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Volume 23, Number 10—October 2017
Research Letter

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

Jens A. HammerlComments to Author , 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)

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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 cholerae isolated 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 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 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 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 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.

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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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References

  1. Mangat  CS, Boyd  D, Janecko  N, Martz  SL, Desruisseau  A, Carpenter  M, et al. Characterization of VCC-1, a novel ambler class A carbapenemase from Vibrio cholerae isolated from imported retail shrimp sold in Canada. Antimicrob Agents Chemother. 2016;60:181925. DOIPubMed
  2. Lutz  C, Erken  M, Noorian  P, Sun  S, McDougald  D. Environmental reservoirs and mechanisms of persistence of Vibrio cholerae. Front Microbiol. 2013;4:375. DOIPubMed
  3. Deshayes  S, Daurel  C, Cattoir  V, Parienti  JJ, Quilici  ML, de La Blanchardière  A. Non-O1, non-O139 Vibrio cholerae bacteraemia: case report and literature review. Springerplus. 2015;4:575. DOIPubMed
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  5. Bier  N, Schwartz  K, Guerra  B, Strauch  E. 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. Front Microbiol. 2015;6:1179. DOIPubMed
  6. Jolley  KA, Maiden  MC. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 2010;11:595. DOIPubMed
  7. Sambrook  JF, Russell  DW. Molecular cloning: a laboratory manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2001.
  8. EFSA Panel on Biological Hazards (BIOHAZ). Scientific opinion on carbapenem resistance in food animal ecosystems. EFSA Journal. 2013;11:3501 [cited 2017 Aug 2]. http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2013.3501/pdf
  9. Jean  SS, Lee  WS, Lam  C, Hsu  CW, Chen  RJ, Hsueh  PR. Carbapenemase-producing Gram-negative bacteria: current epidemics, antimicrobial susceptibility and treatment options. Future Microbiol. 2015;10:40725. DOIPubMed
  10. Woodford  N, Wareham  DW, Guerra  B, Teale  C. Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: an emerging public health risk of our own making? J Antimicrob Chemother. 2014;69:28791. DOIPubMed

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Cite This Article

DOI: 10.3201/eid2310.161625

1Current affiliation: European Food Safety Authority, Parma, Italy.

Table of Contents – Volume 23, Number 10—October 2017

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Jens A. Hammerl, Department of Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn Strasse 8-10, D-10589 Berlin, Germany

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Page created: September 19, 2017
Page updated: September 19, 2017
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The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
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