Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 Vibrio cholerae Isolated from German Retail Seafood

Abstract

Non-O1 and non-O139 Vibrio cholerae (NOVC) can cause gastrointestinal infections in humans. Contaminated food, especially seafood, is an important source of human infections. In this study, the virulence potential of 63 NOVC strains isolated from retail seafood were characterized at the genotypic and phenotypic levels. Although no strain encoded the cholera toxin (CTX) and the toxin-coregulated pilus (TCP), several virulence factors, including the HlyA hemolysin, the cholix toxin ChxA, the heat-stable enterotoxin Stn, and genes coding for the type 3 and type 6 secretion systems, were detected. All strains showed hemolytic activity against human and sheep erythrocytes: 90% (n = 57) formed a strong biofilm, 52% (n = 33) were highly motile at 37 °C, and only 8% (n = 5) and 14% (n = 9) could resist ≥60% and ≥40% human serum, respectively. Biofilm formation and toxin regulation genes were also detected. cgMLST analysis demonstrated that NOVC strains from seafood cluster with clinical NOVC strains. Antimicrobial susceptibility testing (AST) results in the identification of five strains that developed non-wildtype phenotypes (medium and resistant) against the substances of the classes of beta-lactams (including penicillin, carbapenem, and cephalosporin), polymyxins, and sulphonamides. The phenotypic resistance pattern could be partially attributed to the acquired resistance determinants identified via in silico analysis. Our results showed differences in the virulence potential of the analyzed NOVC isolated from retail seafood products, which may be considered for further pathogenicity evaluation and the risk assessment of NOVC isolates in future seafood monitoring.

Keywords: antimicrobial resistance; genetic and phenotypic characterization; non-O1/non-O139 Vibrio cholerae; seafood; virulence potential.

Figure 1

cgMLST analysis of seafood-associated NOVC, clinical NOVC, and pandemic O1/O139 V. cholerae strains. Seafood-associated NOVC are labeled in green, clinical NOVC are labeled in orange, and pandemic O1/O139 V. cholerae are labeled in red. A green background shows a clustering of only seafood-associated NOVC. An orange background shows clusters containing clinical NOVC or pandemic O1/O139 V. cholerae and seafood-associated NOVC. A red background shows a clustering of only pandemic O1/O139 V. cholerae. The clusters were defined based on a clustering threshold ranging from 1 to 1000 allelic differences to identify a phylogenetic relationship according to Liang et al., 2020 [42]. The minimum spanning tree was created using grapetree [28] and visualized using Geneious software (v2022.1.1).

Figure 2

(a). The presence/absence of toxin genes, genes involved in secretion systems, and genes included in pathogenicity islands of seafood-associated NOVC (n = 63) based on WGS data. Black boxes represent gene presence, and white boxes represent gene absence. Detailed information about the genes can be found in Table S3. (b). The presence/absence of genes that play roles as putative virulence factors or genes involved in systems that play roles in the virulence of seafood-associated NOVC (n = 63) based on WGS data. Black boxes represent gene presence, and white boxes represent gene absence. Detailed information about the genes can be found in Table S3.

Figure 3

Antibiotic resistance pattern of NOVC strains (n = 63) at genetic and phenotypic level. SXT/R391: conserved region of SXT/R391. bcr: Bcr/CflA family efflux transporter. dps: member of ferritin-like diiron-carboxylate proteins encoding gene. vcaM: ATP binding cassette encoding gene. varG: VarG beta-lactamase Subclass B1 encoding gene. EF-Tu: elfamycin resistance gene. parE: fluoroquinolone resistance gene. almG: lipid A acyltransferase encoding gene. MATE: multidrug and toxic compound extrusion pump encoding gene. qnrVC: quinolone resistance gene. dfrA31: trimethoprim resistant dihydrofolate reductase encoding gene. catB9: chloramphenicol acetyltransferase encoding gene. blaCARB-7: beta-lactamase encoding gene. In genetic result, black boxes represent gene presence, and white boxes represent gene absence. In phenotypic result, black boxes represent resistance, grey boxes represent intermediate, and white boxes present susceptibility. Detail information about breakout points is shown in Table S2.