Advertisement

Emergence of mcr-1 and mcr-3 variants coding for plasmid-mediated colistin resistance in Escherichia coli isolates from food- producing animals in South Korea

Open AccessPublished:May 24, 2018DOI:https://doi.org/10.1016/j.ijid.2018.05.011

      Highlights

      • First report of colistin-resistance E. coli isolates harboring mcr-3 from South Korea.
      • Distribution and molecular characterization of isolates harboring mcr-1 gene.
      • All colistin resistant isolates were multidrug-resistant.
      • mcr-1 and mcr-3 genes could readily transfer to recipient strain by conjugation.

      Abstract

      We hereby report the first characterization of mcr-3 gene from healthy animals in South Korea. Out of 636 E. coli isolates, collected between 2014- 2017, nine colistin resistant isolates were screened for the presence of mcr-1 and mcr-3 genes. Nine (1.4%) isolates had shown resistance for colistin and among them three and two isolates were mcr-1 harboring and mcr-3 harboring strains, respectively. All the colistin-resistant isolates were multidrug-resistant. mcr-1 and mcr-3 genes were confirmed to be transferred to a recipient E. coli J53 AZR.

      Graphical abstract

      Keywords

      Since it was first described in China in 2015 (
      • 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.
      ), the plasmid-mediated mcr-1 gene has been gaining huge attention worldwide, and the awareness and the extent of the problem also increased gradually. Following the first report, another mcr-variant of the plasmid-mediated colistin resistance called mcr-2 gene was identified in E. coli strain recovered from swine and cattle in Belgium (
      • Xavier B.B.
      • Lammens C.
      • Ruhal R.
      • Kumar-Singh S.
      • Butaye P.
      • Goossens H.
      • et al.
      Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016.
      ), and in June 2017, mcr-3 was detected on the 261 kb plasmid (IncHl2) of E. coli isolated from swine in China (
      • Yin W.
      • Li H.
      • Shen Y.
      • Liu Z.
      • Wang S.
      • Shen Z.
      • et al.
      Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli.
      ). This third variant showed nucleotide sequence similarities of 45% and 47% with previously detected mcr-1 and mcr-2 genes, respectively. There is a need for strict regulation on the use of colistin in veterinary medicine to reduce the danger of transferable mcr genes to humans via the food chain (
      • 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.
      ). Here, we report the first detection of the mcr-3 gene variant in South Korea and describe the molecular characteristics of mcr-1 and mcr-3 harboring E. coli isolates recovered from healthy animals.
      A total of 636 commensal E. coli isolates recovered from fecal samples obtained from clinically healthy animals (341 from cattle, 265 from swine, and 30 from chickens) during a nationwide surveillance study conducted between 2014 and 2017 on antimicrobial susceptibility. MIC testing was conducted according to the recommendation of the Clinical Laboratory Standards Institute (
      • Clinical Laboratory and Standards Institute
      Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI M100-S25. Clinical and Laboratory Standards Institute.
      ), and resulting MIC values were interpreted using CLSI and European Committee for Antimicrobial Susceptibility Testing (

      European Committee on Antimicrobial Susceptibility Testing (EUCAST). [Internet]. [Accessed 1 October 2017]. Available from: http://www.eucast.org.

      ) breakpoints. PCR amplification to investigate the mcr-1 and mcr-3 encoding genes was conducted on isolates exhibiting colistin resistance using previously employed primer pairs and PCR conditions (
      • 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.
      ;
      • Yin W.
      • Li H.
      • Shen Y.
      • Liu Z.
      • Wang S.
      • Shen Z.
      • et al.
      Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli.
      ). The transferability of colistin resistant genes between donors (mcr-1 and mcr-3 positive isolates) and recipient bacteria (E. coli J53 azide resistant) was assessed by conjugation experiments using the broth mating method (
      • Wang M.
      • Tran J.H.
      • Jacoby G.A.
      • Zhang Y.
      • Wang F.
      • Hooper D.C.
      Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China.
      ).
      Of the 636 investigated E. coli isolates, 9 (1.4%) were resistant to colistin, and the mcr-3 gene was detected in 2 (MIC of 4 μg/ml) of these 9 isolates, whereas the mcr-1 gene was detected in 3 isolates (MICs 4 μg/ml to 8 μg/ml). Although, to the best of our knowledge, this is the first report of the mcr-3 gene in South Korea, the mcr-1 gene has been previously detected in human and livestock (
      • Lim S.K.
      • Kang H.Y.
      • Lee K.
      • Moon D.C.
      • Lee H.S.
      • Jung S.C.
      First detection of the mcr-1 gene in Escherichia coli isolated from livestock between 2013 and 2015 in South Korea.
      ;
      • Kim E.S.
      • Chong Y.P.
      • Park S.J.
      • Kim M.N.
      • Kim S.H.
      • Lee S.O.
      • et al.
      Detection and genetic features of mcr-1-producing plasmid in human Escherichia coli infection in South Korea.
      ). The emergence of colistin resistance resulted due to excessive consumption of colistin in veterinary medicine. In the years 2005 to 2015 on average 6-16 tons of colistin was annually applied in food producing animals. In addition, colistin is the main treatment option used to treat infections in pigs. DNA sequencing of the PCR fragments was performed, with following BLASTN against the GenBank database of the National Center for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The results revealed 99% and 98% sequence identities with previously deposited mcr-1 (GenBank accession no. KY964067.1) and mcr-3 (GenBank accession no. KY924928.1) gene sequences, respectively.
      A literature review showed that only 3 reports of mcr-3 in E. coli have been issued, mcr-3 was first described in China in 2017 (
      • Yin W.
      • Li H.
      • Shen Y.
      • Liu Z.
      • Wang S.
      • Shen Z.
      • et al.
      Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli.
      ), and this was followed by a Danish report on a hospitalized patient who had traveled to Thailand (
      • Roer L.
      • Hansen F.
      • Stegger M.
      • Sönksen U.W.
      • Hasman H.
      • Hammerum A.M.
      Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014.
      ), and a Spanish report (
      • Hernández M.
      • Iglesias M.R.
      • Rodríguez-Lázaro D.
      • Gallardo A.
      • Quijada N.M.
      • Miguela-Villoldo P.
      • et al.
      Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.
      ) on the isolation of MDR E. coli recovered from cattle.
      Although the prevalence of the mcr-1 gene is considered to be very low in isolates from healthy animals, a slight increase in the prevalence of this gene has been reported in South Korea mainly among healthy chickens (
      • Lim S.K.
      • Kang H.Y.
      • Lee K.
      • Moon D.C.
      • Lee H.S.
      • Jung S.C.
      First detection of the mcr-1 gene in Escherichia coli isolated from livestock between 2013 and 2015 in South Korea.
      ). However, in our report all isolates carrying the mcr-1 gene were from pigs, which might have been due to the investigation of fewer isolates from chickens or possibly further spread of this gene, which necessitates profound surveillance to determine its prevalence in different animal species.
      The global spread of the mcr-1 gene suggests there might be a similar chance for mcr-3 to spread into different geographical regions (
      • Hernández M.
      • Iglesias M.R.
      • Rodríguez-Lázaro D.
      • Gallardo A.
      • Quijada N.M.
      • Miguela-Villoldo P.
      • et al.
      Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.
      ). For isolates harboring the mcr-1 gene (isolated between 2015 and 2016) colistin MIC values ranging from 4 μg/ml to 8 μg/ml and belonged to phylogenetic groups A and B, whereas mcr-3 (isolated between 2016 and 2017) harboring isolates both had a colistin MIC value of 4 μg/ml and affiliated to D and A phylogenetic groups (Table 1). Interestingly, all isolates harboring mcr-1 and mcr-3 genes were MDR. The isolates showed resistance to at least 3 antimicrobials and carried various antimicrobial resistant genes, which concurs with a previous report (
      • Lim S.K.
      • Kang H.Y.
      • Lee K.
      • Moon D.C.
      • Lee H.S.
      • Jung S.C.
      First detection of the mcr-1 gene in Escherichia coli isolated from livestock between 2013 and 2015 in South Korea.
      ). Moreover, all 5 isolates that harbored mcr-1 or mcr-3 were capable of transferring their genes to the recipient E. coli J53 AZR strain as indicated by broth mating assay. The conjugation efficiencies of the isolates lay between 8.6x10−3 and 1.9x10−4 (Table 2).
      Table 1MICs, Phylogrouping and antimicrobial resistant genes for colistin resistant E. coli isolates of animal origin from South Korea between 2015- 2017.
      IsolatesProvinceOriginYear of isolationPhylo- groupMIC μg/mlmcr gene detectedTransferabilityOther AMR genes
      CL
      EUCAST interpretation was used.
      LEVOXYMINTOBCFNEOSTRAMPTETCIPDOX
      DCA4245ChungnamPig2016B148≥256328≥256128256≥25664≥12832mcr-1YestetA, tetB, TEM, ampC, floR, sul2, sul3, cmlA,strAB, aphA1
      17S-208JeollabukPig2017D48256164323232≥25616≥12816mcr-3YestetA, TEM, ampC, sul2, floR, strAB, aphA1
      16S-251ChungnamPig2016A40.52564321664256≥2568≥12816mcr-3YestetA, OXA, TEM, ampC, floR, sul1, sul2, cat, strAB, aphA1
      15S-103GyeonggiPig2015A882561632161256≥25664≥12816mcr-1YestetA, TEM, floR, ampC, sul1, sul2, strAB
      15S-123ChungnamPig2015A80.5≥256164321128≥25632≥12816mcr-1YestetB, TEM, floR, ampC, sul1, sul2, sul3, cat
      Abbreviations: CL,colistin; LEV, levofloxacin; OXY, oxytetracycline; MIN, minocycline; TOB, tobramycin;CF, cephalothin; NEO, neomycin; STR, streptomycin; AMP, ampicillin; TET,tetracycline; CIP, ciprofloxacin; DOX, doxycycline.
      * EUCAST interpretation was used.
      Table 2Conjugation transfer efficiencies of mcr-3 and mcr-1 positive isolates, and colistin MIC values for transconjugants.
      IsolateTransferabilityMIC (μg/ml)Recipient (
      Colistin MIC value for the recipient strain (E. coli J53 AZR) was 0.5μg/ml.
      E. coli J53 AZR)
      DonorTransconjugantsConjugation efficiency
      DCA4245+41.41x1082.2X1064.1X1021.9X10−4
      17S-208+41.7X1051.47X1038.6X10−3
      16S-251+47X1054.8X1026.9X10−4
      15S-103+41.1X1064.5X1024.1X10−4
      15S-123+89.2X1051.4X1031.5X10−3
      * Colistin MIC value for the recipient strain (E. coli J53 AZR) was 0.5 μg/ml.
      In conclusion, it would appear that the extensive application of colistin to food-producing animals has promoted the emergence of plasmid-mediated colistin resistance in E. coli. Therefore, prudent consideration should be exercised before administering colistin. Furthermore, we advise continuous and regular surveillance by implementing an appropriate monitoring program on antimicrobial resistance and also on antimicrobial use to trace the early dissemination of mcr-3 and for that matter, to identify new mcr variants of mcr-4, -5, -6 and so on.

      Acknowledgements

      This study was supported by the “Cooperative research program for agriculture science and technology development (project No. PJ00897001)”, RDA, the Research Institute of Veterinary Science and BK21 PLUS program, Seoul National University, Republic of Korea.
      Conflict of interest: None to declare.

      References

        • 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.
        Lancet Infect Dis. 2016; 16: 161-168
        • Xavier B.B.
        • Lammens C.
        • Ruhal R.
        • Kumar-Singh S.
        • Butaye P.
        • Goossens H.
        • et al.
        Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016.
        Euro Surveill. 2016; 21: 30280
        • Yin W.
        • Li H.
        • Shen Y.
        • Liu Z.
        • Wang S.
        • Shen Z.
        • et al.
        Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli.
        MBio. 2017; 83: e00543-e617
        • Clinical Laboratory and Standards Institute
        Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI M100-S25. Clinical and Laboratory Standards Institute.
        Clinical and Laboratory Standards Institute, Wayne, PA2015
      1. European Committee on Antimicrobial Susceptibility Testing (EUCAST). [Internet]. [Accessed 1 October 2017]. Available from: http://www.eucast.org.

        • Wang M.
        • Tran J.H.
        • Jacoby G.A.
        • Zhang Y.
        • Wang F.
        • Hooper D.C.
        Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China.
        Antimicrob Agents Chemother. 2003; 47: 2242-2248
        • Lim S.K.
        • Kang H.Y.
        • Lee K.
        • Moon D.C.
        • Lee H.S.
        • Jung S.C.
        First detection of the mcr-1 gene in Escherichia coli isolated from livestock between 2013 and 2015 in South Korea.
        Antimicrob Agents Chemother. 2016; 60: 6991-6993
        • Kim E.S.
        • Chong Y.P.
        • Park S.J.
        • Kim M.N.
        • Kim S.H.
        • Lee S.O.
        • et al.
        Detection and genetic features of mcr-1-producing plasmid in human Escherichia coli infection in South Korea.
        Diagn Microbiol Infec Dis. 2017; 89: 158-160
        • Roer L.
        • Hansen F.
        • Stegger M.
        • Sönksen U.W.
        • Hasman H.
        • Hammerum A.M.
        Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014.
        Euro Surveill. 2017; 22: 30584
        • Hernández M.
        • Iglesias M.R.
        • Rodríguez-Lázaro D.
        • Gallardo A.
        • Quijada N.M.
        • Miguela-Villoldo P.
        • et al.
        Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.
        Euro Surveill. 2017; 22: 30586