Coproduction of MCR-1 and NDM-1 by Colistin-Resistant Escherichia coli Isolated from a Healthy Individual

LETTER

The first transferable plasmid-mediated colistin resistance gene, mcr-1 was reported in Escherichia coli isolates from food animals, food, and patients in China and now has been reported worldwide (1). Furthermore, cocarriage of mcr-1 and bla_NDM has been reported in E. coli and other members of the family Enterobacteriaceae from a chicken meat sample; patients with peritonitis, urinary tract infections, and rectal cancer; and a Muscovy duck, all in China. Most recently, E. coli carrying mcr-1 and bla_NDM-5 was isolated from a patient with a urinary tract infection in the United States (2–7). Here, we report asymptomatic carriage of E. coli harboring both mcr-1 and bla_NDM-1 in an otherwise healthy individual.

A total of 151 nonduplicate, serial fecal specimens were collected from 98 inpatients and 53 healthy individuals at Guangdong General Hospital in Guangzhou, China, during the first week of January 2016 for the purpose of detecting extended-spectrum β-lactamase-producing Enterobacteriaceae. Each sample was screened on a Columbia blood agar plate without any antibiotics and then subcultured on MacConkey agar with 2 μg/ml cefotaxime. Colonies selected from the MacConkey agar were identified to the species level by the API 20E system (bioMérieux, Marcy l'Etoile, France) and 16S rRNA gene sequencing (8). As a result, 73 nonduplicate E. coli isolates were collected from 58 inpatients and 15 healthy individuals. Of these isolates, 17 were found to harbor mcr-1 by PCR assay and sequencing performed as previously described (1, 8). Of the 17 mcr-1-carrying E. coli isolates, 2 were from clinical cultures of inpatients, 12 were from rectal surveillance cultures of inpatients, and 3 (E. coli GB049, GB090, and GB135) were from healthy individuals who provided rectal cultures with stool specimens after consent during outpatient visits for their annual physical examinations. Since carriage of mcr-1 by healthy individuals is of particular epidemiologic interest, we analyzed these three strains further.

E. coli GB049 was recovered from a 23-year-old male, E. coli GB090 was from a 68-year-old female, and E. coli GB135 was from a 56-year-old female. These healthy individuals were all nonvegetarian, living in the city >10 km from commercial animal farms, drinking the municipal water, had received a secondary or tertiary education, had a mid to high socioeconomic status, and had traveled overseas. The individual with E. coli GB049 traveled in India for 5 days in September 2015; and the other two individuals with E. coli GB090 and GB135 traveled to the United States for 9 and 15 days, respectively, in December 2015. In addition, the individual with E. coli GB090 had taken oral amoxicillin for several days for her respiratory symptoms 3 months earlier.

Antimicrobial susceptibility testing was performed by the agar dilution method and interpreted according to the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2016) for colistin, polymyxin B, and tigecycline and those of the Clinical and Laboratory Standards Institute (CLSI, 2015) for other antimicrobials. All three isolates were resistant to colistin and polymyxin B but susceptible to tigecycline and amikacin. In addition, E. coli GB090 was also resistant to cephalosporins, ertapenem, fosfomycin, and ciprofloxacin.

PCR was performed to identify β-lactamase, plasmid-mediated quinolone resistance, and 16S rRNA methyltransferase genes as described previously (9–11). Sanger sequencing of both strands of all PCR products was performed. As a result, in addition to mcr-1, E. coli GB090 was positive for bla_NDM-1, bla_TEM-1, bla_SHV-12, bla_CTX-M-15, bla_CTX-M-14, fosA3, qnrB, qnrS, and aac(6′)-Ib-cr.

All three mcr-1-positive E. coli isolates from healthy individuals were subjected to pulsed-field gel electrophoresis (PFGE) as previously described (12, 13). The three isolates were clonally unrelated by PFGE (data not shown). E. coli GB090 was then subjected to phylogenetic typing and multilocus sequence typing (MLST) according to the protocol at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli (9) and classified into phylogenetic group B2 and sequence type 744 (ST744), which belongs to the ST10 complex. E. coli B2 ST744 was also reported in mcr-1-carrying E. coli from a human bloodstream infection and imported chicken meat in Denmark (14). Phylogenetic group B2 ST10 complex strains, along with phylogenetic group B2 ST131 and ST405 strains, have been implicated as vehicles driving the international spread of bla_CTX-M-15 (15).

Rifampin-resistant E. coli EC600 was used as the recipient for the conjugation and transformation experiments. The transconjugants or transformations were selected on lysogeny broth (LB) agar plates containing rifampin (500 μg/ml) plus colistin (4 μg/ml), cefotaxime (16 μg/ml), ertapenem (2 and 4 μg/ml), or fosfomycin (256 μg/ml). As a result, colistin-resistant transconjugants containing mcr-1, bla_CTX-M-15, and bla_TEM-1 were successfully obtained from plates containing either colistin or cefotaxime for E. coli GB090, which was positive for an IncFI replicon by PCR-based replicon typing (16) (Table 1). We could not obtain transconjugants or transformants containing bla_NDM-1. In order to determine the locations of the mcr-1 and bla_NDM genes, DNA linearization of E. coli GB090 with S1 nuclease, followed by PFGE and Southern hybridization using mcr-1 and bla_NDM gene probes, was conducted as previously described (17).The results indicated that mcr-1 was located on an ∼33-kDa plasmid together with the β-lactamase genes bla_CTX-M-15 and bla_TEM-1, whereas bla_NDM was not located on this plasmid (see Fig. S1 in the supplemental material). Taking the results together, we speculate that bla_NDM-1 may be located on the chromosome of E. coli GB090. In addition, colistin-resistant transconjugants containing mcr-1 were successfully obtained for E. coli GB049 but not E. coli GB135.

TABLE 1.

Characterization of three mcr-1-positive E. coli isolates from healthy individuals and their transconjugants

Parameter	E. coli GB049	Transconjugant of E. coli GB049	E. coli GB135	E. coli GB090	Transconjugant of E. coli GB090	E. coli EC600
Isolation date	1 January 2016		4 January 2016	2 January 2016
Source	Healthy individual		Healthy individual	Healthy individual
Isolation site	Feces		Feces	Feces
MLST result	ST1193		ST38	ST744
Phylogenetic group	D		D	B2
Resistance gene(s)a	mcr-1, blaTEM-1	mcr-1, blaTEM-1	mcr-1, blaTEM-1	mcr-1, blaNDM-1, blaTEM-1, blaSHV-12, blaCTX-M-15 blaCTX-M-14, fosA3, qnrB, qnrS, aac(6′)-Ib-cr	mcr-1, blaCTX-M-15, blaTEM-1
Plasmid replicon type(s)	IncI2	IncI2	IncFIB, IncN	IncFI, IncN, IncP, IncK	IncFI
MIC (μg/ml) of:
Colistin	8	16	16	16	16	0.25
Polymyxin B	16	16	16	16	16	0.5
Tigecycline	0.5	0.25	0.5	1	0.5	0.25
Ampicillin	>256	>256	>256	>256	>256	8
Amoxicillin-clavulanic acid	16	4	16	128	8	2
Cefotaxime	≤1	≤1	≤1	128	256	≤1
Ceftazidime	≤1	≤1	≤1	>256	64	≤1
Cefepime	≤0.5	≤0.5	≤0.5	16	32	≤0.5
Gentamicin	2	≤1	≤1	128	4	≤1
Amikacin	4	2	2	8	4	2
Ertapenem	0.125	≤0.063	0.125	4	0.125	≤0.063
Imipenem	0.125	≤0.063	≤0.063	1	0.25	0.125
Meropenem	≤0.063	≤0.063	≤0.063	1	0.125	≤0.063
Fosfomycin	16	≤8	≤8	>512	16	≤8
Nitrofurantoin	≤8	≤8	16	32	16	≤8
Ciprofloxacin	0.125	0.064	0.064	16	0.064	≤0.032

^aDetermined by PCR and sequencing.

The genomic DNA of E. coli GB090 was extracted and digested with EcoRI and XbaI and then ligated to cloning vector pBC-SK(–). The ligation products were transferred to E. coli DH10B by electroporation and cultured on LB agar supplemented with chloramphenicol (30 μg/ml) and colistin (4 μg/ml) or ertapenem (2 and 4 μg/ml), which yielded colistin- and ertapenem-resistant transformants, respectively. Sequencing of the recombinant plasmids carried by these E. coli transformants revealed ∼6.1- and ∼8.1-kb inserts containing the mcr-1 and bla_NDM-1 genes, respectively.

Sequence analysis of the ∼6.1-kb genetic environment surrounding mcr-1 revealed 100% identity with that of pHNSHP45 in our previous study (1), which was also similar to the sequence found in various mcr-1-carrying plasmids, with up to 99% identity with pA31-12 from E. coli strain A31-12 (accession no. KX034083), pAF23 from E. coli strain Af23 (KX032519), pSCS23 from Salmonella enterica strain SC23 (KU934209), and pABC149-MCR-1 from E. coli strain ABC149 (KX013538). The genetic environment surrounding bla_NDM-1 was identical to that found in various bla_NDM-1-carrying plasmids in Enterobacteriaceae in China, including pKP04NDM (KU314941), pNDM-HN380 (JX104760), pKPN5047 (KC311431), and pNDM-SX04 (KC876051) in K. pneumoniae; pYE315203 (JX254913) and p112298-NDM (KP987216) in Citrobacter freundii; pNDM-HF727 in Enterobacter cloacae (KF976405); and a plasmid in Raoultella planticola (KF877335). These results indicate that the genetic structures of both mcr-1 and bla_NDM-1 have likely already spread to different species of Enterobacteriaceae.

Recovery of mcr-1- and bla_NDM-1-carrying E. coli from a healthy individual suggests that Enterobacteriaceae with extreme antimicrobial resistance may already circulate in the community in China. Our findings underline the importance of continuous microbiological and molecular surveillance with regard to further dissemination of mcr-1 in the community. The high rate of mcr-1-carrying E. coli isolation from both hospitalized and nonhospitalized patients also suggests intestinal colonization as a key source in its dissemination.

Accession number(s).

The nucleotide sequences obtained in this study were submitted to the GenBank database and assigned accession no. KX886345 and KX886346.