IncX3 Epidemic Plasmid Carrying bla_NDM-5 in Escherichia coli from Swine in Multiple Geographic Areas in China

ABSTRACT

Six imported pigs originating from Guangdong, Henan, and Hunan provinces in China during October 2015 to February 2017 were cultured and found to be positive for meropenem-resistant Escherichia coli. The samples yielded 9 E. coli isolates of diverse sequence types carrying bla_NDM-5 on IncX3 (8 isolates from 5 farms) or IncFII (1 isolate from 1 farm) plasmids. The mcr-1 gene was coharbored by 4 isolates. The IncX3 plasmids (∼46 kb) carrying bla_NDM-5 were identical or nearly identical to each other.

TEXT

The presence of carbapenemase-producing Enterobacteriaceae (CPE) in livestock animals is of concern because this may facilitate expansion of the gene pool from which pathogenic bacteria can pick up resistance genes, and consumers may subsequently be exposed through the food chain (1, 2). For this reason, there is a need to enhance the monitoring of carbapenem resistance in the food supply (1). In Hong Kong, 80% of the food animals are imported from mainland China and involve farm suppliers from multiple provinces in the country (3).

From September 2008 to February 2017, rectal swabs were obtained from randomly selected fresh pig carcasses at a centralized slaughterhouse in Hong Kong by trained veterinary staff, as part of an ongoing surveillance (3). Each swab was collected from a single animal and was inoculated into nutrient broth with 10 mg/liter vancomycin and 0.5 mg/liter meropenem (4), followed by subculture on a MacConkey agar plate supplemented with 2 mg/liter meropenem. Five to ten colonies from each selective plate were picked. Matrix-assisted laser desorption ionization–time of flight mass spectrometry was used for bacterial identification. The agar dilution (for colistin) and disc diffusion (for other antibiotics) methods were used to determine antimicrobial susceptibility (5, 6). Isolates from the same animal were considered to be unique if the resistance profiles for meropenem and colistin were different.

In total, 856 pigs were cultured over 263 sampling dates (see Table S1 in the supplemental material). Six pigs originating from six different farms were cultured and found to be positive for meropenem-resistant Escherichia coli (Table 1). According to the susceptibility patterns, a total of nine isolates were considered to be unique and were investigated further. All isolates had positive CarbaNP test results and were resistant to ertapenem, imipenem, and meropenem. The presence of carbapenemase genes and mcr-1 was investigated by PCR and sequencing (7–9). The bla_NDM-5 gene was identified in all nine isolates, of which four were resistant to colistin and coharbored mcr-1. The isolates were further investigated by multilocus sequence typing (MLST) and replicon typing (7, 9, 10). Plasmids carrying bla_NDM-5 were X3 (n = 8, ∼45 kb) or F36 (n = 1, ∼100 kb). Of the four mcr-1 genes identified, three were harbored on plasmids of different replicon types (X4, FIB, and Y). In conjugation experiments, no cotransfer of carbapenem and colistin coresistance was seen, but the plasmids carrying bla_NDM or mcr-1 could be transferred separately at frequencies of 10⁻⁴ to 10⁻⁵ and 10⁻¹ to 10⁻⁶ transconjugants per donor cell, respectively (9, 10).

TABLE 1.

Sources and characteristics for nine NDM-positive E. coli isolates

Strain	Specimen	Sourceb	Date collected	MLST	blaNDM-5	mcr-1	Replicon type of plasmid harboring:		Resistance patternd
							blaNDM	mcr-1
P744A	Pig 1	Henan (A1)	October 2015	ST10	+	−	X3	None	Chl, Nit
P744Ta	Pig 1	Henan (A1)	October 2015	ST1602	+	+	X3	X4	Chl, Cip, Nit
P748a	Pig 2	Hunan (B)	January 2016	ST167	+	−	F36	None	Gen, Chl, Cip, Nit
P768	Pig 3	Henan (A2)	May 2016	ST117	+	−	X3	None	Gen, Chl, Cip
P768-11a	Pig 3	Henan (A2)	May 2016	ST871	+	+	X3	FIB	Cip
P785a	Pig 4	Guangdong (C1)	June 2016	ST7512	+	+	X3	Chromosomalc	Gen, Chl, Cip, Nit
P788A	Pig 5	Guangdong (C2)	June 2016	ST1286	+	−	X3	None	Nit
P788A-32a	Pig 5	Guangdong (C2)	June 2016	ST7510	+	+	X3	Y	Gen, Chl, Nit
P855a	Pig 6	Guangdong (C3)	February 2017	ST7511	+	−	X3	None	Gen, Chl, Cip, Nit

^aThe six isolates were investigated further by genome sequencing.

^bProvince (farm) origin of the pig.

^cChromosomal location of mcr-1 in the isolate was confirmed by genome sequencing.

^dResistance patterns for amikacin (Ak), chloramphenicol (Chl), ciprofloxacin (Cip), fosfomycin (Fos), gentamicin (Gen), and nitrofurantoin (Nit).

Six isolates (one from each animal) were sequenced by an Illumina MiSeq platform at >150-fold coverage (Table 1). The plasmids were assembled de novo using a CLC Genomics Workbench (Qiagen, Redwood City, CA), and gaps were closed by additional PCR and Sanger sequencing (7, 9, 10). ISfinder (https://www-is.biotoul.fr/about.php) was used to identify and annotate insertion sequences. In strain P748, bla_NDM-5 was found in a contig (∼32 kb) with 100% coverage and 98% identity to p28078-NDM (GenBank accession no. MF156713).

Complete sequences of the five ∼46-kb IncX3 plasmids were obtained (see Table S2 in the supplemental material). They were found to have plasmid scaffolds typical of IncX3 plasmids (Fig. 1a). The genetic load regions in the five plasmids were compared with two reference IncX3 plasmids (pNDM-HN380 and pIncX-SHV) (Fig. 1b). In the bla_NDM-carrying plasmids, an ISL3 with 8-bp flanking direct repeats (ATATGCAT) was found downstream of the resolvase gene. The umuD gene was split into two fragments (umuDΔ1 and umuDΔ2) at the same position as in pIncX-SHV, resulting in a pair of 3-bp direct repeats (TGT). In pNDM-HN380, bla_NDM was inserted as an IS26-ISAba125 transposon-like structure (Fig. 1b). Subsequently, the upstream ISAba125 was disrupted by IS5 (10). In four plasmids with links to the Guangdong and Henan provinces, the sequences inserted between the two umuD fragments were 100% identical (10,117 bp in length). This inserted sequence differed from that in pNDM-HN380 by a deletion of 7,874 bp (Fig. 1b). The remaining plasmid with a link to Henan had an additional deletion (616 bp) at the junction between the IS5 and ISAba125Δ1 remnants. In the five NDM-5 plasmids, IS5 was inserted at the same position, leading to the flanking 4-bp direct repeats (CTAA). In pNDM-HN380, IS5 was inserted at a different position in the opposite orientation.

FIG 1.

Comparisons of IncX plasmids in this study. (a) Circular map of plasmid pP768-NDM-5. This plasmid was used to illustrate the backbone shared by all the analyzed plasmids and the location of the genetic load region. (b) Comparison of the genetic load region in 5 plasmids harboring bla_NDM-5 with 2 reference plasmids (pIncX-SHV and pNDM-HN380). (c) Alignment of pP768-NDM-5 with 22 plasmids identified in GenBank (last accessed 27 October 2017). The circular maps were generated with the BLAST Ring Image Generator, and each plasmid was colored by the geographic origin (China, blue; Myanmar, yellow; Oman, orange; India, red; Canada, pink; and Kuwait, purple) in the following order (outer to inner circles): pP768-NDM-5, pCREC-A6-NDM, pSCE516-2, pNDM-5_IncX3, pEc1929, pECNDM101, pAD-19R, pNDM-5_WCHEC0215, pK518_NDM-5, pK516_NDM-5, NUHL24835, pNDM-QD28, pNDM-QD29, pEC50-NDM-7, pZHDC40, pJEG027, pM216_X3, pM213_X3 and pM110_X3, pOM26-1, pNDM-MGR194 and pKpN01-NDM7, and pKW53T-NDM (see full details of plasmids in Table S3).

To explore the geographic distribution of potentially related bla_NDM-carrying IncX3 plasmids, the complete sequence of pP768-NDM-5 (chosen as a representative) was used to query the GenBank database. Twenty-two plasmids related to pP768-NDM-5 were identified (Fig. 1c), including 14 plasmids from China, 4 from Myanmar, and 1 each from Canada, India, Kuwait, and Oman (see Table S3 in the supplemental material). The plasmids did not carry resistance genes other than bla_NDM. Multiple NDM variants were carried by the plasmids. These include NDM-1 and variants that differed by one to three amino acids, including NDM-4 (M154L), NDM-5 (V88L, M154L), NDM-7 (D130N, M154L), and NDM-17 (V88L, M154L, E170K).

We identified the occurrence of similar IncX3 plasmids carrying bla_NDM-5 in pigs originating from multiple farms across three different Chinese provinces. The involvement of IncX3 plasmids (represented by pNDM-HN380) in the dissemination of NDM in multiple geographic areas in China was initially reported by our group in 2012 (7). Subsequently, sporadic reports of pNDM-HN380-like plasmids carrying NDM variants have been reported in India, the Arabian Peninsula, Europe, and Australia (11–14). Recently, a Chinese national survey and several provincial studies revealed that IncX3 plasmids harboring different bla_NDM variants were frequently found among clinical isolates of different MLSTs and species, suggesting that they represent an important vector responsible for the wide dissemination of NDM in China (15–17). IncX3 plasmids have a narrow host range and have been found mainly in Enterobacteriaceae (18). Our finding from analysis of complete plasmid sequences indicates that the five IncX3 plasmids originating from pigs are related to pNDM-HN380 and plasmids originating from many other geographic areas, thus confirming that this mobile NDM vector is widespread in the ecosystem.

As carbapenems have never been licensed for use in food animals in China, the NDM-producing pig isolates detected in the present study may have been introduced to the farms via human activity or contaminated feeds. It is worrisome that some of the NDM-producing isolates in the present study were found to coharbor mcr-1 in another plasmid or the chromosome. Nonetheless, our isolates that coharbored mcr-1 were recovered before the ban of colistin in animal feeds was implemented in China in November 2016.

In conclusion, this study identified an epidemic IncX3 plasmid carrying bla_NDM-5 disseminated among E. coli originating from pigs with epidemiological links to geographically segregated areas in China.

Accession number(s).

The complete sequences of the five IncX3 plasmids were deposited in the GenBank database under accession numbers MF547511 (pP744-NDM-5), MF547510 (pP768-NDM-5), MF547509 (pP785-NDM-5), MF547507 (pP788A-NDM-5), and MF547508 (pP855-NDM-5).