Environmental and Clinical Strains of Vibrio cholerae Non-O1, Non-O139 From Germany Possess Similar Virulence Gene Profiles

Abstract

Vibrio cholerae is a natural inhabitant of aquatic ecosystems globally. Strains of the serogroups O1 and O139 cause the epidemic diarrheal disease cholera. In Northern European waters, V. cholerae bacteria belonging to other serogroups (designated non-O1, non-O139) are present, of which some strains have been associated with gastrointestinal infections or extraintestinal infections, like wound infections or otitis. For this study, environmental strains from the German coastal waters of the North Sea and the Baltic Sea were selected (100 strains) and compared to clinical strains (10 isolates) that were from patients who contracted the infections in the same geographical region. The strains were characterized by MLST and examined by PCR for the presence of virulence genes encoding the cholera toxin, the toxin-coregulated pilus (TCP), and other virulence-associated accessory factors. The latter group comprised hemolysins, RTX toxins, cholix toxin, pandemic islands, and type III secretion system (TTSS). Phenotypic assays for hemolytic activity against human and sheep erythrocytes were also performed. The results of the MLST analysis revealed a considerable heterogeneity of sequence types (in total 74 STs). The presence of virulence genes was also variable and 30 profiles were obtained by PCR. One profile was found in 38 environmental strains and six clinical strains. Whole genome sequencing (WGS) was performed on 15 environmental and 7 clinical strains that were ST locus variants in one, two, or three alleles. Comparison of WGS results revealed that a set of virulence genes found in some clinical strains is also present in most environmental strains irrespective of the ST. In few strains, more virulence factors are acquired through horizontal gene transfer (i.e., TTSS, genomic islands). A distinction between clinical and environmental strains based on virulence gene profiles is not possible for our strains. Probably, many virulence traits of V. cholerae evolved in response to biotic and abiotic pressure and serve adaptation purposes in the natural aquatic environment, but provide a prerequisite for infection of susceptible human hosts. These findings indicate the need for surveillance of Vibrio spp. in Germany, as due to global warming abundance of Vibrio will rise and infections are predicted to increase.

Keywords: Baltic Sea; Germany; North Sea; Vibrio cholerae; clinical isolates; multilocus sequence typing; virulence-associated factors; whole genome sequencing.

Figure 1

Genetic relationships of Vibrio cholerae non-O1, non-O139 isolates from German coastal waters and from German clinical samples obtained by goeBURST Full MST analysis based on MLST allelic profiles using PHYLOViZ 1.1a. Each sequence type (ST) is displayed as a circle with a size proportional to the number of isolates by which it is represented. The coloring indicates the origin: environmental/North Sea (dark blue), environmental/Baltic Sea (turquoise), and clinical/Germany (dark red). Circles surrounded by a green halo are (sub-) group founders. Single-locus variants (SLVs) are connected via black lines. Gray lines indicate connections with a higher level. The number of differing loci between two STs is shown next to the connection lines. Clonal complexes (CCs) formed at SLV level by North Sea and German clinical (CC1; A) or Baltic Sea and German clinical (CC2; B) isolates, respectively, as well as the full Minimum Spanning Tree (MST; C) are shown. In the full MST, environmental STs forming SLVs to TLVs (triple-locus variants) with clinical STs are marked with a dark red arrow.

Figure 2

Schematics of genomic islands of the Vibrio cholerae non-O1, non-O139 strains VN-00470 and VN-00459 from the German Baltic Sea. (A) Vibrio seventh pandemic island 2 (VSP-2)-like element identified in strain VN-00470. Light blue areas below the VN-00470 sequence indicate similarity to genes of VSP-2 of V. cholerae O1 reference strain N16961 (gene numbering according to NC_002505.1). (B) Variant Vibrio pathogenicity island 2 (VPI-2) present in strain VN-00459. Gray areas below the VN-00459 sequence indicate similarity to genes of VPI-2 of V. cholerae O1 reference strain N16961 (gene numbering according to NC_002505.1). Blue colored areas below the VN-00459 sequence indicate similarity to the type three secretion system (TTSS) of strain AM-19226 (accession number AATY02000000). Dark blue regions represent the core region of the TTSS. Light blue regions represent the 5′- and 3′-flanking regions of the TTSS. attL, left attachment site; attR, right attachment site; ORF, open reading frame.

Figure 3

SNP-based phylogeny tree of Vibrio cholerae non-O1, non-O139 isolates from German coastal waters (•) and from German clinical samples (). SNP tree was conducted using CSI Phylogeny 1.4 under default settings and the exclusion of heterozygous SNPs. Single nucleotide polymorphisms (SNPs) were called by mapping to the V. cholerae VN-00533 genome as reference (MWZM00000000). Scale bar represents the number of nucleotide substitutions per site and numbers indicate branch length. Basic information about the sample type and the year of isolation are given after the strain identifier: North Sea/seawater (NS-sw), North Sea/bivalve mollusk (NS-bm), Baltic Sea/seawater (BS-sw), and peritoneal fluid (pf).