IncFII Conjugative Plasmid-Mediated Transmission of bla_NDM-1 Elements among Animal-Borne Escherichia coli Strains

ABSTRACT

This study aims to investigate the prevalence and transmission dynamics of the bla_NDM-1 gene in animal Escherichia coli strains. Two IncFII bla_NDM-1-encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of bla_NDM-1 in these strains. The bla_NDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

TEXT

Carbapenems are one of the last-line antibiotics used to treat serious Gram-negative bacterial infections (1, 2). The increasing rate of carbapenem resistance due to dissemination of the bla_NDM-1 element has raised serious public health concerns (3, 4). The metallo-β-lactamase (MBL)-encoding bla_NDM-1 gene was first reported in 2009 (5), and yet, evidence of worldwide dissemination of this resistance determinant emerged soon after (6, 7). This gene has been known to spread to different species of Enterobacteriaceae and other Gram-negative bacteria (8). Animals have been considered a potential reservoir of multidrug-resistant (MDR) Gram-negative organisms due to the extensive use of antibiotics in animals as growth promoters and for treatment purposes. However, detection and transmission of bla_NDM-1 in animals have rarely been reported. The molecular basis of the low prevalence of bla_NDM-1-positive bacteria in animals is currently not understood. It is possible that there is a lack of selective pressure due to carbapenems in animals, since this category of antimicrobial agents are not used in veterinary medicine. Nevertheless, several recent studies have begun to report the detection and isolation of bla_NDM-1-positive strains in animals (9, 10). These reports have raised serious public health concerns, since settlement of the bla_NDM-1 elements or strains carrying such elements in animals may result in an enhanced rate of transmission of both resistance genes and resistant strains among farmed animals, which in turn pose an increased risk of human infections. This study aims to investigate the prevalence and genetic characteristics of mobile elements harboring the bla_NDM-1-like genes that were transmissible among Escherichia coli strains isolated from food animals in China and assess their threat to humans.

Animal fecal samples from 10 swine farms located in seven different provinces in China during the period of May 2014 to May 2015 were collected and subjected to procedures for isolating carbapenem-resistant E. coli, using MacConkey agar containing 0.5 μg/ml of meropenem. A maximum of four colonies on each positive plate were picked, purified, and subjected to species identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-MS) and 16S rRNA gene sequencing (11). A total of 220 pig fecal samples were collected from 10 farms. Carbapenem-resistant E. coli isolates were only detectable on one farm in Henan province, where 11 carbapenem-resistant E. coli strains were isolated from 5 finishing pigs (pigs 1 to 5), and yet, none of the 10 weaning piglets sampled was found to harbor carbapenem-resistant E. coli.

Pulsed-field gel electrophoresis (PFGE) analysis was performed on these isolates as previously described (12); four different patterns were identified in 10 isolates, and one isolate was untypeable (see Fig. S1 in the supplemental material). E. coli strains with the same PFGE pattern could be detected in different pigs, and interestingly, E. coli strains with different PFGE patterns could emerge from the same animal, suggesting that the transmission of carbapenem-resistant E. coli strains on this pig farm was due to both clonal and nonclonal transmission. All carbapenem-resistant E. coli strains were subjected to multilocus sequence typing (MLST) typing, following the method of Wirth et al. (13). The four different PFGE types belonged to sequence type 1695 (ST1695), ST1585, ST1721, and ST359. Except for ST359, which is commonly associated with extended-spectrum β-lactamase-producing human clinical isolates (14) or animal isolates (15), the other three ST types have rarely been reported; they were shown here to be associated with a bla_NDM-1 and the production of a carbapenem phenotype for the first time.

Screening of carbapenemase and extended-spectrum β-lactamase genes was performed in these isolates (16), with the results showing that all test organisms harbored the bla_NDM-1 gene. In addition, all isolates also harbored the fosA3 gene, and some of the isolates were found to harbor different bla_CTX-M variants. S1 nuclease PFGE and Southern hybridization showed that the bla_NDM-1 gene was encoded on two types of plasmids, which had sizes of ∼100 kb and ∼95 kb, and that they were all conjugative (see Table S1 and Fig. S2 in the supplemental material). The plasmids recovered from the transconjugant of strain HNEC55 (T-HNEC55) and T-HNEC46 were subjected to sequencing using both Illumina and PacBio single-molecule real-time sequencing (SMRT) technologies. The plasmid from T-HNEC55 was found to be 81,498 bp in size and to contain 110 open reading frames and was designated pHNEC55-NDM. On the other hand, the plasmid recovered from transconjugant T-HNEC46, designated pHNEC46-NDM, was found to be 74,046 bp in size and to contain 101 open reading frames. Both plasmids belong to the IncFII type, and they share an almost identical backbone, with the only difference being the structure of the MDR-encoding mobile elements that they harbored; this was the only type of mobile element located in these two plasmids (Fig. 1). The pHNEC55-NDM plasmid was found to comprise four gene cassettes, each of which contained different antibiotic resistance genes and was flanked by multiple transposase genes, with IS26 being the most common. These gene cassettes included the mphA, bla_NDM-1, fosA3, and rmtB gene clusters. Compared to the MDR mobile element in pHNEC55-NDM, the one in pHNEC46-NDM was found to contain identical mphA and bla_NDM-1 gene cassettes but lacked those of fosA3 and rmtB.

FIG 1.

Comparative analysis of plasmids pHNEC55, PHENC46, and Phn7A8. Dark-gray shading denotes 100% nucleotide identity, and light gray indicates >99% nucleotide identity. Green arrows denote primers targeting the two plasmids which have been sequenced. The locations of the primers used to identify the plasmid are labeled in the figure.

BLAST analysis of the pHNEC55 sequence revealed that the backbone of pHNEC55-NDM (the plasmid excluding the MDR element) exhibited >99.9% homology to that of pHN7A8 (62.3 kb), a plasmid carried by E. coli strain 7A8, which originated from animal in China (17). The MDR elements of these two plasmids were found to contain several identical gene cassettes, such as the upstream transposase gene and the downstream rmtB and fosA3 cassettes. A major structural difference was that the bla_CTX-M-65 cassette in pHN7A8 was replaced by the mphA and bla_NDM-1 genes in pHNEC55-NDM (Fig. 1 and 2). Plasmids with similar backbones but carrying different bla_CTX-M gene cassettes were also reported previously, including plasmid pKP1034 (GenBank accession number KP893385.1), which was carried by a clinical K. pneumoniae isolate, and plasmid pHNFP460-1 (KJ020575), which was recovered from an E. coli strain isolated from animals.

FIG 2.

Comparative analysis of the MDR region in different plasmids. Dark-gray shading represents 100% nucleotide identity, and the light-gray region depicts >99% nucleotide identity. Cyan arrows represent mobile elements, red arrows denote antibiotic resistance genes, magenta arrows represent nonfunctional genes, gray arrows depict hypothetical proteins, and all other genes are labeled in orange.

The MDR region of pHNEC55 is a 19-kb fragment comprising three major antimicrobial resistance gene cassettes flanked by four IS26 elements. These included an 11-kb cassette harboring the mphA and bla_NDM-1 genes and an 8-kb cassette containing the fosA3, bla_TEM, and rmtB genes. In contrast, the MDR region of pHNEC46 only contained the 11-kb cassette that comprised the bla_NDM-1 gene (Fig. 2). BLAST analysis showed that the MDR region of pHNEC55 was similar to the one found in plasmid pKP1034 (KP893385.1), which was carried by a clinical K. pneumoniae stain isolated from China in 2015, as well as to plasmid pHN7A8 (17). The major difference is that the gene cassette containing bla_CTX-M-65 in these two plasmids was replaced by the bla_NDM-1 gene fragment (Fig. 2). The mobile element harboring the bla_NDM-1 gene in pHNEC55 and pHNEC46 contained a structure identical to the ones found in an Acinetobacter species (18), Citrobacter freundii (19), Providencia rettgeri (20), and a Klebsiella species (GenBank accession number CP010390.1) of human origin. A 2-nucleotide deletion was observed in the aphA6 gene located in pHNEC55 and pHNEC46, resulting in early termination of the protein and inactivation of this gene product; this observation was therefore consistent with the loss of the aminoglycoside resistance phenotype in E. coli strains carrying these two plasmids (see Table S1 in the supplemental material).

To characterize the genetic features of conjugative plasmids encoding bla_NDM-1 genes recovered from other transconjugants, a set of primers targeting various regions of pHNEC55 and pHNEC46, as shown in Table S1 in the supplemental material, was used to screen for plasmids harbored by other transconjugants. The results showed that plasmids from T-HNEC47, T-HNEC62, and T-HNEC70 were structurally similar to pHNEC46, while other transconjugants carried plasmids resembling pHNEC55 (see Table S2 in the supplemental material).

In conclusion, this study showed that the dissemination of bla_NDM-1 in E. coli from animals was due to the capture of bla_NDM-1-encoding mobile elements by conjugative plasmids that circulate among animal E. coli isolates. This plasmid may be easily disseminated within an animal farm and further transmitted to humans. Since animal isolates may serve as a vector for rapid transmission of carbapenemase genes, future studies should focus on depicting factors that determine the efficiency of carriage and transmission of carbapenemase genes in such organisms.

Accession number(s).

The completed plasmid sequences for pHNEC55 and pHNEC46 were deposited in NCBI with accession numbers KT879914.1 and KX503323, respectively.