Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015

The plasmid-mediated colistin resistance gene, mcr-1 , was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2*, an incX4 replicon was found to be linked to


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Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015 The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2*, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.
Very recently, in November 2015, Liu et al. reported the finding of a transferable plasmid-mediated colistin resistance gene, mcr-1, detected in Escherichia coli isolates from animals, food and patients in China. Moreover, they found mcr-1 in Klebsiella pneumoniae isolates from patients [1]. Horizontal gene transfer represents a paradigm shift in colistin resistance, which until then only was found to be mediated by chromosomal mutations and thus spread by vertical transmission.

National surveillance of antimicrobial resistance in food animals, food and humans in Denmark using whole genome sequencing
Since 2012, the national surveillance of antimicrobial resistance in food animals, food and humans in Denmark (www.DANMAP.org) has used whole-genome sequence (WGS) analysis for detection of resistance genes and multilocus sequence typing (MLST) using the open-access bioinformatic web-tools ResFinder and MLST, respectively from www.genomicepidemiology.org for characterisation of extended spectrum beta-lactamase (ESBL)-and AmpC-producing E. coli isolates [2][3][4]. The mcr-1 sequence from China was added on 24 November 2015 to the ResFinder database as soon as it was available from The National Center for Biotechnology Information (NCBI).

Investigation of presence of mcr-1 in E. coli isolates from food animals, food and human bloodstream infections
The updated version of ResFinder was used to analyse the WGS data from ESBL-and AmpC-producing E. coli isolates from food animals and food for the years 2012 to 2014, as well as ESBL-and AmpC-producing E. coli isolates from human bloodstream infections, and carbapenemase-producing organisms (CPOs) from humans, from January 2014 to beginning of November 2015 (Table 1), for the presence of mcr-1. Furthermore, fluoroquinolone resistance determinants were investigated by searching manually for mutations in the GyrA, ParC and ParE. [5].
The mcr-1 gene was detected in one E. coli isolate from a human bloodstream infection from 2015 and in five E. coli isolates obtained from chicken meat of European origin imported to Denmark from 2012, 2013 and 2014 ( Table 2). None of the CPOs were positive for mcr-1 ( Table 1).
The patient infected with the mcr-1-positive E. coli was an elderly man with prostate cancer and repeated urinary tract infections with ESBL-producing E. coli resulting in four positive urine samples over five month prior to the bloodstream infection, all resistant to third generation cephalosporins, gentamicin, sulfamethoxazole, trimethoprim and ciprofloxacin (these isolates were not investigated further). He had been treated empirically with piperacillin/tazobactam and subsequently meropenem after susceptibility testing of the bloodstream isolate, but not with colistin according to the available patient data.
Besides mcr-1, the human isolate from the Danish patient contained 15 different resistance genes including bla CTX-M-55 and bla CMY-2 conferring resistance to extended-spectrum beta-lactam antibiotics as well as two GyrA mutations (S83L, D87N) and a ParC mutation (E62K) leading to high-level fluoroquinolone resistance ( Table 2). The human mcr-1 positive E. coli isolate belonged to ST744, a rare sequence type in both humans and animals in Denmark. The patient had not been travelling abroad recently and the origin of the isolate is unknown.
The bla CMY-2 gene was detected in three of the five mcr-1-positive chicken meat isolates. In addition, three of the chicken meat E. coli isolates carried bla SHV-12 conferring resistance to extended-spectrum beta-lactam antibiotics excluding cephamycins.
One of the mcr-1 positive E. coli isolates from chicken meat belonged to ST131. The other chicken meat isolates belonged to sequence types not frequently found in Denmark ( Table 2). The human MCR-1-producing E. coli isolate was only susceptible to piperacillin/tazobactam, carbapenems and tigecycline according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [6], whereas the chicken meat isolates were less resistant (Table 3).
WGS analysis using the web-tool PlasmidFinder [9] identified an IncI2 replicon present in the human isolate as well as in three of the chicken meat isolates, but this replicon was not detected in the remaining two chicken meat isolates. De novo assembly using CLCbio Genomics Workbench (v8.5.1; Qiagen, Aarhus,   Denmark) of the genomic data produced a direct link between the mcr-1 gene and an IncX4 replicon in one of the two isolates not containing IncI2 replicons. An identical IncX4 replicon was detected in four of the chicken meat isolates including both isolates lacking an IncI2 replicon (but not in the human isolate).

Discussion and conclusion
Here we describe a MCR-1 producing E. coli isolate from a human infection coproducing both an ESBL (CTX-M-55) and an AmpC (CMY-2) cephalosporinase as well as five MCR-1 producing E. coli from chicken meat coproducing either and ESBL (SHV-12) or an AmpC (CMY-2) cephalosporinase, or both. Human and animal CTX-M-55-producing isolates are commonly reported from Asia [10,11], but are relatively rarely seen in Denmark.

Table 3
Antimicrobial susceptibility profiles of the five MCR-1-producing E. coli isolates from chicken meat and the MCR-1producing E. coli isolate from human bloodstream infection, Denmark November 2015 SHV-12-producing [4]. Based on antibiogram data it seems plausible that the bloodstream infection is related to the repeated urinary tract infections, but this will need to be confirmed by additional WGS analysis. At this point in time, the origin of the human isolate is unresolved.
MLST analysis did not show any close clonal relationship between any of the six isolates. However, one of the chicken meat isolates belonged to ST131. This sequence type is commonly associated with human E. coli urinary tract and blood infection isolates worldwide, but are rare in animal E. coli isolates [4,14,15]. The fact that a ST131 MCR-1-producing E. coli isolate was found is of special concern, since ST131 isolates have spread epidemically during the last decade [14,15] and the ability of mcr-1 to be acquired by this sequence type has been demonstrated here.
The mcr-1 gene was initially reported to be located on an IncI2 plasmid without other known resistance markers [1]. Here only four of the isolates were found to contain an IncI2 replicon, suggesting that the mcr-1 gene was either located on the chromosome or on a plasmid type belonging to another group. In support of the latter is the fact that de novo assembly of the genomic data from one of the isolates produced a continuous DNA fragment containing both an IncX4 and the mcr-1 gene, strongly suggesting that the mcr-1 gene is not restricted to the IncI2 plasmid group, but additional studies are needed to clarify this further.
In conclusion, this study is to our knowledge, the first proof of colistin-resistant mcr-1 positive E. coli outside China. The human isolate was only susceptible to very few antimicrobial classes such as carbapenems.
Should an isolate like this acquire carbapenem resistance, it would leave very few, if any, suitable treatment options. Finally, our findings underline the importance of continuous microbiological surveillance programs and not the least the benefit of employing comprehensive WGS-based surveillance of antimicrobial resistance, as it allows for rapid re-analysis of large datasets in silico and thus make early detection and risk assessment possible when new resistance genes emerge.

*Authors' correction
Upon request of the authors, Christina Aaby Svendsen's name was corrected in the Acknowledgement section on 14 December 2015. In addition, the sentence "In addition to IncN2, an incX4 replicon was found to be linked to mcr-1." was corrected to read "In addition to IncI2, an incX4 replicon was found to be linked to mcr-1." on 16 December 2015 upon request of the authors.