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Emergence of a colistin-resistant Escherichia coli clinical isolate harboring mcr-1 in Japan

  • Tatsuya Tada
    Affiliations
    Department of Microbiology, Juntendo University School of Medicine, Tokyo, Japan

    Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
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  • Kohei Uechi
    Affiliations
    Division of Clinical Laboratory and Blood Transfusion, University Hospital of the Ryukyus, Okinawa, Japan

    Department of Infectious Diseases, Respiratory, and Digestive Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
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  • Isamu Nakasone
    Affiliations
    Control and Prevention of Infectious Disease, University Hospital of the Ryukyus, Okinawa, Japan
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  • Kayo Shimada
    Affiliations
    Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
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  • Masashi Nakamatsu
    Affiliations
    Department of Infectious Diseases, Faculty of the Ryukyus, Okinawa, Japan
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  • Teruo Kirikae
    Correspondence
    Corresponding author at: Department of Microbiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: +81 3 5802 1041; fax: +81 3 5684 7830.
    Affiliations
    Department of Microbiology, Juntendo University School of Medicine, Tokyo, Japan

    Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
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  • Jiro Fujita
    Affiliations
    Department of Infectious Diseases, Respiratory, and Digestive Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
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Open AccessPublished:August 02, 2017DOI:https://doi.org/10.1016/j.ijid.2017.07.023

      Highlights

      • A colistin-resistant Escherichia coli isolate harboring mcr-1 was isolated in a university hospital in Japan.
      • Pulsed-field gel electrophoresis and Southern hybridization revealed that mcr-1 was located on a 130-kb plasmid.
      • The genomic environment surrounding mcr-1 had 99.99% identity with those in Citrobacter braakii plasmid from Bolivia and Salmonella enterica serovar Typhimurium plasmid from China.
      • This is the first report of a clinical isolate harboring mcr-1 in Japan.

      Abstract

      The mcr-1 is a gene encoding a phosphoethanolamine transferase, which confers resistance to colistin by transferring phosphoethanolamine to lipid A. We describe here the emergence of a colistin-resistant Escherichia coli clinical isolate harboring plasmid-mediated mcr-1 in Japan. The isolate belonged to ST5702 and is suspected to come from livestock and transmitted to human. This is the first report of a clinical isolate harboring mcr-1 in Japan.
      The mcr-1 was first detected on a plasmid in colistin-resistant Escherichia coli from livestock and patients in China (
      • Liu Y.Y.
      • Wang Y.
      • Walsh T.R.
      • Yi L.X.
      • Zhang R.
      • Spencer J.
      • et al.
      Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.
      ), leading to worldwide dissemination of mcr-1-positive strains in Enterobacteriaceae (
      • Poirel L.
      • Jayol A.
      • Nordmann P.
      Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes.
      ). We describe here the emergence of a colistin-resistant E. coli isolate harboring mcr-1 from a patient in a university hospital in Okinawa, Japan.
      Four E. coli isolates were screened by active surveillance for colistin-resistant Gram-negative bacteria during January to April 2017 at 14 hospitals and 2 clinical examination laboratories in Okinawa, using screening cystine lactose electrolyte deficient agar (Sigma-Aldrich, Tokyo, Japan) containing 2 μg/ml colistin and 3 μg/ml vancomycin. Of them, an isolate, E. coli RYU2912C-1, was positive for mcr-1. E. coli RYU2912C-1 was isolated from feces of a male patient, 18 years old, who twice had extended hospital stays in 2016, with acute leukemia in February 2017. The patient was prescribed oral polymyxin B (1 × 106 units three times per day for 12 days) to prevent infection by decontamination of the digestive tract before obtaining the isolate. He has an international travel history (Taiwan in 2015) and has been living closely to livestock. The MICs of antimicrobial agents were determined using the microdilution method, according to the guidelines of the
      • Clinical and Laboratory Standards Institute
      Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI M100-S25.
      . The genome and extracted plasmids were sequenced by MiSeq (Illumina, San Diego, CA) and analyzed using CLC genomics workbench v.8.0 (CLC bio, Tokyo, Japan) as described previously (
      • Tada T.
      • Nhung P.H.
      • Miyoshi-Akiyama T.
      • Shimada K.
      • Phuong D.M.
      • Anh N.Q.
      • et al.
      IMP-51, a novel IMP-type metallo-β-lactamase with increased doripenem and meropenem hydrolyzing activities, in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.
      ). Drug resistance genes encoding aminoglycosides, β-lactamases and colistin were identified using ResFinder 2.1, a web tool for detecting acquired antimicrobial resistance genes in total or partial sequenced isolates of bacteria (https://cge.cbs.dtu.dk//services/ResFinder/). To determine whether the mcr-1 is located on the chromosome or the plasmid in RYU2912C-1, Southern hybridization analysis was conducted; i.e. genomic DNA of RYU2912C-1 was digested with S1 nuclease, separated by pulsed-field gel electrophoresis, and subjected to Southern hybridization using a probe for mcr-1 as previously described (
      • Liu Y.Y.
      • Wang Y.
      • Walsh T.R.
      • Yi L.X.
      • Zhang R.
      • Spencer J.
      • et al.
      Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.
      ).
      As shown in Table 1, E. coli RYU2912C-1 was resistant to chloramphenicol, colistin, penicillins and tetracyclines, whereas it was susceptible to aminoglycosides, other β-lactams, ciprofloxacin and fosfomycin. In particular, MICs of colistin for the isolate was 16 μg/mL for the broth microdilution method. The isolate belonged to ST5702 and harbored mcr-1, blaSHV-14 blaTEM-1, ampC and aadA1. The mcr-1 was located on a 70-kb plasmid (Figure 1), and the incompatibility of the plasmid was IncI2. The remaining 3 colistin-resistant isolates were negative for mcr-1 and had 4 μg/mL MICs of colistin.
      Table 1MICs of various antibiotics for E. coli RYU2912C-1.
      AntibioticsMICs (μg/ml)
      Broth microdilution
      Ampicillin512
      Ampicillin/sulbactam
      The ratio of ampicillin to sulbactam was 2:1.
      32
      Penicillin>1024
      Aztreonam0.25
      Ceftazidime1
      Cefotaxime≤0.125
      Imipenem0.25
      Meropenem0.063
      Amikacin8
      Arbekacin4
      Gentamicin2
      Ciprofloxacin0.5
      Colistin16
      Tigecycline0.5
      Tetracycline256
      Minocycline64
      Fosfomycin128
      Chloramphenicol256
      a The ratio of ampicillin to sulbactam was 2:1.
      Figure 1
      Figure 1Southern blot analysis with a probe for mcr-1. (A) Pulsed-field gel electrophoresis patterns; the whole genome of RYU2912C-1 was digested with S1. (B) Southern hybridization was performed with a probe for mcr-1. M, MidRange PFG marker (New England Biolabs, Tokyo, Japan).
      The genomic environment surrounding mcr-1 in RYU2912C-1 (accession no. LC228070) was traL-orf1-nikA-nikB-mcr-1-orf2-ofr3-ydfA-topB-orf4 (orf1, orf3 and ofr4; encoding genes of hypothetical proteins, orf2; encoding gene of putative PAP family transmembrane protein). The genomic environment surrounding mcr-1 had 99.99% identity with those in Citrobacter braakii CA-26 plasmid pCA-26 (GenBank accession number KY624633) from Bolivia (
      • Sennati S.
      • Di Pilato V.
      • Riccobono E.
      • Di Maggio T.A.L.
      • Villagran A.L.
      • Pallecchi L.
      • et al.
      Citrobacter braakii carrying plasmid-borne mcr-1 colistin resistance gene from ready-to-eat food from a market in the Chaco region of Bolivia.
      ), and Salmonella enterica serovar Typhimurium plasmid pHSSH22-MCR-1 (GenBank accession number KX856067) from China.
      There are three possible ways which the isolate was transmitted to the patient. One possibility is hospital acquired infection, and another is acquisition of the strain in Taiwan during his travel. The patient most likely acquired the strain from the livestock. However, it is not likely that the strain was acquired from livestock in Taiwan. Unfortunately, the patient’s family did not agree to analyze samples from their livestock; therefore, we could not obtain the direct evidence that the strain came from the livestock. We are planning to conduct active surveillance for mcr-1 positive microorganisms in livestock in Okinawa with veterinarians. The mcr-1 seems to have disseminated among swine pathogenic E. coli in Japan since the first case in 2007 (
      • Kusumoto M.
      • Ogura Y.
      • Gotoh Y.
      • Iwata T.
      • Hayashi T.
      • Akiba M.
      Colistin-resistant mcr-1-positive pathogenic Escherichia coli in swine, Japan, 2007–2014.
      ). Four mcr-1-positive ST457 E. coli isolates from cattle and one isolate of Salmonella enterica from swine were also reported in Japan (
      • Suzuki S.
      • Ohnishi M.
      • Kawanishi M.
      • Akiba M.
      • Kuroda M.
      Investigation of a plasmid genome database for colistin-resistance gene mcr-1.
      ). ST5702 E. coli isolates were detected from livestock in China and human in this study. Therefore, RYU2912C-1 may be from livestock.
      This is the first report of a clinical isolate harboring mcr-1 in Japan.

      Conflict of interest

      None.

      Acknowledgements

      This study was approved by the Ethical Committee, University of the Ryukyus (approval number: 890), and the Biosafety Committee, National Center for Global Health and Medicine (approval number: 28-M-053) and supported by grants of Okinawa Communicable Disease Research Hub Formation Promotion Project, Research Program on Emerging and Re-emerging Infectious Diseases from AMED and JSPS KAKENHI .
      Grant Number 16K19133 .

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