Transmissible colistin resistance encoded by mcr-1 detected in clinical Enterobacteriaceae isolates in Singapore.

Dear Editor,

Following the recent description of transmissible plasmid-mediated colistin resistance encoded by mcr-1 in clinical and veterinary Escherichia coli and Klebsiella pneumoniae isolates in China,[1] several groups have reported mcr-1 in colistin-resistant isolates from humans and food animals across Asia[2, 3, 4] to Europe.[5, 6, 7, 8] It is evident that mcr-1 has disseminated globally.

We performed a prospective study in January 2016 using 306 consecutive clinical Enterobacteriaceae isolates collected from blood, urine and miscellaneous samples (swabs and pus). The species investigated were E. coli (n=166), K. pneumoniae (n=87), Klebsiella oxytoca (n=4), Enterobacter spp. (n=22), Proteus spp. (n=10), Citrobacter spp. (n=9), Morganella morganii (n=5), Providencia rettgeri (n=1), Salmonella enteritidis (n=1) and Serratia marcescens (n=1). Isolates were PCR-screened for mcr-1[1] without prior knowledge of their antibiograms or colistin-resistance status. Three of these isolates (two E. coli and one K. pneumoniae) were mcr-1 positive, with their full-length mcr-1 gene matching the nucleotide identity of the first-described isolate exactly.[1] Multilocus sequence typing (MLST) was performed (http://bigsdb.web.pasteur.fr/index.html). There was no evidence of nosocomial transmission, as the two E. coli isolates were of different sequence types (STs; Table 1).

Table 1

Characteristics of mcr-1-positive clinical Enterobacteriaceae isolates in Singapore

Isolate	Specimen	Species	Date of isolation	MLST	β-lactamases	ISApl1 associated	mcr-1 transmissible via conjugationa	MIC (mg/L)b
								PB	COL	IMP	MEM	ETP	FOX	CAZ	AMP	PTZ	CIP	LEV	GEM	AMK
NM12	Urine	E. coli	11/01/2016	ST460C	TEM-1	Yes	Yes, ≈10−6	4	4	≤0.25	≤0.25	≤0.5	≤4	≤1	≥32	64	2	4	<1	<2
NM4	Urine	E. coli	14/01/2016	ST156	TEM-1	No	Yes, ≈10−7	4	4	≤0.25	≤0.25	≤0.5	≥64	16	≥32	≤4	1	2	>16	<2
NM2	Urine	K. pneumoniae	13/01/2016	ST1535	TEM-1, CTX-M-15	No	Yes, ≈10−3	24	8	≤0.25	≤0.25	≤0.5	≤4	16	≥32	≤4	1	2	<1	<2
Transconjugant NM12	—	E. coli	—	—	TEM-1	—	—	2	2	≤0.25	≤0.25	≤0.5	≤4	≤1	≥32	128	2	4	≤1	≤2
Transconjugant NM4	—	E. coli	—	—	TEM-1	—	—	4	6	≤0.25	≤0.25	≤0.5	≤64	16	≥32	≤4	1	2	≥16	≤2
Transconjugant NM2	—	E. coli	—	—	Not detected	—	—	8	6	≤0.25	≤0.25	≤0.5	≤4	≤1	8	≤4	≤0.25	≤0.12	≤1	≤2
J53	—	E. coli	—	—	—	—	—	0.5	0.25	≤0.25	≤0.25	≤0.5	≤4	≤1	8	≤4	≤0.25	≤0.12	≤1	≤2

Abbreviations: amikacin, AMK; ampicillin, AMP; ceftazidime, CAZ; ciprofloxacin, CIP; colistin, COL; cefotaximase, CTX; ertapenem, ETP; cefoxitin, FOX; gentamicin, GEM; imipenem, IMP; levofloxacin, LEV; minimum inhibitory concentration, MIC; meropenem, MEM; polymyxin B, PB; piperacillin-tazobactam, PTZ.

The conjugation efficiency is calculated as the number of transconjugants per donor cell.

The interpretation of results of susceptibility testing were based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. For colistin, a susceptible breakpoint of ≤2 mg/L and a resistant breakpoint of>2 mg/L was applied. Because no interpretative criteria are available for polymyxin B, colistin breakpoints were applied.

The Institut Pasteur MLST scheme was utilized. The following new allelic profile was obtained: dinB 22; pabB 21; putB 23; trpB 127; icdA 61; polB 109; trpA 41; uidA 164, and was assigned as ST460.

The isolates were urinary specimens (Table 1). Our detection rate was estimated to be 0.98% (95% confidence interval of 0.3%–2.8%, Wilson score interval). This was very close to 1% mcr-1 prevalence in China.[1] Sensitivity testing was performed via E-test for polymyxin B and colistin and with the Vitek2 GNR257 card for all other antimicrobials. Phenotypically, the isolates were resistant to polymyxin B (minimum inhibitory concentration (MIC) 4 mg/L–24 mg/L) and colistin (MIC 4 mg/L–8 mg/L) but were sensitive to carbapenems. There was a variable sensitivity to third-generation cephalosporins and piperacillin-tazobactam (Table 1). PCR screening for carbapenemases (K. pneumoniae carbapenemase (KPC), metallo-β-lactamases (New Delhi Metallo-β-lactamase (NDM), verona integron-encoded metallo-β-lactamase, imipenemase), class D carbapenemases (oxacillinase (OXA)-23, OXA-48-like)) and broad- and extended-spectrum β-lactamases (BSBL and ESBLs) was performed.[9] Only narrow-spectrum β-lactamase (TEM)-1 BSBL was detected in the E. coli isolates (Table 1). This was noteworthy because mcr-1-positive isolates have been found to be associated with cefotaximase (CTX)-M-like ESBLs.[4, 5, 6] Furthermore, in our isolates, carbapenemase genes were not carried with mcr-1, which contrasts growing reports in which mcr-1 has been found together with blaKPC-2 and blaNDM carbapenemase genes[7, 8] and results in colistin-resistant isolates that are also carbapenem resistant.[7, 8] Using liquid-mating assays, mcr-1 was transferable to an E. coli recipient, J53, in all the clinical donor isolates. Transconjugants were selected on Luria Bertani agar containing 50 mg/L of sodium azide and 0.5 mg/L of colistin. All of the transconjugants were phenotypically resistant to colistin and polymyxin B. TEM-1 also transferred in two transconjugants (Table 1). PCR replicon typing[10] indicated that the transconjugants of NM2 and NM12 could not be typed, while transconjugant NM4 carried IncF and IncI1. The genetic environment of mcr-1 is variable and is not always associated with an upstream ISApl1.[7] Here PCR mapping and sequencing based on the initial genetic environment[1] showed that one isolate did not have a flanking ISApI1 (Table 1). This suggests that the dissemination of mcr-1 is likely facilitated by a diverse range of mobile genetic elements. We plan to commence full-genome sequencing in the near future to characterize the plasmids and mobile elements in detail. Because Singapore has limited farming and agricultural activity, mcr-1 is less likely to be acquired through contact with local livestock; although not yet conclusively proven, it appears that imported meat products and vegetables are more likely sources of mcr-1.[11, 12, 13]