Genomic insights into clinical non-O1/non-O139 Vibrio cholera e isolates in Japan

Abstract

Non-O1/non-O139 Vibrio cholerae (NOVC) causes sporadic intestinal infections and systemic symptoms. Recently, global warming has led to an increase in NOVC infections; however, there have been no reports on the genomic analysis of clinical NOVC isolates in Japan, and the molecular understanding of NOVC remains inadequate. We identified three cases of NOVC infection in Nagoya, Japan, in 2020 and performed genomic analysis to reveal the molecular characteristics of these isolates and compared them with those of previously reported clinical cases. Phylogenetic analysis revealed high diversity. This result indicates that the three cases were sporadic within the area over a short period of time from multiple contaminated sources. However, most clinical isolates carried virulence factor genes encoding El Tor type hemolysin, type VI secretion system (T6SS), and repeats-in-toxin toxin, and approximately half of the isolates carried nanH encoded on VPI-2, as well as the type III secretion system (T3SS). Additionally, detailed whole-genome sequencing (WGS) analysis revealed various types of T3SS, T6SS, VPI-2, and VSP-2. Variations in the number of Rep1 units of VopM were observed in the T3SS of the NOVC isolates in this study. The T6SS type identified in this study consisted of a large cluster and auxiliary clusters, showing diversity among the NOVC isolates. We recommend continued monitoring of clinical NOVC isolates as horizontal transmission along with host and environmental recombination may lead to changes in virulence.IMPORTANCEAlthough reports of non-O1/non-O139 Vibrio cholerae (NOVC) infections are rare, their actual incidence remains uncertain. This is partly due to nonspecific symptoms, the absence of a surveillance system in most countries including Japan, and the lack of appropriate laboratory culture techniques. However, NOVCs in the environment are increasing due to global warming, and the risk of NOVC infections is increasing. In this study, we conducted a comprehensive genomic analysis of clinical NOVC isolates from a city in Japan and compared their virulence factor profiles with those of previously reported clinical isolates using whole-genome sequencing (WGS). This result indicates that some sporadic cases have occurred in the area, suggesting that there are multiple sources of NOVC infection. The accumulation of such data will enhance our understanding of the pathogenicity of NOVCs and improve diagnostic accuracy.

Keywords: T3SS; T6SS; non-O1/non-O139 Vibrio cholerae (NOVC); virulence factor gene; whole-genome sequencing.

Fig 1

Phylogenetic tree based on pan-genome analysis using whole-genome sequencing data from the present isolates and previously reported clinical isolates of non-O1/non-O139 Vibrio cholerae, along with a search for virulence factor genes using the Virulence Factor Database. Phylogenetic and virulence factor gene detection analyses using whole-genome sequencing data of 57 clinical non-O1/non-O139 Vibrio cholerae isolates, including the isolates in this study and seven environmental strains as references in the public database, revealed genetic lineage diversity. Among the clinical isolates, 98.2% (56/57) harbored hlyA, 93.0% (53/57) harbored the type IV secretion system, 86.0% (49/57) harbored repeats-in-toxin toxin clusters, 61.4% (35/57) harbored nanH, and 47.4% (27/57) harbored the type III secretion system. The cholera toxin gene was detected in 7.0% (4/57) of the isolates.

Fig 2

Comparison of type III secretion system (T3SS) genes detected in non-O1/non-O139 Vibrio cholerae isolates through whole-genome sequencing reveals predominantly conserved T3SS clusters in clinical isolates. T3SS gene cluster regions were compared using Easyfig. The figures are shown in the order of AM-19226, such as the reference non-O1/non-O139, and Vibrio cholerae strains harboring T3SS, NGY2020-029, NGY2020-031, and NGY2020-056 from the top to the bottom. Red, orange, and dark blue arrows indicate T3SS component genes, identified genes, and hypothetical proteins, respectively. T3SS membrane-associated pore-forming complex genes and ATPases required for secretion, T3SS-associated translocator genes, transcriptional regulator genes, and virulence factor genes were detected in all isolates, although VopM and VopW differed among these isolates.

Fig 3

Genome map comparison of non-O1/non-O139 Vibrio cholerae isolates using the BLAST Ring Image Generator (BRIG) tool. Data comparisons for each draft genome were performed using BRIG. The reference data used to create the genome map were drawn using the N16961 strain, which is an El Tor type O1 Vibrio cholerae strain. N16961 is shown in red; MO10, the O139 V. cholerae strain, in brown; AM-19226, the O1 and O139 V. cholerae strains in blue; NGY2020-029 in sky blue; NGY2020-031 in orange; and NGY2020-056 in green. All strains in this study were found to be deficient in the cholera toxin phage, VPI-1, and VSP-1. In the VPI-2 region, all isolates in this study had a gene distribution similar to that of AM-19226, whereas NGY2020-031 harbored a fragmented VSP-2 region.