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

Table

Technical Appendix

Jens A. Hammerl

, Claudia Jäckel, Valeria Bortolaia, Keike Schwartz, Nadja Bier, Rene S. Hendriksen, Beatriz Guerra1, and Eckhard Strauch

Author affiliations: German Federal Institute for Risk Assessment, Berlin, Germany (J.A. Hammerl, C. Jäckel. K. Schwartz, N. Bier, B. Guerra, E. Strauch); Technical University of Denmark, Lyngby, Denmark (V. Bortolaia, R.S. Hendriksen)

Cite This Article

Abstract

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.

Mangat et al. recently identified a novel ambler class A carbapenemase (VCC-1) in nontoxigenic Vibrio choleraeisolated from imported retail shrimp from India intended for human consumption in Canada (1). Occurrence of blaVCC-1–harboring bacteria in seafood might be caused by uptake of V. cholerae in the aquatic environment. Lutz et al. reported worldwide distribution of V. cholerae non–O1/O139 strains in coastal waters with low salinity (2). Some of these strains were associated with wound infections and with diarrheal diseases after ingestion of contaminated seafood (3,4).

An antimicrobial resistance survey of potentially pathogenic Vibrio spp. recovered from coastal waters of Germany identified 4 carbapenem-resistant V. cholerae non–O1/O139 isolates (5). These isolates were detected in the Baltic Sea (VN-2997, Eckernförde) and North Sea (VN-2808, Büsum; VN-2825, Speicherkoog; VN-2923, unknown). We used whole-genome sequencing to examine the genetic basis of carbapenem resistance in these strains.

We isolated genomic DNA by using the Easy-DNA Kit (Invitrogen, Carlsbad, CA, USA). This DNA was used for preparation of libraries by using the Nextera-XT-DNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced by using an MiSeq-benchtop-sequencer, MiSeq-Reagent version 2 (300 cycles), and two 150-bp paired-end reads (Illumina Inc.). We then performed de novo assemblies of reads by using SPAdes version 3.5.0 (http://spades.bioinf.spbau.ru/release3.5.0/manual.html ). We deposited sequences in GenBank (Table) and preformed genome annotation by using the NCBI Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/).

We found blaVCC-1 in all isolates on contigs of 2,135 bp (VN-2825, VN-2997), 2,139 bp (VN-2923), and 2,737 bp (VN-2808). The blaVCC-1-coding sequences and flanking nucleotide sequences were 100% identical among the strains, as determined by sequence alignments. We also identified the main characteristics of V. cholerae genomes (Table; Technical Appendix[PDF - 326 KB - 1 page] Figure). Overall, the genomes belong to the same multilocus sequence type (ST), ST336 (adk 57, gyrB 76, mdh 14, metE 115, pntA 18, purM 1, pyrC 101) (6).

We performed functional studies of the entire blaVCC-1–harboring region (pVCC-1C, 2.7 kb) and blaVCC-1 gene (pVCC-1G, 0.9 kb) of V. cholerae VN-2808 (Technical Appendix[PDF - 326 KB - 1 page] Figure) by molecular cloning of PCR-amplified regions according to standard procedures (7). After transformation of Escherichia coli GeneHogs (Invitrogen, Darmstadt, Germany) and susceptibility testing for aztreonam, imipenem, and meropenem as described (5), both constructs showed decreased inhibition zone diameters compared with that for E. coli GeneHogs vector. We observed slightly reduced drug susceptibility levels or intermediate resistance levels, as observed in V. cholerae VN-2808 (Technical Appendix[PDF - 326 KB - 1 page] Figure).

On the basis of these results, we conducted blaVCC-1 screening of the V. cholerae collection at the German Federal Institute for Risk Assessment (Berlin, Germany). This collection contains 312 toxigenic and nontoxigenic isolates from human, environmental, and food origin obtained in Europe (n = 218), Africa (n = 20), Asia (n = 18), North America (n = 1), South America (n =1), and of unknown origin (n = 54) during 1941–2015.

We performed PCR by using primers (blaVCC-1-forward/reverse: 5′-ATCTCTACTTCAACAGCTTTCG/CCTAGCTGCTTTAGCAATCAC-3′) with denaturation at 94°C for 120 s; 35 cycles of denaturation at 94°C for 15 s, annealing at 53°C for 30 s, and elongation for 210 s at 72°C; and a final elongation at 72°C for 1 min. This testing detected blaVCC-1 in 3 (1.6%) ctx-negative, non–O1/O139 V. cholerae isolates obtained from waters of the port of Husum, Germany, on the North Sea during 2015. Sanger sequencing confirmed the presence of blaVCC-1. These 3 isolates belong to the new multilocus ST516. This type is divergent from ST336 only for the novel pyrC 150 variant, which was recently deposited in the PubMLST database (https://pubmlst.org ).

In conclusion, this study showed the presence of 7 VCC-1 carbapenemase-producing V. cholerae at different locations on the coastline of Germany. The blaVCC-1 flanking genetic sequences were identical in the 4 sequenced V. cholerae from Germany and appeared to be different from the strain isolated in Canada, which probably originated in India (Technical Appendix[PDF - 326 KB - 1 page] Figure). This finding suggests that blaVCC-1 was acquired by V. cholerae from a yet unknown progenitor on at least 2 occasions. Strains from Germany probably belong to the autochthonous microflora and represent an environmental reservoir of carbapenem resistance in coastal waters. blaVCC-1–encoding V. cholerae might be taken up by mussels, shrimps, and fish and then enter the food chain.

Because carbapenems are needed for treatment of severe infections with multidrug-resistant bacteria in humans, the presence of bacteria with acquired, and thereby potentially transferable, carbapenemases in environments near human activities is a major public health concern (8). To date, acquired carbapenemases were detected mainly in clinical isolates and only rarely in environmental and foodborne bacteria (9,10). Exposure of humans to carbapenemase-producing pathogenic bacteria by ingestion of contaminated food products or by direct contact with contaminated water might pose a threat to public health. Our findings indicate that surveillance for antimicrobial drug resistance should be extended to locations of human activities and foods of aquatic origin.

Dr. Hammerl is a research scientist and deputy at the National Reference Laboratory on Antimicrobial Resistance, Department of Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany. His primary research interests are microbiological and molecular tracing of foodborne pathogens and antimicrobial resistance.

Acknowledgments

This study was supported by the Federal Ministry of Education and Research (VibrioNet grant 01KI1015A). The German Research Program Auswirkungen des Klimawandels auf Wasserstraßen und Schifffahrt was supported the Federal Ministry of Transport and Digital Infrastructure.

The positions and opinions in this article are those of the authors alone and are not intended to represent the views or scientific works of the European Food Safety Authority.

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