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Letter to the Editor

mcr-3 and mcr-4 Variants in Carbapenemase-Producing Clinical Enterobacteriaceae Do Not Confer Phenotypic Polymyxin Resistance

Jeanette W. P. Teo, Marimuthu Kalisvar, Indumathi Venkatachalam, Oon Tek Ng, Raymond T. P. Lin, Sophie Octavia
Nathan A. Ledeboer, Editor
Jeanette W. P. Teo
aNational University Hospital, Department of Laboratory Medicine, Singapore
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Marimuthu Kalisvar
bTan Tock Seng Hospital, Department of Infectious Diseases, Singapore
cNational Centre for Infectious Diseases, Singapore
dNational University of Singapore, Yong Loo Lin School of Medicine, Singapore
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Indumathi Venkatachalam
eSingapore General Hospital, Department of Infectious Diseases, Singapore
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Oon Tek Ng
bTan Tock Seng Hospital, Department of Infectious Diseases, Singapore
cNational Centre for Infectious Diseases, Singapore
fNanyang Technological University, Lee Kong Chian School of Medicine, Singapore
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Raymond T. P. Lin
aNational University Hospital, Department of Laboratory Medicine, Singapore
gNational Public Health Laboratory, Ministry of Health, Singapore
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Sophie Octavia
gNational Public Health Laboratory, Ministry of Health, Singapore
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Nathan A. Ledeboer
Medical College of Wisconsin
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DOI: 10.1128/JCM.01562-17
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LETTER

The worldwide distribution of plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, and mcr-4) coupled to the emerging observation that colistin resistance is more prevalent in carbapenem-resistant Enterobacteriaceae (CRE) (1, 2) presents a daunting challenge in combatting antimicrobial resistance. Undoubtedly, next-generation sequencing approaches have expedited the discovery of mobile colistin resistance determinants (3). In this study, we undertook the in silico screening of 500 phenotypically carbapenem-resistant carbapenemase-producing Enterobacteriaceae whole genomes for the presence of the mcr gene, using CLC Genomics Workbench (CLC Bio-Qiagen, Aarhus, Denmark). The isolates comprised clinical and screening pure cultures submitted to the national reference laboratory for mandatory CRE surveillance. Locally, the presence of mcr-1 as well as its cocarriage with KPC-2 had been previously well described (4, 5); hence, we did not look further into the distribution of mcr-1. mcr-2 was not detected among the genomes analyzed. mcr-3 was identified in one Escherichia coli genome (ENT1955) by the use of both read mapping and de novo assembly. To date, several mcr-3 variants have been observed (6, 7). The mcr-3 gene identified in this study shared 99.94% nucleotide identity with the first mcr-3 gene discovered by Yin et al. (8) (GenBank accession no. KY924928.1) due to a “C” deletion at nucleotide position 218; the deletion was confirmed by Sanger sequencing. This resulted in a truncated protein of only 88 amino acids (Table 1) that was deemed to be nonfunctional (10). This mcr-3-like gene has been deposited in GenBank (see below). The genome of ENT1955 was de novo assembled using all the reads, and the draft genome was annotated. No plasmid-related genes were found in the approximately 9-kb contig containing the mcr-3-like gene. We also performed read mapping using E. coli Y5 (GenBank accession no. CP013483.1) as the reference because it was the most closely related genome available in GenBank. Reads that did not match the E. coli Y5 chromosome were separately assembled. Using this approach, the derived mcr-3-like contig was almost identical to the one obtained from de novo assembly using all reads and, again, no plasmid-related genes were observed. This led us to believe that, in contrast to those reported in several previous studies (8, 11, 12), our mcr-3-like gene was not plasmid associated. Interestingly, downstream of the mcr-3-like gene, a gene encoding IS2 transposase TnpB, matching the gene encoding the transposase carried by mcr-3-bearing plasmid pWJ1 (8), was detected. Phenotypically, the isolate was susceptible to both colistin and polymyxin B (Table 1) as determined by the reference broth microdilution method (13).

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TABLE 1

Characteristics of KPC-2 carbapenemase-producing Enterobacteriaceae cocarrying mcr variants

There were no exact matches to mcr-4 in the screened genomes. Instead, the mcr-4-like gene was found in six Enterobacter cloacae isolates (Table 1), which had 100% nucleotide identity to a putative sulfatase from Shewanella frigidimarina. Compared to mcr-4 (GenBank accession no. MF543359), this mcr-4-like gene carried 2 missense mutations resulting in codon changes at positions V179G and V236F. The mcr-4-like gene was located on a 7.7-kb contig, and a BLAST search showed 95% coverage and 99% similarity to the mcr-4 gene in plasmid pMCR_R3445 (GenBank accession no. MF543359). Thus, we named this mcr-4-like gene “mcr-4.2” (see below).

Interestingly, the genomes of all of the mcr-4.2-positive isolates also possessed another putative phosphoethanolamine transferase (EPT) (GenBank accession no. WP_012477388). This putative EPT was not encoded on the plasmid. An alignment of the MCR-4.2 amino acid sequence and the putative EPT against MCR-1 and MCR-2 showed conservation in active site residues (data not shown) (10). However, it appears that the presence of mcr-4.2 and the predicted novel EPT genes did not confer overt phenotypic resistance to polymyxins (Table 1). The finding of heterogeneously elevated drug MICs for E. cloacae isolates is likely attributable to heteroresistance causing a “skip well” phenomenon observed during the broth microdilution susceptibility testing (Table 1) (9).

Expression vector pET-48b(+) (Novagen, WI, USA) was used for functional cloning of the full coding sequences of the mcr-3-like and mcr-4.2 genes but without the fusion tags. There was essentially no difference in the polymyxin MICs for the cloned mcr-3-like and mcr-4.2 genes, which suggests that they did not confer phenotypic resistance (Table 2). Therefore, we concluded that the mcr-3-like gene carrying a nonsense mutation was nonfunctional and that mcr-4.2 alone was unlikely to be the major mechanism of resistance to polymyxins.

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TABLE 2

Broth microdilution polymyxin susceptibility testing of cloned mcr-3-like and mcr-4.2 expressed in E. coli BL21(DE3) host

In summary, we describe the discovery of new mcr-like elements, although phenotypic susceptibility testing indicates that the presence of these genes alone was unlikely to contribute to colistin resistance.

Accession number(s).The mcr-3-like and mcr-4.2 genes identified in this study have been deposited in GenBank under accession no. MG026622 and MG026621, respectively.

ACKNOWLEDGMENTS

We thank the National Public Health Laboratory (NPHL), Singapore, for providing carbapenemase-producing Enterobacteriaceae isolates and, in particular, Felicia Ong for performing the carbapenemase screening assays and Siti Zulaina for preparing plate cultures. We thank Bernadette Cheng for performing the expression cloning. We also thank the Carbapenemase-producing Enterobacteriaceae in Singapore (CaPES) study group. CaPES study group members are Asok Kurup, Benjamin Cherng, Choong Weng Lam, Deepak Rama Narayana, De Partha Pratim, Hsu Li Yang, Indumathi Venkatachalam, Jeanette Teo, Kalisvar Marimuthu, Koh Tse Hsien, Micehlle Ang, Nancy Tee, Nares Smitasin, Ng Oon Tek, Ooi Say Tat, Prabha Unny Krishnan, Raymond Fong, Raymond Lin Tzer Pin, Surinder Kaur Pada, Tan Thean Yen, and Thoon Koh Cheng.

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REFERENCES

  1. 1.↵
    1. Giani T,
    2. Antonelli A,
    3. Caltagirone M,
    4. Mauri C,
    5. Nicchi J,
    6. Arena F,
    7. Nucleo E,
    8. Bracco S,
    9. Pantosti A
    ; AMCLI-CoSA survey participants, Luzzaro F, Pagani L, Rossolini GM. 2017. Evolving beta-lactamase epidemiology in Enterobacteriaceae from Italian nationwide surveillance, October 2013: KPC-carbapenemase spreading among outpatients. Euro Surveill 22:30583. doi:10.2807/1560-7917.ES.2017.22.31.30583.
    OpenUrlCrossRef
  2. 2.↵
    1. Huang TD,
    2. Bogaerts P,
    3. Berhin C,
    4. Hoebeke M,
    5. Bauraing C,
    6. Glupczynski Y
    . 2017. Increasing proportion of carbapenemase-producing Enterobacteriaceae and emergence of a MCR-1 producer through a multicentric study among hospital-based and private laboratories in Belgium from September to November. Euro Surveill 22:30530. doi:10.2807/1560-7917.ES.2017.22.19.30530.
    OpenUrlCrossRef
  3. 3.↵
    1. Kluytmans J
    . 2017. Plasmid-encoded colistin resistance: mcr-one, two, three and counting. Euro Surveill 22:30588. doi:10.2807/1560-7917.ES.2017.22.31.30588.
    OpenUrlCrossRef
  4. 4.↵
    1. Teo JQM,
    2. Ong RTH,
    3. Xia E,
    4. Koh TH,
    5. Khor CC,
    6. Lee SJY,
    7. Lim TP,
    8. Kwa AL
    . 2016. mcr-1 in multidrug-resistant blaKPC-2-producing clinical Enterobacteriaceae isolates in Singapore. Antimicrob Agents Chemother 60:6435–6437. doi:10.1128/AAC.00804-16.
    OpenUrlFREE Full Text
  5. 5.↵
    1. Teo JW,
    2. Chew KL,
    3. Lin RT
    . 2016. Transmissible colistin resistance encoded by mcr-1 detected in clinical Enterobacteriaceae isolates in Singapore. Emerg Microbes Infect 5:e87. doi:10.1038/emi.2016.85.
    OpenUrlCrossRef
  6. 6.↵
    1. Roer L,
    2. Hansen F,
    3. Stegger M,
    4. Sönksen UW,
    5. Hasman H,
    6. Hammerum AM
    . 2017. Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014. Euro Surveill 22:22846. doi:10.2807/1560-7917.ES.2017.22.31.30584.
    OpenUrlCrossRef
  7. 7.↵
    1. Ling Z,
    2. Yin W,
    3. Li H,
    4. Zhang Q,
    5. Wang X,
    6. Wang Z,
    7. Ke Y,
    8. Wang Y,
    9. Shen J
    . 24 October 2017. Chromosome-mediated mcr-3 variants in Aeromonas veronii from chicken meat. Antimicrob Agents Chemother doi:10.1128/AAC.01272-17.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Yin W,
    2. Li H,
    3. Shen Y,
    4. Liu Z,
    5. Wang S,
    6. Shen Z,
    7. Zhang R,
    8. Walsh TR,
    9. Shen J,
    10. Wang Y
    . 2017. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli. mBio 8:e00543-17. doi:10.1128/mBio.00543-17.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Landman D,
    2. Salamera J,
    3. Quale J
    . 2013. Irreproducible and uninterpretable polymyxin B MICs for Enterobacter cloacae and Enterobacter aerogenes. J Clin Microbiol 51:4106–4111. doi:10.1128/JCM.02129-13.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Hu M,
    2. Guo J,
    3. Cheng Q,
    4. Yang Z,
    5. Chan EWC,
    6. Chen S,
    7. Hao Q
    . 2016. Crystal structure of Escherichia coli originated MCR-1, a phosphoethanolamine transferase for colistin resistance. Sci Rep 6:38793. doi:10.1038/srep38793.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Hernández M,
    2. Iglesias MR,
    3. Rodríguez-Lázaro D,
    4. Gallardo A,
    5. Quijada N,
    6. Miguela-Villoldo P,
    7. Campos MJ,
    8. Píriz S,
    9. López-Orozco G,
    10. de Frutos C,
    11. Sáez JL,
    12. Ugarte-Ruiz M,
    13. Domínguez L,
    14. Quesada A
    . 2017. Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015. Euro Surveill 22:30586. doi:10.2807/1560-7917.ES.2017.22.31.30586.
    OpenUrlCrossRef
  12. 12.↵
    1. Litrup E,
    2. Kiil K,
    3. Hammerum AM,
    4. Roer L,
    5. Nielsen EM,
    6. Torpdahl M
    . 2017. Plasmid-borne colistin resistance gene mcr-3 in Salmonella isolates from human infections, Denmark, 2009–17. Euro Surveill 22:30587. doi:10.2807/1560-7917.ES.2017.22.31.30587.
    OpenUrlCrossRef
  13. 13.↵
    Clinical and Laboratory Standards Institute. 2015. M07-A10. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: ninth edition. Clinical and Laboratory Standards Institute, Wayne, PA.
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mcr-3 and mcr-4 Variants in Carbapenemase-Producing Clinical Enterobacteriaceae Do Not Confer Phenotypic Polymyxin Resistance
Jeanette W. P. Teo, Marimuthu Kalisvar, Indumathi Venkatachalam, Oon Tek Ng, Raymond T. P. Lin, Sophie Octavia
Journal of Clinical Microbiology Feb 2018, 56 (3) e01562-17; DOI: 10.1128/JCM.01562-17

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mcr-3 and mcr-4 Variants in Carbapenemase-Producing Clinical Enterobacteriaceae Do Not Confer Phenotypic Polymyxin Resistance
Jeanette W. P. Teo, Marimuthu Kalisvar, Indumathi Venkatachalam, Oon Tek Ng, Raymond T. P. Lin, Sophie Octavia
Journal of Clinical Microbiology Feb 2018, 56 (3) e01562-17; DOI: 10.1128/JCM.01562-17
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KEYWORDS

bioinformatics
Enterobacteriaceae
next-generation sequencing
PCR
antibiotic resistance
plasmid
polymyxin

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