Characterization of a Nonagglutinating Toxigenic Vibrio cholerae Isolate

Abstract

Toxigenic Vibrio cholerae serogroup O1 is the etiologic agent of the disease cholera, and strains of this serogroup are responsible for pandemics. A few other serogroups have been found to carry cholera toxin genes-most notably, O139, O75, and O141-and public health surveillance in the United States is focused on these four serogroups. A toxigenic isolate was recovered from a case of vibriosis from Texas in 2008. This isolate did not agglutinate with any of the four different serogroups' antisera (O1, O139, O75, or O141) routinely used in phenotypic testing and did not display a rough phenotype. We investigated several hypotheses that might explain the recovery of this potential nonagglutinating (NAG) strain using whole-genome sequencing analysis and phylogenetic methods. The NAG strain formed a monophyletic cluster with O141 strains in a whole-genome phylogeny. Furthermore, a phylogeny of ctxAB and tcpA sequences revealed that the sequences from the NAG strain also formed a monophyletic cluster with toxigenic U.S. Gulf Coast (USGC) strains (O1, O75, and O141) that were recovered from vibriosis cases associated with exposures to Gulf Coast waters. A comparison of the NAG whole-genome sequence showed that the O-antigen-determining region of the NAG strain was closely related to those of O141 strains, and specific mutations were likely responsible for the inability to agglutinate. This work shows the utility of whole-genome sequence analysis tools for characterization of an atypical clinical isolate of V. cholerae originating from a USGC state. IMPORTANCE Clinical cases of vibriosis are on the rise due to climate events and ocean warming (1, 2), and increased surveillance of toxigenic Vibrio cholerae strains is now more crucial than ever. While traditional phenotyping using antisera against O1 and O139 is useful for monitoring currently circulating strains with pandemic or epidemic potential, reagents are limited for non-O1/non-O139 strains. With the increased use of next-generation sequencing technologies, analysis of less well-characterized strains and O-antigen regions is possible. The framework for advanced molecular analysis of O-antigen-determining regions presented herein will be useful in the absence of reagents for serotyping. Furthermore, molecular analyses based on whole-genome sequence data and using phylogenetic methods will help characterize both historical and novel strains of clinical importance. Closely monitoring emerging mutations and trends will improve our understanding of the epidemic potential of Vibrio cholerae to anticipate and rapidly respond to future public health emergencies.

Keywords: MAG; NAG; USGC Vibrio cholerae; Vibrio cholerae; nonagglutinating Vibrio strain; toxigenic Vibrio surveillance.

FIG 1

Core genome SNP phylogeny of toxigenic ctx (+) Vibrio cholerae serogroup O1, O139, O37, O75, and O141 strains and the NAG strain, generated using Parsnp version 1.5.2. 3566-08 served as the reference genome to build this phylogeny. Nontoxigenic ctx (−) variants of O141 and O1 were also included for comparison.

FIG 2

Neighbor-joining phylogeny of ctxAB sequences (1,148 bp) from toxigenic (ctx⁺) strains of Vibrio cholerae: the NAG strain and strains from O1, O75, O141, and other toxigenic serogroups. The phylogeny was generated using MEGA7 with the Kimura 2-parameter method and 500 replications of the interior branch test. Vibrio cholerae O139 strain F9993 was not included (its ctxAB sequence is identical to that of Vibrio cholerae O1 strain N16961). ctxB alleles and SNP ranges are displayed (e.g., number of differences). Bootstrap values greater than 50 are shown.

FIG 3

Maximum likelihood phylogeny of tcpA sequences (675 bp) from the Vibrio cholerae NAG strain and strains from O1, O75, O141, and other representative serogroups. The phylogenetic tree was generated using MEGA7 with the general time-reversible model and 500 bootstrap replications. Vibrio mimicus was also included for comparison. Bootstrap values greater than 50 are shown.

FIG 4

Evidence of mutations in the NAG strain O antigen. The illustration shows the approximate locations of genes with mutations in the NAG strain 3528-08 O-antigen region—an unknown gene that is wbfA-like and the pglJ gene—as well as those in O141 reference strain 3566-08 and O75 cluster I and II reference strains 3541-04 and 2011V-1043. The location of the O-antigen junction genes gmhD and rjg are labeled in red and blue, respectively. The O-antigen region lengths are shown on the right. While the O75 sequences for these genes were not examined in detail, the wbfA-like gene and pglJ gene are also present in O75, as shown.