Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014

A novel variant of the plasmid-borne colistin resistance gene mcr-3 was detected on an IncHI2 plasmid in an ST131 CTX-M-55-producing Escherichia coli isolate from a Danish patient with bloodstream infection in 2014. The discovery of novel plasmid-borne genes conferring resistance to colistin is of special interest since colistin has reemerged as an important drug in the treatment of infections with multidrug-resistant Gram-negative bacteria.

A novel variant of the plasmid-borne colistin resistance gene mcr-3 was detected on an IncHI2 plasmid in an ST131 CTX-M-55-producing Escherichia coli isolate from a Danish patient with bloodstream infection in 2014.The discovery of novel plasmid-borne genes conferring resistance to colistin is of special interest since colistin has reemerged as an important drug in the treatment of infections with multidrug-resistant Gram-negative bacteria.
Very recently, in June 2017, Yin et al. reported a new transferable plasmid-borne colistin resistance gene, mcr-3, detected on an IncHI2-type plasmid in an Escherichia coli isolate from pig faeces in China [1].The mcr-3 gene showed 45% and 47% nucleotide sequence similarity to mcr-1 and mcr-2, respectively [1].Yin et al. also compared the mcr-3 sequence to data from GenBank and found 100% nucleotide similarity to mcr-3 sequences from a porcine E. coli in Malaysia, a human Klebsiella pneumoniae isolate in Thailand and a human Salmonella Typhimurium in the United States.Furthermore, 99.94% nucleotide similarity was seen in two human K. pneumoniae isolates from Thailand [1].
Here we report an mcr-3 variant from an extendedspectrum beta-lactamase-producing (ESBL) E. coli isolated from a bloodstream infection in 2014 in Denmark.

Escherichia coli isolates from human bloodstream infections and clinical carbapenemase-producing organisms
Since 2014, ESBL/AmpC-producing E. coli isolates from bloodstream infections and all clinical carbapenemaseproducing organisms (CPOs) from patients in Denmark have on a voluntary basis been referred to at Statens Serum Institut for whole genome sequencing (WGS) as part of the national surveillance programme DANMAP (www.DANMAP.org).
The 872 ESBL/AmpC-producing E. coli isolates from human bloodstream infections collected in the years 2014 to 2016, as well as the 317 human CPOs collected from January 2014 until May 2017 were investigated in silico for the presence of mcr-3 using MyDbFinder (https://cge.cbs.dtu.dk/services/MyDbFinder/). None of the CPOs were positive for mcr-3.
An mcr-3-variant was detected in one ST131 E. coli isolate (isolate id SNTR36B6, short read archive (SRA) ID ERR1971735).The isolate was obtained from a male patient admitted to hospital under the clinical diagnosis of pyelonephritis.An ESBL-producing E. coli with the same resistance patterns as SNTR36B6 was isolated from catheter-urine (not included in the study).The patient had no former history of hospitalisation and was without known somatic comorbidity.Upon admission, he informed about travel to Thailand two months earlier, where he had stayed locally.Antibiotic treatment with intravenously administered cefuroxime, ciprofloxacin and gentamicin was started at admission.On day 3, the patient had fully recovered, the treatment was changed to monotherapy with perioral ciprofloxacin and the patient was discharged.
In the Sensititre broth microdilution method, the isolate, SNTR36B6, was only susceptible to piperacillin/ tazobactam, meropenem, and tigecycline and intermediate resistant to ciprofloxacin according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [2] (Table 1).
The ST131 ESBL-producing E. coli isolate, SNTR36B6, had 99.94% nucleotide similarity to the first mcr-3 gene reported by Yin et al.This mcr-3 variant differed by one amino acid (T488I) from MCR-3 (Table 2).The two Klebsiella pneumoniae with 99.94% nucleotide identity to mcr-3 reported by Yin et al. also differed by one amino acid, but at different positions compared with the mcr-3 (D295E and G373V) in SNTR36B6 [1].
Thus, the mcr-3 we report here is a novel mcr-3 variant (Table 2).
We also investigated our isolates for the presence of mcr-1 and mcr-2.None of the CPOs were positive for either gene.One ESBL-producing E. coli strain carried the mcr-1 gene and has been described earlier [3].No other mcr-1-positive ESBL-producing E. coli were detected and none of the isolates were positive for mcr-2.

Discussion
This study is to our knowledge, the first report of mcr-3 in E. coli outside Asia.The fact that an ST131 MCR-3producing and CTX-M-55 producing E. coli isolate was found is of particular concern, since ST131 E. coli isolates have spread epidemically during the last decade

SNTR36B6 scaffolds
The mcr-3-positive E. coli SNTR36B6 from human bloodstream infection was compared with the mcr-3 plasmid pWJ1 using the GView Server.
The concentric rings display similarity between the pWJ1 reference plasmid in the inner ring and the SNTR36B3 scaffolds in the outer ring.The red and blue arrows indicate resistance genes of pWJ1, according to the orientation on the DNA strand.and the isolate only was susceptible to very few antimicrobial classes such as carbapenems [7,8].
The sul3 gene was originally detected in a porcine E. coli isolate from Switzerland, where 33% of the sulfonamide-resistant porcine E. coli isolates carried sul3 [13].An investigation of sulfonamide-resistant E. coli in Danish pigs, pork and patients from 2002 to 2003 only detected sul3 in isolates from pigs and pork, but not in human isolates [14].Between 2014 and 2016, however, the sul3 gene was detected in 1.5% of the ESBL/ AmpC-producing E. coli isolates from Danish patients, and was also observed in the SNTR36B6 strain in the present study.
The mcr-3 gene was initially reported to be located on an IncHI2-type plasmid named pWJ1.An IncHI2 replicon was also detected in SNTR36B6, and our BLAST analysis suggested that the mcr-3 variant could be located on a plasmid with a similar backbone belonging to this type, but this will have to be confirmed by further plasmid analysis.However, the lack in SNTR36B6 of several resistance markers which are present on pWJ1 suggests that the plasmid from SNTR36B6 is not completely identical to pWJ1.
The ST131 E. coli isolate carrying the mcr-3 gene variant in this study, had the fimH22 allele.Only two isolates with this allele were found among the 122 invasive ST131 ESBL/AmpC-producing E. coli isolates in a study from 2017 by Roer et al. [5].The origin of the ST131 MCR-3-producing and CTX-M-55-producing E. coli isolate is unknown, but might be related to travel to Thailand and, based on the presence of the sul3 resistance gene, it might be of porcine origin.
In conclusion, with the re-emergence of colistin as an important drug in the treatment of infections with multidrug-resistant Gram-negative bacteria [15], the discovery of a plasmid-borne gene conferring resistance to colistin in an E. coli of human origin is of special concern.Our findings underline the usefulness of WGS-based surveillance of antimicrobial resistance for detection of new resistance genes by re-analysis of large datasets in silico.

Table 2
mcr-3 and mcr-3 variants and their deduced MCR-3 and MCR-like proteins in relation to a patient isolate, Denmark, September 2014 The ID number for SNTR36B6 refers to the short read archive.The other ID numbers refer to GenBank. a