The first human report of mobile colistin resistance gene, mcr‐1, in Finland

Colistin resistance mediated by mobile mcr‐1 gene has raised concern during the last years. After steep increase in mcr‐1 reports, other mcr‐gene variants (mcr‐2 to mcr‐5) have been revealed as well. In 2016, a clinical study was conducted on asymptomatic stool carriage of extended spectrum beta‐lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae among Finnish adults. All suspected ESBL producing bacterial isolates were first tested by phenotypic ESBL‐confirmation methods, and then further analyzed with whole genome sequencing to identify the resistance genes. We found one study subject carrying a colistin resistant E. coli with a transferrable mcr‐1 gene. This multi‐drug resistant isolate, although initially suspected to be an ESBL producer, did not carry any ESBL genes, but was proven to carry several other resistance genes by using whole genome sequencing. Sequence type was ST93. The mcr‐1 gene was connected to IncX4 plasmid which suggests that the colistin resistance gene locates in the respective plasmid. Here, we report the finding of a mcr‐1 harboring human E. coli isolate from Finland. Clinical antimicrobial resistance (AMR) rates are low in Finland, and mobile colistin resistance has not been reported previously. This highlights the importance of AMR surveillance also in populations with low levels of resistance.

The global spread of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBL) and carbapenemases (CPE) has limited the options of suitable antibiotics for the treatment of infections caused by these gram-negative bacteria. A new chapter in the era of emerging antimicrobial resistance started in 2015 when Liu et al. reported the first finding of transferable plasmid-encoded colistin resistance in Enterobacteriaceae isolated from humans and animals (1). Colistin is considered as the last resort of treatment against multi-drug resistant bacteria. Since the first report from China, several countries from five continents, have detected the mcr-1 gene in Enterobacteriaceae isolated from humans, animals or the environment (2,3). Sporadic findings of mcr-1 carrying isolates have previously been reported from three other Nordic countries, namely Sweden, Norway and Denmark (4)(5)(6). After the first wave of mcr-1 reports, several countries including Denmark from the Nordic countries, have identified isolates carrying new gene variants of mcr gene (currently mcr-1 to mcr-5) (7,8). In Finland, the overall antimicrobial resistance level has remained relatively low, and use of colistin is reserved for only very rare clinical indications. Now we report the first Escherichia coli isolate from Finland with colistin resistance mediated by mcr-1 gene.

MATERIALS AND METHODS
During 2016, we conducted a prospective, clinical study to investigate asymptomatic fecal ESBL carriage in Received 28 November 2017. Accepted 8 February 2018 Southwest Finland as part of the Northern Dimension Antibiotic Resistance Study (NoDARS, http://www.nd phs.org/?database,view,project,1468). Fecal samples were collected from 176 healthy, anonymous volunteers, and the samples were screened for ESBL producing E. coli and K. pneumoniae from ESBL specific chromogenic media (unpublished data). Antimicrobial susceptibility testing (AST) was performed for all isolates according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints (version 6.0, http:// www.eucast.org/clinical_breakpoints/). Minimal inhibitory concentration for colistin was preliminarily determined with a gradient strip test (Liofilchem, Roseto degli Abruzzi, Italy) and confirmed by broth microdilution (Thermo Scientific TM Sensititre TM System, Thermo Scientific, East Grinsted, England). Strains with reduced susceptibility to third generation cephalosporins were tested for ESBL production with the combination disk test according to EUCAST recommendations.
To analyze the molecular mechanisms of ESBL, all phenotypically confirmed E. coli and K. pneumoniae ESBL isolates were analyzed using paired-ended (2 9 150 bp) whole-genome sequencing (WGS) with Illumina MiSeq platform. ResFinder (database version 2.1) (9) and in silico PCR, using previously reported primers (10), were used to identify the resistance genes and plasmid replicons from the WGS data. The reads of the E. coli isolate carrying mcr-1 gene have been deposited in NCBI SRA database under the accession numbers SRR6656065.

RESULTS
During our clinical screening study (Ny et al. unpublished data), we decided to extend our analysis by whole genome sequencing all phenotypically confirmed ESBL isolates (E. coli and K. pneumoniae). The analysis revealed that one of the E. coli isolates carried the mcr-1 gene. The WGS analysis also revealed that the only acquired b-lactam resistance genes found from this isolate were bla TEM-1b and bla CMY-2 .
Mcr-1 gene and the IncX4 replicon were connected in the de Bruijn graph of the assembly which was visualized with Bandage (11). This strongly suggests that mcr-1 is located in an IncX4 plasmid.
The close proximity region of mcr-1 gene is illustrated in Fig. 1.
The strain belonged to sequence type (ST) 93. Colistin resistance was confirmed both by the gradient strip test and microdilution methods. All detected plasmid replicons, antimicrobial resistance genes and the AST panel of this isolate are listed in Table 1.
The mcr-1 carrying E. coli was isolated from a fecal sample of a 26 year old healthy male. The exclusion criteria for the study included antibiotic treatment 3 months prior to sampling. Previous usage of antibiotics was not recorded. A background questionnaire revealed that he had travelled to Central Europe and South-America within 6 months before the sampling.

DISCUSSION
During the last years there has been a vast expansion of reports on findings of colistin resistant organisms in humans without prior colistin therapy or clonal transmission (12). Many of these arise from retrospective surveys that have screened past clinical or environmental sample collections for the mcr-1 gene. Our finding was isolated from a healthy volunteer in 2016. Stool carriage of mcr-1 gene has been reported from healthy individuals in Europe at least from the Netherlands, Switzerland and Sweden (4,13,14). Other reports are from China, Laos and Thailand (12). Of the Nordic countries, Norway and Denmark have both also reported of finding of Enterobactericeae isolates carrying mcr-1 from clinical sample collections (5,6). A recent publication from Norway, a Nordic country with very low usage of colistin, detected two ESBL producing E. coli isolates carrying mcr-1 gene from environmental samples collected in 2010 (15).
Although the phenotypic characterization of the isolate reported here suggested it to be an ESBL and AmpC producer, it did not carry any classical ESBL genes based on WGS analysis. However, the isolate can be considered as multi-drug resistant based on criteria by Magiorakos et al. (16). Instead of ESBL genes, the isolate carried a bla CMY-2 gene which encodes for a plasmid-mediated AmpC b-lactamase. This specific bla CMY-2 gene has especially been reported from animal isolates, mainly from poultry (17). ST-93 is found both from human and animal sources (https://enterobase.warwick.ac.uk/). The plasmid composition and location of the mcr-1 gene in IncX4 resembles an isolate that has been reported from a pig in Austria (18). Recent studies from Sweden (19) and Norway (20) have identified close genetic relatedness of E. coli isolates carrying the bla CMY-2 gene both from human samples and retail poultry meat, which suggests a possible common source. The source of the Finnish mcr-1 E. coli isolate, however, remains unknown.
shown an increase during the last few years. National Institute for Health and Welfare has retrospectively screened all CPE isolates (n = 145) found from Finland between 2008-2015 for the mcr-1 gene. No such isolates have been found (unpublished data). Our current mcr-1 finding is not from a clinical case but from a healthy volunteer. AST for colistin is routinely often limited to the most resistant strains, i.e. those with reduced susceptibility to carbapenems. In the lack of an easy screening test, the presence of transferrable colistin resistance may remain undetected in strains fully susceptible to carbapenems. We detected the presence of mcr-1 only in WGS, which may prove a reliable means to detect transferrable colistin resistance. Our report clearly highlights the presence of transferrable colistin resistance among human non-ESBL producing E.coli isolates also in countries with proven history of low rates of antimicrobial resistance or colistin use.

ETHICS
The study was conducted in accordance with the Declaration of Helsinki and national and institutional standards. Written informed consent was obtained from all study participants. Study design was approved by Ethics Committee, Hospital District of Southwest Finland (ETMK 157/2015).
We thank Mari Virta for excellent technical assistance.

FUNDING
This work was supported by Ministry of Social Affairs and Health; Hospital District of Southwest Finland and partly by European Union: Regional East Action Programme 2013 Part 2, Northern Dimension funds (Grant Contract 2014/344-660 (NoDARS)).

TRANSPARENCY DECLARATIONS
None to declare.