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

Genotypische und phänotypische Charakterisierung von Vibrio vulnificus Isolaten aus der Umwelt und klinischen Proben der Nordsee- und Ostseeregion

Table of Contents ...................................................................................................................... i List of Abbreviations ................................................................................................................ v 1 Introduction ...................................................................................................................... 1 1.1 Taxonomy and Biochemical Characteristics of Vibrio vulnificus ............................... 1 1.2 Ecology of Vibrio vulnificus ........................................................................................ 3 1.3 Disease and Epidemiology of Vibrio vulnificus .......................................................... 6 1.4 Virulence Factors and Pathogenesis of Vibrio vulnificus .......................................... 11 1.5 Enigma of Vibrio vulnificus ....................................................................................... 14 1.6 Clinical versus Environmental Isolates of Vibrio vulnificus ..................................... 15 1.6.1 Multilocus Sequence Typing .............................................................................. 16 1.6.2 16S rRNA ........................................................................................................... 17 1.6.3 Virulence Correlated Gene (vcg) ........................................................................ 17 1.6.4 Mannitol Fermentation and Associated Genes ................................................... 18 1.6.5 pilF ..................................................................................................................... 19 1.6.6 Sialic Acid Catabolism and Biosynthesis ........................................................... 19 1.6.7 Genomic Region XII .......................................................................................... 20 1.7 Aims of the Study ...................................................................................................... 21 2 Publications ..................................................................................................................... 22 2.1 List of Publications and Own Contribution ............................................................... 22 2.2 Publication 1: Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region ...................... 24 2.3 Publication 2: Multiplex PCR for Detection of Virulence Markers of Vibrio vulnificus ................................................................................................................................... 36 2.4 Publication 3: Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates .................................................. 43 2.5 Publication 4: 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 ............................................................................. 60 2.6 Publication 5: PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus ................................................................................................................................... 71 3 General Discussion ......................................................................................................... 76 3.1 Background ................................................................................................................ 76 3.2 Genotypic and Phenotypic Characterization of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region .......................................................... 77 3.2.1 Resistance to Human Serum .............................................................................. 77 3.2.2 Hemolysis ........................................................................................................... 78 3.2.3 Cytotoxicity to Caco-2 Cells .............................................................................. 80 3.2.4 Detection of Potential Virulence-Associated Genes .......................................... 82 3.2.5 Genotyping of 16S rRNA, vcg and pilF Genes .................................................. 82 3.2.6 Multilocus Sequence Typing .............................................................................. 83 3.2.7 Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry ............................................................................................................................ 84 3.2.8 Estimation of the Pathogenicity Potential of Vibrio vulnificus .......................... 85 3.3 Multiplex PCR for Simultaneous Detection of Putative Pathogenicity Markers of Vibrio vulnificus .................................................................................................... 86 3.4 Prevalence of Virulence-Associated Characteristics among Environmental Vibrio vulnificus Isolates from the North Sea and Baltic Sea ............................................... 86 3.5 Antimicrobial Resistance among Vibrio vulnificus Isolates from German Coastal Waters ........................................................................................................... 87 3.6 Molecular Cloning in Vibrio vulnificus and Potential Applications.......................... 90 4 Summary ......................................................................................................................... 92 5 Zusammenfassung .......................................................................................................... 94 6 References ....................................................................................................................... 96 7 Appendix ....................................................................................................................... 113 7.1 Supplementary Material .......................................................................................... 113 7.1.1 Publication 1 ..................................................................................................... 113 7.1.2 Publication 2 ..................................................................................................... 121 7.1.3 Publication 3 ..................................................................................................... 123 7.1.4 Publication 4 ..................................................................................................... 145 7.2 Publications and Congress Presentations ................................................................ 162 7.3 Eidesstattliche Erklärung ......................................................................................... 164The Gram-negative bacterium V. vulnificus is ubiquitously distributed in coastal and estuarine waters with moderate salinity and sporadically causes severe foodborne or wound infections. Infections with this fast-growing pathogen are rare in Germany. However, forecasts assume a rising occurrence of V. vulnificus in German coastal waters, as well as increasing incidences of infection in the course of climate change. In view of these predictions, fundamental research and characterization of V. vulnificus present in German coastal waters regarding its pathogenic potential is of great importance for public health concerns. Furthermore, methods to identify potentially pathogenic strains of this bacterium are strongly needed to estimate the risk emanating from this pathogen. In this work, the distribution of potential virulence genes, as well as of virulenceassociated pheno- and genotypes was examined in clinical and environmental isolates from the Baltic Sea region to assess their suitability as potential pathogenicity markers. The most promising results were seen for genomic region XII, the nanA gene, and a mannitol fermentation operon, which were found in most clinical and relatively few environmental isolates. Genotypes that are described to correlate with the clinical origin in other studies, such as vcg-type C or 16S rRNA-type B were only rarely detected in clinical isolates from cases of the Baltic Sea region. However, these genotypes are strong indicators for phylogenetic lineages with a high pathogenicity potential. Phenotypic traits associated with virulence of V. vulnificus, such as resistance to human serum, strong hemolytic activity or cytotoxicity were exhibited by most isolates, underlining the destructive character of this pathogenic species. The results led to the development and validation of a multiplex PCR that allows simultaneous amplification of genomic region XII, the nanA gene, the mannitol fermentation operon, as well as of the vcg-type C and 16S rRNA-type B alleles. The prevalence of these potential pathogenicity markers was investigated in recent environmental isolates from the Baltic Sea as well as the North Sea. Together with phylogenetic analyses using multilocus sequence typing, this study showed that V. vulnificus populations in both areas vary considerably. The majority of North Sea isolates have to be considered as potential risk for human infection, although no V. vulnificus infections have been reported from this region so far. Therefore, increased awareness of this pathogen is needed in both, the Baltic Sea as well as the North Sea area, especially in summer when exposition to the pathogen is at its highest. The characterization of V. vulnificus isolates was complemented by investigations on antimicrobial resistance patterns. All V. vulnificus isolates - irrespective of their origin - were susceptible to clinically relevant agents, such as tetracyclines, third-generation cephalosporins, and fluoroquinolones. No multidrug resistance was observed. Reduced susceptibility was exclusively found for streptomycin, which might be the result of an intrinsic resistance. Finally, the applicability of a newly designed shuttle vector for molecular cloning in Vibrio spp. was demonstrated. The developed system represents an efficient tool for functional analyses of putative virulence genes.V. vulnificus ist ein Gram-negatives Bakterium, das ubiquitär in Küstengewässern und Ästuarien mit mäßigem Salzgehalt verbreitet ist und schwere Lebensmittel- und Wundinfektionen verursachen kann. Infektionen mit diesem schnell wachsenden Bakterium sind selten in Deutschland. Im Zuge des Klimawandels wird jedoch sowohl mit einem Anstieg des Vorkommens von V. vulnificus in deutschen Küstengewässern als auch mit einer steigenden Inzidenz an Infektionen gerechnet. In Anbetracht dieser Vorhersagen ist die grundlegende Untersuchung und Charakterisierung von V. vulnificus Isolaten in deutschen Küstengewässern in Bezug auf deren pathogenes Potential von großer Bedeutung für die öffentliche Gesundheit. Darüber hinaus werden dringend Methoden benötigt, die die Identifizierung von pathogenen Stämmen ermöglichen, um das von diesen Bakterien ausgehende Gesundheitsrisiko beurteilen zu können. In dieser Arbeit wurde das Vorkommen mögliche Virulenzgene und virulenzassoziierter Phäno- und Genotypen in klinischen Isolaten und Umweltisolaten der Ostseeregion untersucht, um ihre Eignung als potentielle Pathogenitätsmarker einzuschätzen. Die aussichtsreichsten Ergebnisse zeigten Untersuchungen der genomischen Region XII, des Gens nanA und des Mannitol Fermentations Operons. Diese Gene/Genregionen waren in den meisten klinischen Isolaten, aber nur in verhältnismäßig wenigen Umweltisolaten zu finden. Genotypen, die laut Literatur mit dem klinischen Ursprung von V. vulnificus Isolaten korrelieren sollten, wie z.B. der vcg-Typ C oder der 16S rRNA-Typ B, wurden nur selten in klinischen Isolaten aus dem Ostseeraum nachgewiesen. Jedoch deuten diese Genotypen stark auf eine Zugehörigkeit zu phylogenetischen Linien mit hohem Pathogenitätspotential hin. Die meisten Isolate wiesen phänotypische Merkmale auf, die mit der Virulenz von V. vulnificus assoziiert werden. Die weit verbreitete Resistenz gegenüber humanem Serum sowie die starke hämolytische Aktivität und Zytotoxizität heben den stark destruktiven Charakter dieser Spezies hervor. Die Ergebnisse legten die Entwicklung und Validierung einer Multiplex PCR nahe, die die simultane Amplifikation der genomischen Region XII, des Gens nanA, des Mannitol Fermentations Operons und der vcg-Typ C und 16S rRNA-Typ B Allele erlaubt. Schließlich wurde das Vorkommen dieser potentiellen Pathogenitätsmarker in kürzlich isolierten Umweltstämmen aus der Nordsee und Ostsee untersucht. Zusammen mit den phylogenetischen Analysen mittels Multilokus-Sequenz-Typisierung zeigten diese Untersuchungen, dass sich die V. vulnificus Populationen in beiden Regionen wesentlich unterscheiden. Obwohl bisher noch keine Infektionen aus dem Nordseeraum berichtet wurden, geht von den in dieser Studie untersuchten Nordseeisolaten ein potentielles Risiko für humane Infektionen aus. Daher ist erhöhte Aufmerksamkeit für die Gesundheitsrisiken dieses pathogenen Erregers im Ostseeraum und Nordseeraum geboten. Dies gilt besonders in den Sommermonaten, wenn die Exposition mit diesem Bakterium am größten ist. Die Charakterisierung der V. vulnificus Isolate wurde durch Antibiotikaresistenzuntersuchungen ergänzt. Alle V. vulnificus Isolate - unabhängig von deren Ursprung - waren empfindlich gegenüber klinisch relevanten antimikrobiellen Substanzen, wie z.B. Tetrazyklinen, Cephalosporinen der 3. Generation und Fluorochinolonen. Es traten keine Multiresistenzen auf und geringere Empfindlichkeit wurde nur gegenüber dem Aminoglycosid Streptomycin beobachtet, wobei es sich hierbei um eine intrinsische Resistenz handeln könnte. Schließlich wurde die Anwendbarkeit eines neu konstruierten “Shuttle”-Vektors für die effiziente Klonierung von Vibrio Spezies nachgewiesen. Das entwickelte Klonierungssystem stellt damit ein effizientes Werkzeug für funktionelle Analysen potentieller Virulenzgene dar

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

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

Prevalence of Alaria alata mesocercariae in wild boars from Brandenburg, Germany

Since 2002, Alaria (A.) alata mesocercariae (AM) have been found during routine Trichinella inspection of wild boars in many European countries. To date, human infection with AM through consumption of undercooked or raw AM infested wild boar meat cannot be excluded. In Germany, data on the parasite’s prevalence in wild boars are scarce. To better understand temporal and spatial fluctuations of this parasite, this study investigated the prevalence of AM in wild boars in the German federal state of Brandenburg during three hunting seasons from 2017 to 2020. In total, 28.3% (100/354, 95% CI: 23.3–33.3%) of all wild boars sampled in eight counties of Brandenburg were tested positive for AM by Alaria alata mesocercariae migration technique (AMT). AM were detected in wild boars from seven different counties. Samples from one county (Havelland) tested completely negative for AM (0/16). Prevalences of the seven AM positive counties of Brandenburg ranged from 11.5 (3/26, 95% CI: 2.5–30.1%) in Märkisch-Oderland to 64.1% (25/39, 95% CI: 47.2–78.8%) in Uckermark. An association between sex and A. alata positivity could not be determined. A statistically significant increase in frequency of older AM positive wild boars was observed (p = 0.001). For a nationwide assessment of the prevalence of A. alata in wild boars and the risk for consumers of ingesting viable AM by consumption of raw or undercooked AM infested wild boar meat, further long-term studies in different regions of Germany are needed

Survival time of Leptospira kirschneri on strawberries.

In the past decade, two leptospirosis outbreaks occurred among strawberry harvesters in Germany, with 13, and 45 reported cases respectively. In both outbreaks, common voles (Microtus arvalis) infected with Leptospira kischneri serovar Grippotyphosa were identified as the most likely outbreak source. In an univariate analysis, eating unwashed strawberries was identified as one of the risk factors associated with Leptospira infection. The aim of this study was to evaluate the survival time of L. kirschneri serovar Grippotyphosa on strawberries under varying conditions. Strawberries were spiked with 5x109 of both a laboratory reference strain (strain Moskva V) and an outbreak field strain (94-6/2007) of L. kirschneri serovar Grippotyphosa sequence type 110. Survival times were investigated in a fully crossed design with three incubation times (2h, 4h, 6h and 8h) and three temperatures (15°C, 21°C and 25°C) with three replicated for each condition. A wash protocol was developed and recovered Leptospira were determined by qPCR, dark field microscopy and culturing. Viable L. kirschneri of both the reference strain and the field strain were identified in all samples at 25°C and an incubation time of 2h, but only 1/9 (11%) and 4/9 (44%) of the samples incubated at 15°C were positive, respectively. Both reference and field strain were viable only in 2/9 (22%) at 25° after 6h. After an 8h incubation, viable Leptospira could not be identified on the surface of the strawberries or within the fruit for any of the tested conditions. Based on these results, the exposure risk of consumers to viable Leptospira spp. through the consumption of strawberries bought at the retail level is most likely very low. However, there is a potential risk of Leptospira infection by consumption of strawberries on pick-your-own farms

Molecular Methods for the Detection of Toxoplasma gondii Oocysts in Fresh Produce: An Extensive Review

Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure

Molecular Methods for the Detection of <i>Toxoplasma gondii</i> Oocysts in Fresh Produce: An Extensive Review

Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure