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mcr-1 colistin resistance in ESBL-producing Klebsiella pneumoniae, France.

Resistance to colistin in gram-negative bacteria stems mainly from structural modifications of bacterial lipopolysaccharide. These modifications include addition of 4-amino-4-deoxy-L-arabinose or phosphoethanolamine caused by chromosomal mutations in genes encoding the 2-component systems PhoPQ and PmrAB, or mutations in the mgrB gene, a negative regulator of PhoPQ (1).

The recent discovery of a horizontally transferable plasmid-mediated mcr-1 gene encoding a phosphoethanolamine transferase is a cause for concern, but few mcr-1 -positive clinical strains of Klebsiella pneumoniae have been reported so far in Europe (2). Colocalization of carbapenemases or extended-spectrum p-lactamase (ESBL) genes and the mcr-1 gene on the same plasmids is of concern because it might lead to pandrug resistance (1,3). We report mcr-1 colistin resistance in ESBL-producing K. pneumoniae isolated from a patient in France.

The patient was a 38-year-old man who had chronic granulomatous disease that was diagnosed when he was 8 months old. Since then, he has had several minor and major diseases and conditions, including primitive femoral osteitis, hepatic abscesses, disseminated candidiasis, and bacteremia, which required several treatments with antimicrobial drugs. However, the patient was never given colistin.

In April 2016, he was hospitalized for surgical removal of a thyroid abscess. Fungal cultures of the abscess grew Aspergillus fumigatus. Despite antifungal treatment with amphotericin B and flucytosine, fungal meningitis, cerebral arterial vasospasm at the Willis polygon, and hydrocephalus developed. The patient also received immunosuppressive therapy (methylprednisolone and anakinra) and empiric antimicrobial drug therapy, including cotrimoxazole, clindamycin, meropenem, and vancomycin successively.

In August 2016, systematic culture of a rectal swab specimen showed an ESBL-producing strain of K. pneumoniae, 2 previous rectal screenings showed negative results. The strain was resistant to colistin (MIC >4 mg/L). Resistance was determined by using a broth microdilution assay (BD Phoenix Instrument; Becton Dickinson, Franklin Lakes, NJ, USA) (online Technical Appendix, https:// An MIC of 24 mg/L was found for polymyxin B by using the E-test method (Polymyxin B E-test strip; bioMerieux, Marcy L'Etoile, France).

The strain was sent to the French National Reference Center for Antibiotic Resistance in Enterobacteriaceae (Hopital Gabriel Montpied, Clermont-Ferrand, France), which confirmed phenotypic resistance to colistin and identified the mcr-1 gene by using PCR and previously described primers (2). Whole-genome sequencing showed that the K pneumoniae strain had genotype ST15 and confirmed the presence of the mcr-1 gene on a 33,303-kb transferable plasmid of incompatibility group IncX4 (online Technical Appendix). This plasmid differed by only 4 mutations from mcr1.2-encoding plasmid pMCR-1.2.IT (GenBank accession no. KX236309) previously characterized in Italy (4). Conjugation of the plasmid into Escherichia coli K12 conferred colistin resistance (MIC increased from 0.25 mg/L to 4 mg/L) to the E. coli strain.

Other resistance genes were also identified (Table), including the ESBL-encoding gene [bla.sub.SHV106] (online Technical Appendix). None of them were localized with the mcr-1 gene on the IncX4 plasmid. Moreover, insertion of mobile element IS5 in the mgrB gene was detected, which is also associated with colistin resistance (5). No mutations were found in the prmA, prmB, phoP, and phoQ genes.

There is currently no commercial medium to screen gram-negative bacteria harboring the mcr-1 gene. Nordmann et al. (6) described an in-house SuperPolymyxin medium composed of eosin methylene blue agar, 3.5 mg/L of colistin sulfate, 10 mg/L of daptomycin, and 5 mg/L amphotericin B, which showed excellent sensitivity and specificity. Colistin resistance can be confirmed within 2 h by using an in-house rapid polymyxin Nordmann-Poirel test (7). The mcr-1 gene can be rapidly detected by real-time PCR of DNA extracts obtained from bacterial strains or directly from stool samples (2,8,9).

We obtained subcultures of the strain from the patient on Columbia CNA agar containing 10 mg/L of colistin and 15 mg/L of nalidixic acid and 5% sheep blood ([CNA.sup.+]; bio-Merieux) but not on Thayer-Martin agar medium containing unknown concentrations of vancomycin, colistin, amphotericin B, and trimethoprim (VCA3; bioMerieux). Lack of growth on this medium might be related to a high colistin concentration or the presence of vancomycin, which can potentiate colistin activity (6). Further investigations using [CNA.sup.+] medium did not identify intestinal carriage of ES-BL-negative but mcr-1-positive enterobacteria in the index case-patient. On the basis of these results, rectal screening of 39 contacts was performed by using an ESBL-screening medium (BLSE agar [MacConkey agar and Drigalski agar]; bioMerieux). All of the tests showed negative results.

The origin of the mcr-1 strain remains unknown. Nosocomial acquisition cannot be ruled out because colistin-resistant strains harboring the mcr-1 gene might have been isolated in the hospital but not identified because this resistance mechanism was initially reported in February 2016. Food might also be incriminated (1); one study identified a 21% mcr-1 prevalence among ESBL-producing E. coli in calves in France (10).

Multiple antimicrobial drug therapy for this patient might have selected for this multidrug-resistant bacteria. The presence of a plasmid containing the mcr-1 and ESBL or other resistance genes in the same strain might be involved in selection of colistin-resistant strains during administration of any ineffective antimicrobial drug (3). Development of efficient tools for rapid detection of mcr-1-harboring strains should be a priority to prevent dissemination of these strains in hospital settings.

Dr. Caspar is a clinical microbiologist in the Bacteriology Laboratory, Grenoble-Alpes University Hospital, and a probationary lecturer in the Medicine Faculty, Grenoble-Alpes University, Grenoble, France. His primary research interests are bacterial resistance or treatment failures for tularemia and evaluation of novel antimicrobial compounds.



(1.) Baron S, Hadjadj L, Rolain J-M, Olaitan AO. Molecular mechanisms of polymyxin resistance: knowns and unknowns. Int J Antimicrob Agents. 2016;48:583-91.

(2.) Liu Y-Y, Wang Y, Walsh TR, 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;16:161-8.

(3.) Schwarz S, Johnson AP. Transferable resistance to colistin: a new but old threat. J Antimicrob Chemother. 2016;71:2066-70.

(4.) Di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici De Angelis L, Fortunato S, et al. mcr-1.2, a new mcrvariant carried on a transferable plasmid from a colistin-resistant KPC carbapenemase-producing Klebsiella pneumoniae strain of sequence type 512. Antimicrob Agents Chemother. 2016;60:5612-5.

(5.) Cannatelli A, D'Andrea MM, Giani T, Di Pilato V, Arena F, Ambretti S, et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother. 2013;57:5521-6.

(6.) Nordmann P, Jayol A, Poirel L. A universal culture medium for screening polymyxin-resistant gram-negative isolates. J Clin Microbiol. 2016;54:1395-9.

(7.) Nordmann P, Jayol A, Poirel L. Rapid detection of polymyxin resistance in Enterobacteriaceae. Emerg Infect Dis. 2016;22:1038-43.

(8.) Nijhuis RH, Veldman KT, Schelfaut J, Van Essen-Zandbergen A, Wessels E, Claas EC, et al. Detection of the plasmid-mediated colistin-resistance gene mcr-1 in clinical isolates and stool specimens obtained from hospitalized patients using a newly developed real-time PCR assay. J Antimicrob Chemother.2016;71:2344-6.

(9.) Bontron S, Poirel L, Nordmann P. Real-time PCR for detection of plasmid-mediated polymyxin resistance (mcr-1) from cultured bacteria and stools. J Antimicrob Chemother. 2016;71:2318-20.

(10.) Haenni M, Metayer V, Gay E, Madec J-Y. Increasing trends in mcr-1 prevalence among ESBL-producing E. coli in French calves despite decreasing exposure to colistin. Antimicrob Agents Chemother.2016;60:6433-4.

Yvan Caspar, Mylene Maillet, Patricia Pavese, Gilles Francony, Jean-Paul Brion, Marie-Reine Mallaret, Richard Bonnet, Frederic Robin, Racha Beyrouthy, Max Maurin

Author affiliations: Centre Hospitalier Universitaire Grenoble-Alpes, Grenoble, France (Y. Caspar, M. Maillet, P. Pavese, G. Francony, J.-P. Brion, M.-R. Mallaret, M. Maurin); University Grenoble Alpes, CNRS, Grenoble; Centre Hospitalier Universitaire Clermont-Ferrand, Clermont-Ferrand, France (R. Bonnet, F. Robin, R. Beyrouthy); Centre National de Reference de la Resistance aux Antibiotiques, Clermont-Ferrand (R. Bonnet, F. Robin, R. Beyrouthy); Universite Clermont Auvergne, Clermont-Ferrand (R. Bonnet, F. Robin, R. Beyrouthy); Institut National de la Sante et de la Recherche Medicale, Clermont-Ferrand (R. Bonnet, F. Robin, R. Beyrouthy); Institut National de la Recherche Agronomique, Clermont-Ferrand (R. Bonnet, F. Robin, R. Beyrouthy)

Address for correspondence: Yvan Caspar, Laboratoire de Bacteriologie, Institut de Biologie et Pathologie, Centre Hospitalier Universitaire Grenoble Alpes, CS10217, 38043 Grenoble CEDEX 9, France; email:
Table. Resistance genes identified by whole-genome sequencing of an
ESBL-producing mcr-1-positive Klebsiella pneumoniae strain isolated
from a 38-year-old man, France *

Resistance gene                Target antimicrobial drug
mcr-1 and inactivation of              Colistin
  mgrB by IS5 insertion
  [bla.sub.SHV-106]                 [beta]-lactams
aac(3)-IId and aadA16-like          Aminoglycoside
aac(6')Ib-cr                 Quinolone and aminoglycoside
fosA5                                 Fosfomycin
sulI and folP                         Sulfonamide
dfrA27                               Trimethoprim
tetD                                 Tetracycline

* ESBL, extended-spectrum [beta]-lactamase.
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Article Details
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Author:Caspar, Yvan; Maillet, Mylene; Pavese, Patricia; Francony, Gilles; Brion, Jean-Paul; Mallaret, Marie
Publication:Emerging Infectious Diseases
Article Type:Letter to the editor
Geographic Code:4EUFR
Date:May 1, 2017
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