An increase in the occurrence of potentially pathogenic Vibrio species is expected for waters in Northern Europe as a consequence of global warming. In this context, a higher incidence of Vibrio infections is predicted for the future and forecasts suggest that people visiting and living at the Baltic Sea are at particular risk. This study aimed to investigate antimicrobial resistance patterns among Vibrio vulnificus and Vibrio cholerae non-O1/non-O139 isolates that could pose a public health risk. Antimicrobial susceptibility of 141 V. vulnificus and 184 V. cholerae non-O1/non-O139 strains isolated from German coastal waters (Baltic Sea and North Sea) as well as from patients and retail seafood was assessed by broth microdilution and disk diffusion. Both species were susceptible to most of the agents tested (12 subclasses) and no multidrug-resistance was observed. Among V. vulnificus isolates, non-susceptibility was exclusively found towards aminoglycosides. In case of V. cholerae, a noticeable proportion of strains was non-susceptible to aminopenicillins and aminoglycosides. In addition, resistance towards carbapenems, quinolones, and folate pathway inhibitors was sporadically observed. Biochemical testing indicated the production of carbapenemases with unusual substrate specificity in four environmental V. cholerae strains. Most antimicrobial agents recommended for treatment of V. vulnificus and V. cholerae non-O1/non-O139 infections were found to be effective in vitro. However, the occurrence of putative carbapenemase producing V. cholerae in German coastal waters is of concern and highlights the need for systematic monitoring of antimicrobial susceptibility in potentially pathogenic Vibrio spp. in Europe.

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.

Vibrio cholerae belonging to the non-O1, non-O139 serogroups are present in the coastal waters of Germany and in some German and Austrian lakes. These bacteria can cause gastroenteritis and extraintestinal infections, and are transmitted through contaminated food and water. However, non-O1, non-O139 V. cholerae infections are rare in Germany. We studied 18 strains from German and Austrian patients with diarrhea or local infections for their virulence-associated genotype and phenotype to assess their potential for infectivity in anticipation of possible climatic changes that could enhance the transmission of these pathogens. The strains were examined for the presence of genes encoding cholera toxin and toxin-coregulated pilus (TCP), as well as other virulence-associated factors or markers, including hemolysins, repeats-in-toxin (RTX) toxins, Vibrio seventh pandemic islands VSP-1 and VSP-2, and the type III secretion system (TTSS). Phenotypic assays for hemolysin activity, serum resistance, and biofilm formation were also performed. A dendrogram generated by incorporating the results of these analyses revealed genetic differences of the strains correlating with their clinical origin. Non-O1, non-O139 strains from diarrheal patients possessed the TTSS and/or the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which were not found in the strains from ear or wound infections. Routine matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS) analysis of all strains provided reliable identification of the species but failed to differentiate between strains or clusters. The results of this study indicate the need for continued surveillance of V. cholerae non-O1, non-O139 in Germany, in view of the predicted increase in the prevalence of Vibrio spp. due to the rise in surface water temperatures.

During antimicrobial drug resistance testing for Vibrio spp. from coastal waters of Germany, we identified 4 nontoxigenic, carbapenem-resistant V. cholerae isolates. We used whole-genome sequencing to identify the carbapenemase gene blaVCC-1. In addition, a molecular survey showed that more blaVCC-1–harboring isolates are present in coastal waters of Germany.

Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Whole-genome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.