The distinction matters because non-O1/non-O139 V. cholerae, often abbreviated NOVC, behave very differently from their pandemic cousins. These organisms are ubiquitous in brackish and coastal waters worldwide and typically lack the two hallmark weapons of cholera: the cholera toxin genes ctxAB and the toxin-coregulated pilus, tcp, which allows intestinal colonization. What they can do, however, is cause serious extraintestinal illness, including bacteremia, wound infections and peritonitis, especially in people whose defenses are already compromised. In China, a survey covering 2005 to 2019 documented 82 cases of NOVC bacteremia, most occurring in patients with liver cirrhosis, diabetes or immunocompromising conditions, and mortality in that cohort was substantial. The incidence of such infections has been rising along the Chinese coast, yet the population structure and genomic diversity of the clinical isolates behind this trend remain poorly charted.

The Xiamen case illustrates the classic clinical setting. The isolate, named HL2601, was recovered from a blood culture of a 56-year-old man with biliary tract infection-associated bacteremia, and he carried a constellation of well-recognized risk factors: poorly controlled diabetes, chronic heavy alcohol use and structural abnormalities of the biliary tract. Biliary disease most likely allowed bacteria to move retrograde from the gut into the bloodstream, with secondary sepsis developing against a backdrop of hepatic dysfunction and alcohol-impaired immunity. Notably, the patient had no cholera-like diarrhea, reinforcing that NOVC infections follow an entirely different disease pattern from the toxigenic O1 and O139 strains and deserve a place in the differential diagnosis of bacteremia in high-risk patients even without gastrointestinal symptoms.

Phenotypically, HL2601 presented a reassuring face. Tested against 11 routinely used antibiotics under Clinical and Laboratory Standards Institute guidelines, the strain proved susceptible to every agent, from imipenem and third-generation cephalosporins to ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole. Quality-control strains validated each run. Genomic screening told a consistent story: no plasmid-borne or acquired transferable resistance genes were found. The genome did contain intrinsic determinants, including the almEFG operon, which modifies lipid A and confers resistance to polymyxins, a class not included in the testing panel, plus a chromosomal parE homolog without known resistance-conferring mutations and the catB9 chloramphenicol acetyltransferase gene. None of these confer clinical resistance to the drugs tested, but the authors caution that such intrinsic machinery, including efflux pump regulators, can serve as raw material for the evolution of higher-level resistance under antimicrobial pressure, and efflux pumps in the Vibrionaceae also contribute to virulence, persistence and biofilm formation.

The genome itself was assembled to high quality: a 4,296,868-base-pair draft across 50 contigs with an N50 above 300 kilobases, 99.98 percent completeness and only 0.03 percent contamination, characteristics that made reliable detection of large-scale structural rearrangements possible. Average nucleotide identity analysis against representative vibrios firmly anchored HL2601 within V. cholerae, yielding 98.11 percent identity to the pandemic reference strain N16961 and 98.04 percent to V. cholerae ATCC 14035, both comfortably above the 95 percent species threshold, while values against V. vulnificus, V. alginolyticus and V. harveyi fell near 79 percent, clearly below it.

The virulence profile is where the strain becomes genuinely concerning. Annotation against the Virulence Factor Database identified 136 virulence-associated genes spanning eight functional categories, all chromosomally encoded. HL2601 carries complete, intact type II and type VI secretion system loci and a partial type III secretion system 2 cluster. It possesses the full RTX toxin gene cluster rtxA through rtxD, thermolabile hemolysin, and the V. cholerae cytolysin hlyA, alongside fully encoded enterobactin and vibriobactin iron-uptake systems, the MSHA type IV pilus, partial capsular polysaccharide genes, 56 flagellar and chemotaxis genes, extracellular enzymes and quorum-sensing systems. Targeted searches confirmed the absence of every major toxigenic locus: ctxAB, tcp, zot, cholix toxin chxA and the thermostable enterotoxin stn. The type VI secretion system is of particular interest because it mediates interbacterial competition, immune evasion and host cell cytotoxicity, functions that support colonization and survival outside the gut, while hemolysins and RTX toxin may drive tissue damage in the bloodstream and iron-uptake systems allow nutrient scavenging in the iron-poor host environment.

Comparative analysis across five additional NOVC strains of clinical, environmental and aquaculture origin defined a conserved core virulence backbone for non-toxigenic V. cholerae: all examined strains uniformly lacked the six classical toxigenic markers and consistently carried rtxA and hlyA. Adherence determinants varied more, with the mshA pilus gene present in most clinical and environmental isolates but absent from HL2601 and an aquaculture water strain. Functional pathway annotation through KEGG showed the expected architecture of a free-living bacterium, with metabolism dominating at 23.4 percent of annotated genes, and at finer resolution HL2601 showed mild relative enrichment in carbohydrate metabolism, membrane transport and secondary metabolite biosynthesis compared with its reference panel, differences that are quantitative rather than qualitative and may aid niche adaptation in aquatic environments and on host mucosal surfaces.

The phylogenetic story is perhaps the most epidemiologically revealing. Core-genome SNP calling across 38 isolates, using the pandemic O1 strain N16961 as an outgroup, placed HL2601 squarely within a well-supported non-pandemic NOVC lineage in which clinical, environmental and foodborne isolates were phylogenetically intermingled rather than segregated by source. Its closest relative was a clinical isolate from Bangladesh, M647, at a distance of 23,720 core SNPs, roughly 0.76 percent nucleotide divergence, with distances of about 0.84 to 0.85 percent to an Indian environmental isolate and a Chinese clinical isolate. This pattern, the authors argue, is consistent with recent local diversification of a circulating clone, in which rapid point mutations in housekeeping genes generate novel sequence types without broad genomic change. GoeBURST analysis positioned ST2040 as a peripheral node connected to neighboring types ST1878, ST2024 and ST2042 through single- and double-locus variants, supporting that interpretation, though the study did not formally exclude homologous recombination as a contributing mechanism. The close mingling of clinical and environmental strains also strengthens the view that aquatic reservoirs act as a persistent source of opportunistic infection, a pattern previously observed for NOVC bloodstream isolates elsewhere in China.

Whole-genome collinearity analysis added a structural dimension to the phylogeny. Synteny conservation was highest, at 89.79 percent, between HL2601 and the clinical isolate NOVC05, intermediate at 85.11 percent with a foodborne isolate, and 85.52 percent with the pandemic O1 strain, the latter comparison revealing 20 large inversions totaling more than 273,000 base pairs. Within the NOVC lineage, by contrast, unaligned segments consisted mainly of mobile genetic elements and strain-specific accessory sequences, with no acquired resistance genes or major pathogenicity islands detected, indicating that horizontal transfer of key determinants is not driving structural differentiation among these strains. The deep structural gulf between O1 and NOVC genomes, meanwhile, points to their long-term independent evolution.

ST2040 joins a growing roster of novel sequence types recently described from Chinese clinical NOVC isolates, including ST1553, ST1538 and the fatal ST1665, underscoring that these emerging clones are diversifying and reaching vulnerable patients. The study’s limitations are acknowledged: it rests on a single isolate that cannot capture regional prevalence, environmental source tracing was unavailable so the reservoir hypothesis rests on phylogenetic clustering, short-read assemblies limit confidence in structural variants smaller than 10 kilobases, and the partial T3SS2 locus awaits functional characterization. Even so, the authors conclude that the picture supports integrating whole-genome sequencing and multilocus sequence typing into routine V. cholerae surveillance in coastal regions, alongside targeted health education for high-risk populations, while conventional beta-lactams and fluoroquinolones remain appropriate first-line empirical therapy for extraintestinal vibriosis in the region as long as susceptibility profiles like this one hold.

Subject of Research: Genomic and phenotypic characterization of a novel non-toxigenic non-O1/non-O139 Vibrio cholerae sequence type causing bacteremia in southern China

Article Title: Novel sequence type 2040 non-O1/non-O139 Vibrio cholerae from southern China: genomic and phenotypic features with molecular epidemiological implications

Article References: Xia, Y., Ding, J., Kai, J., Xie, C., Nie, Y., Ru, J., Huang, L., & Wen, J. (2026). Novel sequence type 2040 non-O1/non-O139 Vibrio cholerae from southern China: genomic and phenotypic features with molecular epidemiological implications. Frontiers in Microbiology, 17, Article 1955993. https://doi.org/10.3389/fmicb.2026.1955993

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1955993

Keywords: Vibrio cholerae, non-O1/non-O139, ST2040, bacteremia, whole-genome sequencing, virulence factors, type VI secretion system, multilocus sequence typing, antimicrobial susceptibility, environmental reservoir, coastal China, molecular epidemiology