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

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

Charakterisierung von Vibrio spp. Isolaten aus Deutschland anhand humaner klinischer Isolate mittels phänotypischer und WGS-gestützter genotypischer Methoden

V. cholerae bacteria are distributed in aquatic environments with low to moderate salinity throughout the world. Toxigenic serogroup O1/O139 V. cholerae strains can cause the epidemic disease cholera. In northern European waters, non-O1, non-O139 V. cholerae strains occur, some of which have been associated with gastrointestinal or extraintestinal infections. So far, infections caused by these bacteria are rare in Germany. However, due to climate change, an increase in non-O1, non-O139 V. cholerae prevalence is expected. Given the popularity of German coastal regions as a travel destination, an increased exposure of the population to strains of these serogroups must be assumed for future years. Therefore, by comparing MLST sequence types (ST) and virulence factor profiles of environmental isolates and clinical isolates, a first risk assessment for non-O1, non-O139 V. cholerae strains from German coastal waters was carried out in this work. The similarity of the virulence gene profiles of environmental strains and clinical strains - irrespective of the ST - as well as the hemolytic activity against human erythrocytes could indicate a human pathogenic potential of many environmental strains in the North Sea and Baltic Sea. Data comparison to a first follow-up study conducted in the German Bight suggests that environmental changes resulting from climate change possibly foster V. cholerae strains with a higher infective risk. The whole genome sequencing (WGS)-based typing scheme that was developed and established at the Vibrio consiliary laboratory in the context of this dissertation can serve as a tool for monitoring and deeper investigation of virulence gene profiles of future non-O1, non-O139 V. cholerae isolates obtained from public health-relevant sources. While Vibrio species such as V. cholerae have been intensively studied for decades as a result of the global disease burden, other Vibrio species such as the presumed environmental species V. navarrensis are hardly explored. Recently, V. navarrensis strains were detected in human clinical specimens as well as veterinary abortion specimens from livestock in Germany. For identification of new potential environmental reservoirs in Germany and against the background of a possible zoonotic potential, comparative genotypic analyses of V. navarrensis isolates from environmental, veterinary, and human clinical specimens were performed in this work. The results of SNP and MLSA analyses suggest the occurrence of V. navarrensis in German freshwater environments, which would extend the range of this species from coastal waters of the North Sea and Baltic Sea to inland waters in Germany. Ingestion of V. navarrensis by farm animals through uptake of surface water seems possible. The presence of an MLSA subcluster encompassing all veterinary and human clinical strains could indicate a human pathogenic potential of the former. The design of a WGS-based analytical procedure for typing of virulence-associated genes in V. navarrensis isolates provided deeper insights into the genetic makeup of all strains. Phenotypic traits such as the hemolytic activity of the investigated environmental and veterinary strains against sheep erythrocytes and human red blood cells underline the pathogenic character of this Vibrio species. A new case of a human infection in summer 2020 supports this assessment. The developed V. navarrensis-specific vvhA and tlh singleplex PCRs represent promising tools for rapid species diagnostics and may thereby facilitate future monitoring and research studies on Vibrio bacteria in the context of the “One Health” approach.V. cholerae-Bakterien sind in aquatischen Umwelten mit geringer bis moderater Salinität weltweit verbreitet. Toxigene V. cholerae-Stämme der Serogruppe O1/O139 können die epidemische Erkrankung Cholera verursachen. In nordeuropäischen Gewässern kommen non-O1, non-O139 V. cholerae-Stämme vor, von denen einige mit gastrointestinalen oder extraintestinalen Infektionen in Verbindung gebracht wurden. Bislang sind durch diese Bakterien verursachte Infektionen in Deutschland selten. Aufgrund des Klimawandels wird jedoch ein Anstieg in der non-O1, non-O139 V. cholerae-Prävalenz erwartet. Angesichts der Beliebtheit deutscher Küstenregionen als Reiseziel muss in den kommenden Jahren von einer erhöhten Exposition der Bevölkerung gegenüber Stämmen dieser Serogruppen ausgegangen werden. Daher wurde in dieser Arbeit über den Vergleich von MLST-Sequenztypen (ST) und Virulenzfaktorprofilen von Umweltisolaten und klinischen Isolaten eine erste Risikobewertung für non-O1, non-O139 V. cholerae-Stämme aus deutschen Küstengewässern durchgeführt. Die Ähnlichkeit der Virulenzgenprofile von Umweltstämmen und klinischen Stämmen - unabhängig vom ST - sowie die hämolytische Aktivität gegenüber humanen Erythrozyten könnten auf ein humanpathogenes Potential vieler Umweltstämme in der Nordsee und Ostsee hindeuten. Ein Vergleich der Daten mit denen einer ersten Folgestudie, die in der Deutschen Bucht durchgeführt wurde, legt nahe, dass sich aus dem Klimawandel ergebende Umweltveränderungen möglicherweise V. cholerae-Stämme mit einem höheren Infektionsrisiko fördern. Das whole genome sequencing (WGS)-basierte Typisierungsschema, das im Rahmen dieser Dissertation entwickelt und am Vibrio-Konsiliarlabor etabliert wurde, kann als Werkzeug zur Überwachung und tieferen Untersuchung von Virulenzgenprofilen künftiger non-O1, non-O139 V. cholerae-Isolate aus für die öffentliche Gesundheit relevanten Quellen dienen. Während Vibrio-Arten wie V. cholerae infolge der globalen Krankheitslast seit Jahrzehnten intensiv untersucht werden, sind andere Vibrio-Arten wie die mutmaßliche Umweltspezies V. navarrensis kaum erforscht. Kürzlich wurden V. navarrensis-Stämme in humanen klinischen Proben sowie veterinären Abortproben aus Nutztieren in Deutschland nachgewiesen. Zur Identifizierung neuer potentieller Umweltreservoire in Deutschland und vor dem Hintergrund eines möglichen zoonotischen Potentials wurden in dieser Arbeit vergleichende genotypische Analysen von V. navarrensis-Isolaten aus Umweltproben, Veterinärproben und humanen klinischen Proben durchgeführt. Die Ergebnisse der SNP- und MLSA-Analysen deuten auf das Vorkommen von V. navarrensis in deutschen Süßwasserumwelten hin, was das Verbreitungsgebiet dieser Spezies von Küstengewässern der Nord- und Ostsee auf Binnengewässer in Deutschland erweitern würde. Eine Aufnahme von V. navarrensis über Oberflächenwasser-Aufnahme durch Nutztiere scheint möglich. Das Vorliegen eines MLSA-Subclusters, welches alle veterinären und humanen klinischen Stämme umfasst, könnte auf ein humanpathogenes Potential der Ersteren hinweisen. Das Design eines WGS-basierten analytischen Verfahrens zur Typisierung Virulenz-assoziierter Gene in V. navarrensis-Isolaten bot tiefere Einblicke in die genetische Ausstattung aller Stämme. Phänotypische Merkmale wie die hämolytische Aktivität der untersuchten Umwelt- und Veterinärstämme gegenüber Schaferythrozyten und humanen Erythrozyten betonen den pathogenen Charakter dieser Vibrio-Art. Ein neuer Fall einer Humaninfektion im Sommer 2020 stützt diese Bewertung. Die entwickelten V. navarrensis-spezifischen vvhA- und tlh-Singleplex-PCRs stellen vielversprechende Werkzeuge für eine schnelle Speziesdiagnostik dar und dürften dadurch künftige Überwachungs- und Forschungsstudien zu Vibrio-Bakterien im Rahmen des “One Health”-Ansatzes erleichtern

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

Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 <i>Vibrio cholerae</i> 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