German coasts harbor non-O1/non-O139 Vibrio cholerae with clinical virulence gene profiles.

Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This ability is associated with various virulence factors. The presence of NOVC in German North Sea and Baltic Sea was observed in previous studies. However, data on virulence characteristics are still scarce. Therefore, this work aimed to investigating the virulence potential of NOVC isolated in these two regions. In total, 31 NOVC strains were collected and subjected to whole genome sequencing. In silico analysis of the pathogenic potential was performed based on the detection of genes involved in colonization and virulence. Phenotypic assays, including biofilm formation, mobility and human serum resistance assays were applied for validation. Associated toxin genes (hlyA, rtxA, chxA and stn), pathogenicity islands (Vibrio pathogenicity island 2 (VPI-II) and Vibrio seventh pathogenicity island 2 (VSP-II)) and secretion systems (Type II, III and VI secretion system) were observed. A maximum likelihood analysis from shared core genes revealed a close relationship between clinical NOVCs published in NCBI and environmental strains from this study. NOVC strains are more mobile at 37 °C than at 25 °C, and 68% of the NOVC strains could form strong biofilms at both temperatures. All tested strains were able to lyse erythrocytes from both human and sheep blood. Additionally, one strain could survive up to 60% and seven strains up to 40% human serum at 37 °C. Overall, the genetic virulence profile as well as the phenotypic virulence characteristics of the investigated NOVC from the German North Sea and Baltic Sea suggest potential human pathogenicity.

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

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.

Prevalence and Distribution of Potentially Human Pathogenic Vibrio spp. on German North and Baltic Sea Coasts.

Global ocean warming results in an increase of infectious diseases including an elevated emergence of Vibrio spp. in Northern Europe. The European Centre for Disease Prevention and Control reported annual periods of high to very high risks of infection with Vibrio spp. during summer months along the North Sea and Baltic Sea coasts. Based on those facts, the risk of Vibrio infections associated with recreational bathing in European coastal waters increases. To obtain an overview of the seasonal and spatial distribution of potentially human pathogenic Vibrio spp. at German coasts, this study monitored V. cholerae, V. parahaemolyticus, and V. vulnificus at seven recreational bathing areas from 2017 to 2018, including the heat wave event in summer 2018. The study shows that all three Vibrio species occurred in water and sediment samples at all sampling sites. Temperature was shown to be the main driving factor of Vibrio abundance, whereas Vibrio community composition was mainly modulated by salinity. A species-specific rapid increase was observed at water temperatures above 10°C, reaching the highest detection numbers during the heat wave event with abundances of 4.5 log10 CFU+1/100 ml of seawater and 6.5 log10 CFU+1/100 g of sediment. Due to salinity, the dominant Vibrio species found in North Sea samples was V. parahaemolyticus, whereas V. vulnificus was predominantly detected in Baltic Sea samples. Most detections of V. cholerae were associated with estuarine samples from both seas. Vibrio spp. concentrations in sediments were up to three log higher compared to water samples, indicating that sediments are an important habitat for Vibrio spp. to persist in the environment. Antibiotic resistances were found against beta-lactam antibiotics (ampicillin 31%, cefazolin 36%, and oxacillin and penicillin 100%) and trimethoprim-sulfamethoxazole (45%). Moreover, isolates harboring pathogenicity-associated genes such as trh for V. parahaemolyticus as well as vcg, cap/wcv, and the 16S rRNA-type B variant for V. vulnificus were detected. All sampled V. cholerae isolates were identified as non-toxigenic non-O1/non-O139 serotypes. To sum up, increasing water temperatures at German North Sea and Baltic Sea coasts provoke elevated Vibrio numbers and encourage human recreational water activities, resulting in increased exposure rates. Owing to a moderate Baltic Sea salinity, the risk of V. vulnificus infections is of particular concern.

Complete Genome Sequence of an Aeromonas rivuli Strain Isolated from Ready-to-Eat Food.

Aeromonads can be associated with diseases in animals and humans. Knowledge regarding Aeromonas rivuli, a species recently discovered in creek water in Germany, is still fragmentary. Here, we announce the complete genome sequence of Aeromonas rivuli strain 20-VB00005, which was recovered from ready-to-eat food.

Case Report: Vibrio fluvialis isolated from a wound infection after a piercing trauma in the Baltic Sea.

Vibrio spp. are Gram-negative bacteria found in marine ecosystems. Non-cholera Vibrio spp. can cause gastrointestinal infections and can also lead to wound infections through exposure to contaminated seawater. Vibrio infections are increasingly documented from the Baltic Sea due to extended warm weather periods. We describe the first isolation of Vibrio fluvialis from a wound infection acquired by an impalement injury in the shallow waters of the Baltic Sea. The severe infection required amputation of the third toe. Whole genome sequencing of the isolate was performed and revealed a genome consisting of two circular chromosomes with a size of 1.57 and 3.24 Mb.

The Acquisition of the scr Gene Cluster Encoding Sucrose Metabolization Enzymes Enables Strains of Vibrio parahaemolyticus and Vibrio vulnificus to Utilize Sucrose as Carbon Source.

Most strains of Vibrio parahaemolyticus are unable to utilize sucrose as carbon source, though few exceptions exist. We investigated a sucrose-positive V. parahaemolyticus strain by whole-genome sequencing (WGS) and confirmed the presences of a genomic island containing sucrose utilization genes. A 4.7 kb DNA cluster consisting of three genes: scrA encoding a sucrose uptake protein, scrK encoding a fructokinase, and scrB coding for a sucrose-6-phosphate hydrolase, was PCR amplified and inserted into the Vibrio/Escherichia coli shuttle vector pVv3. Two recombinant plasmids, only differing in the orientation of the insert with respect to the pVv3-lacZα-fragment, conferred the E. coli K12 transformants the ability to utilize sucrose. The introduction of the two plasmids into sucrose-negative V. parahaemolyticus and V. vulnificus strains also results in a change of the sucrose utilization phenotype from negative to positive. By performing a multiplex PCR targeting scrA, scrK, and scrB, 43 scr-positive V. parahaemolyticus isolates from our collection of retail strains were detected and confirmed to be able to use sucrose as carbon source. Strains unable to utilize the disaccharide were negative by PCR for the scr genes. For in-depth characterization, 17 sucrose-positive V. parahaemolyticus were subjected to WGS. A genomic island with a nucleotide identity of >95% containing scrA, scrB, scrK and three additional coding sequences (CDS) were identified in all strains. The additional genes were predicted as a gene coding for a transcriptional regulator (scrR), a porin encoding gene and a CDS of unknown function. Sequence comparison indicated that the genomic island was located in the same region of the chromosome II in all analyzed V. parahaemolyticus strains. Structural comparison of the genomes with sequences of the sucrose utilizing species V. alginolyticus revealed the same genomic island, which indicates a possible distribution of this genetic structure by horizontal gene transfer. The comparison of all genome sequences based on SNP differences reveals that the presence of sucrose utilizing genes is found in genetically diverse V. parahaemolyticus strains and is not restricted to a subset of closely related strains.

Complete and Circularized Genome Assembly of a Human Isolate of Vibrio navarrensis Biotype pommerensis with MiSeq and MinION Sequence Data.

Vibrio navarrensis is a rare human pathogen. Strains of Vibrio navarrensis biotype pommerensis were isolated from seawater of the Baltic Sea. Recently, a strain of this biotype was recovered from a human patient. The isolate contains two circular chromosomes and a large plasmid with a size of 180 kb.

Draft Genome Sequences of Vibrio cholerae Non-O1, Non-O139 Isolates from Common Tern Chicks (Sterna hirundo) following a Mass Mortality Event.

Vibrio cholerae is an inhabitant of aquatic environments worldwide. Here, we report the draft genome sequences of eight V. cholera non-O1, non-O139 isolates that were recovered from the corpses of two seabird chicks (common terns) following a mass mortality event in a German breeding colony in 2019.

Phenotypic and Genotypic Properties of Vibrio cholerae non-O1, non-O139 Isolates Recovered from Domestic Ducks in Germany.

Vibrio cholerae non-O1, non-O139 bacteria are natural inhabitants of aquatic ecosystems and have been sporadically associated with human infections. They mostly lack the two major virulence factors of toxigenic V. cholerae serogroups O1 and O139 strains, which are the causative agent of cholera. Non-O1, non-O139 strains are found in water bodies, sediments, and in association with other aquatic organisms. Occurrence of these bacteria in fecal specimens of waterfowl were reported, and migratory birds likely contribute to the long-distance transfer of strains. We investigated four V. cholerae non-O1, non-O139 isolates for phenotypic traits and by whole genome sequencing (WGS). The isolates were recovered from organs of domestic ducks with serious disease symptoms. WGS data revealed only a distant genetic relationship between all isolates. The isolates harbored a number of virulence factors found in most V. cholerae strains. Specific virulence factors of non-O1, non-O139 strains, such as the type III secretion system (TTSS) or cholix toxin, were observed. An interesting observation is that all isolates possess multifunctional autoprocessing repeats-in-toxin toxins (MARTX) closely related to the MARTX of toxigenic El Tor O1 strains. Different primary sequences of the abundant OmpU proteins could indicate a significant role of this virulence factor. Phenotypic characteristics such as hemolysis and antimicrobial resistance (AMR) were studied. Three isolates showed susceptibility to a number of tested antimicrobials, and one strain possessed AMR genes located in an integron. Knowledge of the environmental occurrence of V. cholerae non-O1, non-O139 in Germany is limited. The source of the infection of the ducks is currently unknown. In the context of the 'One Health' concept, it is desirable to study the ecology of V. cholerae non-O1, non-O139, as it cannot be excluded that the isolates possess zoonotic potential and could cause infections in humans.

Phenotypic and Genotypic Characterization of Veterinary Vibrio cincinnatiensis Isolates.

Vibrio cincinnatiensis is a halophilic species which has been found in marine and estuarine environments worldwide. The species is considered a rare pathogen for which the significance for humans is unclear. In this study, nine veterinary isolates were investigated that were obtained from domestic animals in Germany. The isolates were mostly recovered from abortion material of pigs, cattle, and horse (amnion or fetuses). One isolate was from a goose. A human clinical strain from a case of enteritis in Germany described in the literature was also included in the study. Whole-genome sequencing (WGS) of all isolates and MALDI-TOF MS (matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry) were performed to verify the species assignment. All strains were investigated for phenotypic traits including antimicrobial resistance (AMR), biochemical properties, and two virulence-associated phenotypes (hemolytic activity and resistance to human serum). WGS data and MS spectra confirmed that all veterinary isolates are closely related to the type strain V. cincinnatiensis NCTC12012. An exception was the human isolate from Germany which is related to the other isolates but could belong to another species. The isolates were similar in most biochemical phenotypes. Only one strain showed a very weak hemolytic activity against sheep erythrocytes, and serum resistance was intermediate in two strains. AMR phenotypes were more variable between the isolates. Resistances were observed against ß-lactams ampicillin and cefoxitin and against tetracycline and the sulfonamide antibiotics trimethoprim and sulfamethoxazole. Some acquired AMR genes were identified by bioinformatics analyses. WGS and MALDI-TOF MS data reveal a close relationship of the veterinary isolates and the type strain V. cincinnatiensis NCTC12012, which is a clinical human isolate. As the veterinary isolates of this study were mostly recovered from abortion material (amnions and fetuses), a zoonotic potential of the veterinary isolates seems possible.

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

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.

Cell-free production of pore forming toxins: Functional analysis of thermostable direct hemolysin from Vibrio parahaemolyticus.

The pore forming characteristic of TDH1 and TDH2 variants of thermostable direct hemolysin (TDH), a major toxin involved in the pathogenesis of Vibrio parahaemolyticus, was studied on a planar lipid bilayer painted over individual picoliter cavities containing microelectrodes assembled in a multiarray. Both proteins formed pores upon insertion into the lipid bilayer which was shown as a shift in the conductance from the baseline current. TDH2 protein was able to produce stable currents and the currents were influenced by external factors like concentration, type of salt and voltage. The pore currents were influenced and showed a detectable response in the presence of polymers which makes them suitable for biotechnology applications.

Carbapenemase VCC-1-Producing Vibrio cholerae in Coastal Waters of Germany.

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.

Diversity of Vibrio navarrensis Revealed by Genomic Comparison: Veterinary Isolates Are Related to Strains Associated with Human Illness and Sewage Isolates While Seawater Strains Are More Distant.

Strains of Vibrio navarrensis are present in aquatic environments like seawater, rivers, and sewage. Recently, strains of this species were identified in human clinical specimens. In this study, V. navarrensis strains isolated from livestock in Germany were characterized that were found in aborted fetuses and/or placentas after miscarriages. The veterinary strains were analyzed using phenotypical and genotypical methods and compared to isolates from marine environments of the Baltic Sea and North Sea. The investigated phenotypical traits were similar in all German strains. Whole genome sequencing (WGS) was used to evaluate a phylogenetic relationship by performing a single nucleotide polymorphism (SNP) analysis. For the SNP analysis, WGS data of two American human pathogenic strains and two Spanish environmental isolates from sewage were included. A phylogenetic analysis of concatenated sequences of five protein-coding housekeeping genes (gyrB, pyrH, recA, atpA, and rpoB), was additionally performed. Both phylogenetic analyses reveal a greater distance of the environmental seawater strains to the other strains. The phylogenetic tree constructed from concatenated sequences of housekeeping genes places veterinary, human pathogenic and Spanish sewage strains into one cluster. Presence and absence of virulence-associated genes were investigated based on WGS data and confirmed by PCR. However, this analysis showed no clear pattern for the potentially pathogenic strains. The detection of V. navarrensis in human clinical specimens strongly suggests that this species should be regarded as a potential human pathogen. The identification of V. navarrensis strains in domestic animals implicates a zoonotic potential of this species. This could indicate a potential threat for humans, as according to the "One Health" concept, human, animal, and environmental health are linked. Future studies are necessary to search for reservoirs of these bacteria in the environment and/or in living organisms.

Genome Sequence of the K139-Like Phage VcP032 Originating from the Vibrio cholerae O1 El Tor Ogawa Serotype.

Vibrio cholerae is the cause of large cholera outbreaks, especially in endemic regions with high poverty and inadequate sanitation. Here, we announce the complete genome sequence of the virulence-associated broad host range V. cholerae phage VcP032, including a brief summary of its genotypic and phenotypic features.

Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates.

Vibrio vulnificus is a halophilic bacterium of coastal environments known for sporadically causing severe foodborne or wound infections. Global warming is expected to lead to a rising occurrence of V. vulnificus and an increasing incidence of human infections in Northern Europe. So far, infections in Germany were exclusively documented for the Baltic Sea coast, while no cases from the North Sea region have been reported. Regional variations in the prevalence of infections may be influenced by differences in the pathogenicity of V. vulnificus populations in both areas. This study aimed to compare the distribution of virulence-associated traits and genotypes among 101 V. vulnificus isolates from the Baltic Sea and North Sea in order to assess their pathogenicity potential. Furthermore, genetic relationships were examined by multilocus sequence typing (MLST). A high diversity of MLST sequences (74 sequence types) and differences regarding the presence of six potential pathogenicity markers were observed in the V. vulnificus populations of both areas. Strains with genotypes and markers associated with pathogenicity are not restricted to a particular geographic region. This indicates that lack of reported cases in the North Sea region is not caused by the absence of potentially pathogenic strains.

Survey on antimicrobial resistance patterns in Vibrio vulnificus and Vibrio cholerae non-O1/non-O139 in Germany reveals carbapenemase-producing Vibrio cholerae in coastal waters.

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 toward aminoglycosides. In case of V. cholerae, a noticeable proportion of strains was non-susceptible to aminopenicillins and aminoglycosides. In addition, resistance toward 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.

The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis (Paris, 11-12th March 2015).

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.

Characterization of trh2 harbouring Vibrio parahaemolyticus strains isolated in Germany.

BACKGROUND: Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. RESULTS: Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2ß effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. CONCLUSION: Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2ß components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics.