Widespread Prevalence of Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli from Père David's Deer in China

The mcr-1 gene is widely reported around the world and has been identified on various plasmids with different replicon types. Resistance to the last-line antibiotic colistin mediated by mcr-1 still represents a threat to global public health.

replicons, including IncX3, IncX4, IncH1, IncHI1, IncHI2, IncP, IncI2, IncF, IncFII, and IncY (3)(4)(5)(6). Resistance to the last-line antibiotic colistin mediated by mcr-1 represents a threat to global public health. Strains positive for mcr-1 in various sources have been reported. Père David's deer is a highly endangered species originating in China, and many deer are currently being raised in nature reserve settings. Dissemination of antimicrobial resistance (AMR) bacteria among animals poses a potential threat to the environment. Therefore, research on the dissemination of mcr-1-positive Escherichia coli from Père David's deer is of great significance. To our knowledge, the distribution of mcr-1 in Père David's Deer National Nature Reserve remained to be investigated up to now. To further learn the transmission characteristics of mcr-1 among nature reserve sites, we studied the prevalence of mcr-1 in E. coli from Père David's deer and proved that mcr-1 has been widespread in the nature reserve.
Among 97 samples, 55 (56.70%) yielded 67 mcr-1-positive strains, and no other mcr variants were found. All the mcr-1-positive strains were identified as E. coli. This indicated that mcr-1-positive E. coli strains existed in nature reserve environments at a high prevalence. All 67 mcr-1-positive isolates conferred resistance to colistin (MICs ranging from 4 to 8 mg/liter) (see Table S1 in the supplemental material). The antibiotic resistance rates of b-lactams amoxicillin, aztreonam, and ceftiofur were very high. We further identified genes encoding b-lactamases through multiplex PCR methods, as previously reported (7). There were 65 (97.01%) mcr-1-positive isolates containing b-lactamase genes (Tables S2 and S3), with bla CTX-M (57 strains) and bla TEM (19 strains) most widely distributed in these mcr-1-positive isolates (Tables S2 and S3). bla CTX-M and bla TEM were prevalent extended-spectrum-b-lactamase (ESBL) genes conferring resistance to most b-lactam antibiotics (8). In addition, genes encoding OXA-1-like broadspectrum b-lactamases were detected in 7 strains harboring IncX4 type plasmids (Table S2). The mcr-1 gene from 66 of 67 isolates and the resistance phenotype were successfully transferred to E. coli J53 (Table S1), suggesting that the mcr-1 gene was located in conjugative plasmids or other mobilizable genetic elements (MGEs) in the 66 isolates. PCR-based replicon typing (PBRT) was performed for all 67 mcr-1-positive isolates and 66 transconjugants. Results showed that 44 transconjugants harbored only an IncI2 plasmid, but corresponding parental strains contained 1 to 4 replicon types, including IncI2. Eight transconjugants harbored only an IncX4 plasmid, and corresponding parental strains also had only an IncX4 plasmid (Table S2). It was concluded that mcr-1 was located in IncI2 or IncX4 plasmids in these strains. Two replicon types of IncHI2 and IncN from 13 transconjugants were identified, and 2 to 4 replicon types, including IncHI2 and IncN, were detected from their corresponding parental strains. It has been reported that the mcr-1-positive IncHI2 plasmid pMCR1_1943 (265,538 bp) also had an IncN replicon (9). S1 nuclease-based pulsed-field gel electrophoresis (S1-PFGE) of 8 transconjugants showed that only one plasmid was visible with a size similar to pMCR1_1943. Therefore, we considered that mcr-1 was located in the IncHI2 plasmid in the 13 strains. However, there were no replicon types detected in strain LD27-1, which implied a possible chromosomal location of mcr-1. In addition, replicon types of one transconjugant were exactly the same as those of its parental strain LD91-1, with four replicon types, IncHI2, IncN, IncFIB, and IncF, detected simultaneously. According to the plasmid size in the S1-PFGE fingerprint of TLD91-1 (transconjugant of LD91-1), we speculated that the plasmid of TLD91-1 was a fused plasmid formed during conjugation whose molecular mechanism was reported previously (10).
PFGE patterns with a cutoff at 90% similarity were considered to belong to the same phylogenetic cluster and were indicated as groups A to W, implying diverse strain clones. Two different mcr-1-positive strains with diverse PFGE types were detected in the same sample among 12 samples according to their colony difference in morphology and color (Table 1; Fig. S1). A single sample containing multiple mcr-1-positive isolates suggested that mcr-1 had spread in the same microbiota. We also found that the mcr-1-harboring plasmids carried by these strains were diverse. IncX4 and IncI2 type mcr-1-bearing plasmids appeared in two strains, respectively, in samples LD4 and LD9, indicating that coexistence of different mcr-1-bearing plasmids in diverse bacteria of the same microbiota sample would exacerbate the transmission of resistance genes. In samples LD26, LD39, LD54, LD75, and LD91, mcr-1-positive plasmids with IncHI2 and IncI2 types were found simultaneously in two different strains. Ten strains from samples LD24, LD36, LD38, LD70, and LD94 carried mcr-1-positive IncI2 plasmids (Table 1). In these samples, strains with the extension "-2" were distributed mainly in two PFGE types (11 strains belonging to group A and 1 strain belonging to group F), while the strains with names ending in "-1" were classified into 8 PFGE types (B, C, E, G, H, K, L, and S). This result indicates that the samples are highly diverse. Partial mcr-1-positive strains isolated from Père David's deer were shown to exhibit genetically similar PFGE types, implying that clonal spread occurred in the nature reserve. mcr-1-positive plasmids with the same replicon type were found in different PFGE clusters, indicating that horizontal dissemination of the mcr-1 gene by plasmids also existed (Fig. S1).
The global spread of ESBL producers is of great concern to human and animal health, and CTX-Ms are the most predominant ESBLs worldwide (15). CTX-M-producing E. coli isolates were recognized as a major cause of hospital-and community-onset infections (16,17). The coexistence of ESBL genes and mcr-1 in E. coli with multidrug resistance was first reported in China in 2016 (18). Evidence showed that ESBL-producing E. coli was more likely to recruit the mcr-1 gene than non-ESBL-producing E. coli (19). Given the fact that colistin is one of the last-line antibiotics for managing multidrug-resistant infections, cotransfer of mcr-1 with ESBL genes by a single mobile plasmid might compromise clinical treatment considerably. In our work, a high prevalence of IncI2 and IncHI2 plasmids harboring both bla CTX-M and mcr-1 ( Table 2; Table S2) indicates a widespread situation of such notorious MDR plasmids among animals. The emergence of the IncI2 plasmid harboring bla CTX-M-132 and mcr-1 reported here should arouse our attention.
The limitations of this study are that geographical distribution and the number of samples collected are relatively confined. Although we collected fresh fecal samples from areas as scattered as possible to guarantee that fecal samples were derived from different individuals of Pere David's deer, duplicate sampling of the same animal was possible.
To conclude, this research is the first report of the prevalence of colistin resistance gene mcr-1 in E. coli from Père David's deer in China that was mainly mediated by plasmids. Mobilizable mcr-1-positive plasmids (IncI2, IncX4, and IncHI2) were widespread in Père David's Deer National Nature Reserve, which may be an important reservoir of mcr-1. The IncI2 mcr-1-bearing plasmid was the most frequent plasmid type (44/67, 65.67%) in the nature reserve, and we are the first to report the IncI2 plasmid harboring both bla CTX-M-132 and mcr-1. Wide spread of mcr-1-harboring IncHI2 plasmids carrying multiple resistance genes in the nature reserve constitutes an easily missed source of mcr-1-bearing IncI2 plasmid pLD67-1-MCR1 in strain LD67-1 in this study and three similar IncI2 plasmids in the NCBI nr database. pLD67-1-MCR1 was used as the reference in the outermost ring. (b) Circular comparison between mcr-1-carrying IncX4 plasmid pLD93-1-MCR1 in strain LD93-1 in this study and two similar IncX4 plasmids in the NCBI nr database. The outer circle with red arrows denotes annotation of reference plasmid pLD93-1-MCR1. (c) Circular comparison between three mcr-1-bearing IncHI2 plasmids and two similar IncHI2 plasmids in the NCBI nr database. (d) Linear comparison of the partial sequence containing mcr-1 of IncHI2 type plasmids (pLD22-1, pLD26-1, and pLD39-1) with two similar structures of plasmids pMCR_WCHEC050613 and p14EC029b in the NCBI nr database.
Characterization of mcr-1-Bearing E. coli Strains multidrug-resistant Gram-negative bacteria. The horizontal dissemination of the mcr-1 gene by plasmids needs to be further investigated, and monitoring of other important emerging resistance genes in nature reserves of animals should be performed.
Bacterial isolates and identification. On 18 August 2018, a total of 97 fresh fecal samples were collected from Père David's Deer National Nature Reserve in Dafeng, Jiangsu Province, China. Individual fresh fecal samples were collected using a sterile swab, subsequently diluted in brain heart infusion broth containing 2 mg/liter colistin, and incubated for 6 h at 37°C. The cultures were plated on MacConkey agar and incubated for 12 h at 37°C. One to three colonies with different morphological characteristics from each MacConkey agar plate were purified and subsequently screened for mcr-1 to mcr-5 genes by multiplex PCR method as previously reported (20). 16S rRNA gene sequencing was performed to confirm bacterial species.
Conjugation experiments and plasmid replicon typing. To investigate the transferability of mcr-carrying plasmids, conjugation assays were performed using MCR-producing strains as donors and E. coli J53 (Azi r ) as the recipient. Bacterial strains were streaked onto LB agar plates, followed by inoculation into LB broth overnight. Cultures of donors and the recipient were mixed 1:3, and then 100 ml of mixed culture was applied onto LB agar plates, followed by incubation at 37°C for 16 to 20 h. After incubation, we subsequently diluted the mixed culture on LB agar plates in sterile saline. LB agar plates supplemented with colistin (2 mg/liter) and sodium azide (150 mg/liter) were used to recover transconjugants. The presence of mcr genes in transconjugants was confirmed by PCR and antimicrobial susceptibility testing as described above. The plasmid contents of mcr-positive strains and transconjugants were determined by PBRT (22) with the addition of PCR detections of IncX4 and IncI2 plasmids, as previously described (3).
XbaI-PFGE and S1-PFGE. XbaI pulsed-field gel electrophoresis (PFGE) was performed to assess the genetic relatedness of all mcr-positive isolates. Briefly, the wholecell DNA of E. coli isolates was digested with the XbaI restriction enzyme for 3 h at 37°C . Electrophoresis was conducted on a CHEF-DR III apparatus (Bio-Rad, Hercules, CA, USA) through a 1% agarose gel in 0.5Â Tris-borate-EDTA buffer using an initial pulse time of 4 s and a final pulse time of 45 s at a voltage of 200 V for 20 h at 14°C (23). Genomic DNA of the Salmonella enterica serovar Braenderup strain H9812 restricted with XbaI (TaKaRa, Osaka, Japan) was used as the reference standard. Cluster analysis of XbaI-PFGE fingerprints was typically performed by using BioNumerics 7.6 (Applied Maths, Sint-Martens-Latem, Belgium). Bacterial DNA was prepared in agarose plugs and digested with 1 U of S1 nuclease (New England Biolabs) for S1 nuclease-based PFGE (S1-PFGE).
Plasmid sequencing and bioinformatics analyses. The genomic DNA of several mcr-1-positive E. coli strains was extracted using a TIANamp bacteria DNA kit (Tiangen, Chain) in accordance with the manufacturer's recommendation. Whole-genome sequencing was performed via Illumina HiSeq and Oxford Nanopore Technologies (ONT) MinION platforms. De novo assembly was performed by Unicycler, combining short-read and long-read data (24). The Rapid Annotation using Subsystems Technology annotation website server (https://rast.nmpdr.org/rast.cgi) was then used to annotate the genomes (25). Online tools including PlasmidFinder 2.1, ResFinder 3.2, and MLST 2.0 (multilocus sequence typing) were utilized to assemble and characterize the mcr-1-bearing genomes (https://cge.cbs.dtu.dk/services/). Comparisons with highly homologous complete plasmid sequences available in NCBI for the plasmids in the study were performed with BRIG (26). To visualize the genetic comparison features, Easyfig was used to generate linear figures (27).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.