosa (De Sales et al ., 2020; Martak et al ., 2020; Stanton et al ., 2020), Streptococcus mutans (Liu et al ., 2020) and Vibrio cholera (Liang et al ., 2020). cgMLST schemes consist of a fixed set of conserved genome‐wide genes.…”

Multilevel Genome Typing Describes Short- and Long-Term Vibrio cholerae Molecular Epidemiology

“…MLST compares alleles in a set of shared loci, and a sequence type (ST) is assigned to represent a unique combination of alleles for those loci ( 10 ). Both the classic seven-gene MLST scheme and larger core genome MLST (cgMLST) schemes have been developed for V. cholerae ( 11 , 12 ). The seven-gene MLST scheme defines nearly the entire 7P as a single type, ST69, while cgMLST divides the 7P into hundreds of STs with little to no population structure defined by each ST ( 12 ).…”

“…Both the classic seven-gene MLST scheme and larger core genome MLST (cgMLST) schemes have been developed for V. cholerae ( 11 , 12 ). The seven-gene MLST scheme defines nearly the entire 7P as a single type, ST69, while cgMLST divides the 7P into hundreds of STs with little to no population structure defined by each ST ( 12 ). Additionally, clustering of cgMLST data using suitable allelic thresholds can group cgMLST STs with informative divisions.…”