Various Sequence Types of Escherichia coli Isolates Coharboring bla_NDM-5 and mcr-1 Genes from a Commercial Swine Farm in China

ABSTRACT

Sixteen different sequence types (STs) of Escherichia coli isolates from a commercial swine farm in China were confirmed to coharbor the carbapenem resistance gene bla_NDM-5 and the colistin resistance gene mcr-1. Whole-genome sequencing revealed that bla_NDM-5 and mcr-1 were located on a 46-kb IncX3 plasmid and a 32-kb IncX4 plasmid, respectively. The two plasmids can transfer together with a low fitness cost, which might explain the presence of various STs of E. coli coharboring bla_NDM-5 and mcr-1.

TEXT

Carbapenemase-producing Enterobacteriaceae has become a major public health threat around the world (1). The recently identified carbapenemase New Delhi metallo-β-lactamase confers resistance to all β-lactam antimicrobials except monobactam (2). The NDM-5-encoding gene bla_NDM-5 was first identified in an Escherichia coli strain recovered from a patient in the United Kingdom in 2011 (3). Since then, bla_NDM-5 was identified in many countries, such as Algeria (4–6), the United States (7), Australia (8), China (9–12), Denmark (13), Japan (14), India (15), and the United Kingdom (3). The widespread occurrence of NDM-5 in recent years should arouse our attention. Colistin is a critically important medication for humans in the treatment of carbapenemase-producing Enterobacteriaceae, and it has been widely used in veterinary medicine in China (16, 17). The first plasmid-mediated colistin resistance gene, mcr-1, was reported in E. coli in 2015 (18). In a short period, colistin-resistant E. coli carrying the mcr-1 gene were reported worldwide (19, 20). Recently, mcr-1 was reported to coexist with bla_NDM (21–23) and bla_CTX-M (24), which brought great challenges for the treatment of bacterial infection. In the present study, we are the first to report the presence of isolates of various sequence types of E. coli coharboring bla_NDM-5 and mcr-1 genes from a commercial pig farm in China.

A total of 105 anal swabs samples from swine were collected from a commercial pig farm on 1 October 2015 in Sichuan province. E. coli strains were selected by eosin-methylene blue agar, and only 1 isolate was picked up from each sample. All 105 isolates were identified by BD Phoenix 100 diagnostic systems (Sparks, MD). Sixty-four strains were nonsusceptible to imipenem and polymyxin B, identified by the agar dilution method according to Clinical and Laboratory Standards Institute guidelines (25). Isolates were divided into 16 different clones by pulsed-field gel electrophoresis after XbaI digestion according to the standard PulseNet conditions (26) (Fig. 1). Phylogenetic group (A, B1, B2, and D) typing showed that all 16 different clones belonged to group A (n = 13) and B1 (n = 3) (27). Multilocus sequence typing (MLST) was performed as previously described (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). All 16 clones belonged to different sequence types. The antimicrobial resistance profile, phylogenetic group, and sequence type of each different clone are shown in Figure 1.

FIG 1.

PFGE profiles, antimicrobial resistance phenotypes, phylogenetic groups, and sequence types (STs) of 16 different E. coli strains. ¹Failure to find any corresponding ST type with MLST database via BLAST. Seven housekeeping gene allele types, i.e., adk, fumC, gyrB, icd, mdh, purA, recA, were identified as follows: 10, 11, 4, 8, 274, 8, 42. IPM, imipenem; MEM, meropenem; AMC amoxicillin-clavulanate; FOX, cefoxitin; CTX, cefotaxime; ATM, aztreonam; CIP, ciprofloxacin; DO, doxycycline; FFC, florfenicol; FOS, fosfomycin; GEN, gentamicin; SXT, trimethoprim-sulfamethoxazole; PB, polymyxin B.

The carbapenemase-encoding genes bla_GES, bla_KPC, bla_IMP, bla_NDM, bla_OXA-48, and bla_VIM and the colistin resistance gene mcr-1 were screened in all 16 different clones by PCR as described previously (18, 28, 29). The results showed that they all carried bla_NDM-5 and mcr-1. Very recently, mcr-1 and bla_NDM-5 coharbored by E. coli ST156 and ST648 were found in a Chinese hospital (23). To the best of our knowledge, this is the first report of the presence of diverse E. coli strains coharboring bla_NDM-5 and mcr-1 on a commercial swine farm.

Whole-genome sequencing for 16 different clones was performed on the Illumina MiSeq (Majorbio, Shanghai, China) using a 400-bp paired-end TruSeq library with a 2 × 300 run. The paired-end reads were assembled de novo using the SOAP v2.04 and GapCloser v1.12. The gaps between different contigs were closed by PCR and sequencing. The genetic environments of bla_NDM-5 and mcr-1 were analyzed by using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence analysis revealed that bla_NDM-5 and mcr-1 were located on two different plasmids, which showed 100% nucleotide identity in all 16 strains. The plasmid carrying bla_NDM-5 belonged to the IncX3 group and was designated pECNDM101. It had a length of 46,165 bp with 46.66% G+C content, which had a 9-bp nucleotide substitution, 1-bp insertion, and 3-bp deletion compared with pNDM_MGR194 (GenBank accession number KF220657). An IS5 was inserted with ISAba125 upstream of bla_NDM-5, which was also found in isolates in India (15), Japan (14), and China (9, 10).The plasmid carrying mcr-1 belonged to the IncX4 group and was designated pECMCR-1101. It had a length of 32,751 bp with 41.96% G+C content, which had a 559-bp deletion between 1,170 bp and 1,171 bp, an 8-bp nucleotide substitution, and a 6-bp insertion compared with pICBEC72Hmcr (GenBank accession number CP015977), which was recovered from a clinical E. coli strain in Brazil.

Conjugation experiments were carried out among the 16 different clones with rifampin-resistant E. coli EC600 as the recipient. Transconjugants were selected on three different Mueller-Hinton agar (Oxoid) plates that contained 400 μg/ml rifampin with 10 μg/ml imipenem or 2 μg/ml polymyxin B. Positive transconjugants were identified by detection of antimicrobial resistance profiles and screening for the presence of bla_NDM-5 and mcr-1. Conjugation frequencies were calculated as the number of transconjugants per recipient cell and are shown in Table 1. When imipenem was used as the selection pressure, transfer frequencies of bla_NDM-5 varied from 7.7 × 10⁻² to 5.7 × 10⁻⁴. When polymyxin B was used as the selection pressure, transfer frequencies of mcr-1 varied from 5.8 × 10⁻⁵ to 5.3 × 10⁻⁷, and similar transfer frequencies of 2.9 × 10⁻⁷ to 2.5 × 10⁻⁵ were detected when imipenem and polymyxin B were used as the selection pressure. It was very interesting that the cotransfer of bla_NDM-5 and mcr-1 was detected in a few transconjugants when using polymyxin B or imipenem alone. Three different transconjugants were obtained, MYNDM-5 carrying pECNDM101, MYmcr-1 carrying pECMCR-1101, and MYNDM-5+mcr-1 carrying pECNDM101 and pECMCR-1101.

TABLE 1.

Transconjugative frequencies of pECNDM101 and pECmcr-1101 plasmids in 16 different E. coli strains

E. coli strain (transconjugant)	Transconjugative frequenciesa
	pECNDM101b	pECmcr-1101c	pECNDM101 and pECmcr-1101d
ECMY1	1.1 × 10−3	8.7 × 10−6	4.0 × 10−6
ECMY2	1.1 × 10−2	9.2 × 10−7	5.5 × 10−7
ECMY3	7.7 × 10−2	4.6 × 10−6	2.8 × 10−6
ECMY4a	5.3 × 10−2	1.6 × 10−5	5.7 × 10−6
ECMY4b	1.3 × 10−2	9.4 × 10−7	4.3 × 10−7
ECMY5	1.7 × 10−2	5.3 × 10−6	1.2 × 10−6
ECMY6	8.0 × 10−3	6.3 × 10−6	8.9 × 10−7
ECMY9	5.7 × 10−4	5.8 × 10−5	2.5 × 10−5
ECMY10	1.1 × 10−3	7.1 × 10−7	4.3 × 10−7
ECMY11	5.3 × 10−3	5.6 × 10−5	2.3 × 10−5
ECMY12	1.8 × 10−3	3.0 × 10−5	1.7 × 10−5
ECMY13	5.6 × 10−3	5.3 × 10−7	2.9 × 10−7
ECMY14	1.4 × 10−3	3.0 × 10−6	1.3 × 10−6
ECMY16	9.1 × 10−4	6.6 × 10−6	3.9 × 10−6
ECMY17	2.2 × 10−3	1.7 × 10−5	1.2 × 10−5
ECMY18	1.8 × 10−3	5.0 × 10−5	1.6 × 10−5

^aThe results shown are presented as the average value of 3 parallel experiments.

^bImipenem was used as selection pressure in conjugation experiments.

^cPolymyxin B was used as selection pressure in conjugation experiments.

^dImipenem and polymyxin B were used as selection pressure in conjugation experiments.

The fitness costs of pECNDM101 and pECMCR-1101 were determined by growth curves and competition experiments as previously described (30, 31). Growth curves of transconjugant MYNDM-5 and recipient E. coli EC600 were similar, and they arrived at the same concentration at the stationary phase (optical density at 600 nm [OD₆₀₀] = 1.166). However, transconjugants MYmcr-1 and MYNDM-5+mcr-1 had a slight growth disadvantage and a lower concentration at the stationary phase (OD₆₀₀ = 1.132 and 1.130, respectively) (Fig. 2A). For the competition experiment, a constant increase in the proportion of transconjugant MYNDM-5 was observed from day 3 on. In contrast, constant decreases in the proportion of transconjugants MYmcr-1 and MYNDM-5+mcr-1 were observed from day 2 on and day 1 on, respectively (Fig. 2B). Transconjugants MYmcr-1 and MYNDM-5+mcr-1 presented competitive disadvantages of ∼11.45% and ∼9.67% per 10 generations relative to those of E. coli EC600. Interestingly, transconjugant MYNDM-5 showed a competitive advantage (∼3% per 10 generations) (Fig. 2C), which might contribute to the propagation of bla_NDM-5.

FIG 2.

Fitness costs of pECNDM101 and pECmcr-1101 in E. coli EC600. (A) Comparison of the growth kinetics of recipient E. coli EC600 and three transconjugants (MYNDM-5, MYmcr-1, and MYNDM-5+mcr-1) without any antibiotics. Growth curves represent the average results of three independent experiments. (B) Growth competition between recipient E. coli 600 and three transconjugants (MYNDM-5, MYmcr-1, and MYNDM-5+mcr-1). The initial ratio was 1:1. Competition experiments were repeated three times. (C) The selection coefficient (S) used to estimate the difference between the relative fitness of two competitors over the entire competition experiment, and S was calculated as the slope of the linear regression model In(CI)/In(d), where CI is the CFU ratio of the resistant and susceptible strains and d is the dilution factor.

In conclusion, to our knowledge, our study is the first to report that a bla_NDM-5-carrying IncX3 plasmid (pECNDM101) and an mcr-1-carrying IncX4 plasmid (pECmcr-1101) coexist in various sequence types of E. coli on a commercial pig farm, and they can transfer together at a low fitness cost. Note that the cotransfer of bla_NDM-5 and mcr-1 by an IncX3-X4 hybrid plasmid was detected in a clinical E. coli isolate in China very recently (32). The results highlight that a swine farm is an important reservoir of E. coli carrying bla_NDM-5 and mcr-1, which presents a serious challenge for public health via food-chain transmission.

Accession number(s).

The complete nucleotide sequences of the bla_NDM-5-carrying IncX3 plasmid (pECNDM101) and the mcr-1-carrying IncX4 plasmid (pECmcr-1101) characterized in this study were submitted to GenBank and assigned accession numbers KX507346 and KX570748, respectively.