Characteristics of Carbapenem-Resistant and Colistin-Resistant Escherichia coli Co-Producing NDM-1 and MCR-1 from Pig Farms in China

The emergence of carbapenem-resistant and colistin-resistant Enterobacteriaceae represents a great risk for public health. In this study, the phenotypical and genetic characteristics of eight carbapenem-resistant and colistin-resistant isolates from pig farms in China were determined by the broth microdilution method and whole genome sequencing. Antimicrobial susceptibility testing showed that the eight carbapenem-resistant and colistin-resistant strains were resistant to three aminoglycosides, twelve β-lactams, one of the phenicols, one of the tetracyclines, and one of the fluoroquinolones tested, simultaneously. The prediction of acquired resistant genes using the whole genome sequences revealed the co-existence of blaNDM-1 and mcr-1 as well as the other genes that were responsible for the multidrug-resistant phenotypes. Bioinformatics analysis also showed that the carbapenem-resistant gene blaNDM-1 was located on a putative IncFII-type plasmid, which also carried the other acquired resistant genes identified, including fosA3, blaTEM-1B and rmtB, while the colistin-resistant gene mcr-1 was carried by a putative IncX4-type plasmid. Finally, we found that these resistant genes/plasmids were conjugative, and they could be co-conjugated, conferring resistance to multiple types of antibiotics, including the carbapenems and colistin, to the recipient Escherichia coli strains.


Introduction
Escherichia coli has become a great concern for global public health. On the one hand, the prevalence and outbreak of some intestinal pathogenic E. coli clones, particularly the well-known O157/ST11 and, more recently, the O104/ST678, has caused high levels of morbidity and mortality worldwide [1][2][3][4]; on the other hand, E. coli has had a great capacity to accumulate resistance genes, representing a natural reservoir of resistance genes, which may therefore contribute to the dissemination of antibiotic resistance and lead to treatment failures in both human and veterinary medicine [5]. It is of great importance to know how resistance genes are acquired and evaluate their capacity of dissemination between bacteria.
Recently, the emergence of the plasmid-mediated New Delhi metallo-β-lactamase-1 encoding gene bla NDM-1 and/or the plasmid-mediated colistin-resistance gene mcr-1 represents a great concern to global public health [6,7]. The plasmid-mediated bla NDM-1 confers resistance to carbapenems [6], which is considered as the last resort for treating multidrug-resistant (MDR) Enterobacteriaceae [8], while mcr-1 mediates the resistance to colistin [9], the key antibiotic used for treating carbapenem-resistant Enterobacteriaceae [8]. The emergence of carbapenems and colistin co-resistant Enterobacteriaceae means there will be little and/or no antibiotic available for the infections caused by such strains in most cases of infections caused by MDR Enterobacteriaceae. However, the co-existence of bla NDM and mcr-1 in Enterobacteriaceae originated from both humans and animals has been increasingly reported worldwide [10][11][12][13]. These isolates are involved in the co-harboring of mcr-1 and different members of bla NDM such as bla NDM-1 [11], bla NDM-4 [13], bla NDM-5 [12], and bla NDM-9 [10,14]. Of particularly concern is the recovery of an E. coli strain co-producing MCR-1 and NDM-9 from a patient with a catheter-associated urinary tract infection (UTI); the infection of such an E. coli led to the failure of antibiotic treatment and finally killed the patient [10]. Therefore, it is of great importance to monitor Enterobacteriaceae strains with co-resistance to carbapenems and colistin and understand how this resistance is accumulated. In this study, we report several carbapenem-resistant and colistin-resistant E. coli co-producing NDM-1 and MCR-1 from pig farms in China.

Bacterial Strains and Antimicrobial Susceptibility Testing
Fecal and environmental swabs from 7 pig farms located in Hubei Province and Henan Province in China during June 2018 and June 2019 were collected for the isolation of carbapenem-resistant and colistin-resistant E. coli, using MacConkey agar containing 4 µg/ml of imipenem and 2 µg/ml of colistin. E. coli isolates were confirmed by PCR detection of the 16S rRNA gene and the seven house-keeping genes (adk, fumC, gyrA, icd, mdh, purA, and recA) of E. coli [15]. A total of 538 samples were collected and carbapenem-resistant and colistin-resistant E. coli were only detectable on samples from one farm in Henan province (sows ≥ 4000), where eight isolates were recovered from swabs of the floor and barrier of different pigsties (RDX007, RDX012), swabs of different food troughs (RDX020, RDX024), fecal samples of different pigs suffered from diarrhea (RDX033, RDX035, RDX100), and water troughs (RDX115).

Detection of the Carbapenem-Resistant and Colistin-Resistant Genes
PCR assays were initially performed to determine the presence and the location of the carbapenem-resistant gene bla NDM-1 and colistin-resistant mcr-1 with primers "F: GGTTTGGCGATCTGGTTTTC; R: CGGAATGGCTCATCACGATC" (for bla NDM-1 , annealing temperature 55 • C, product size: 621 bp) [16] and "F: CGGTCAGTCCGTTTGTTC; R: CTTGGTCGGTCTGTAGGG" (for mcr-1, annealing temperature 55 • C, product size: 309 bp) [9], respectively. PCR reactions were performed in a 25 µl amplification mixture containing 2 µl of template DNA, 12.5 µl of 2×Taq Master Mix (Dye Plus, Vazyme Biotech Co., Ltd; Nanjing, China), 1 µl each of 10 µM forward and reverse primer, and 8.5 µl of nuclease-free water. The reaction was performed under the following cycling conditions: an initial denaturation at 95 • C for 10 min, followed by 35 cycles of denaturation at 94 • C for 45 s, annealing at 55 • C for 45 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min. PCR products were analyzed by electrophoresis on 1% agarose gel. Two rounds of PCR assays were performed. The first round of PCR assays was performed using the genomic DNA isolated from the strains as the template to determine the presence of the two resistant genes. An additional round of PCR assays was performed using the plasmids isolated from the strains to determine whether the detected resistant genes were located on the plasmids.

DNA Extraction and Whole Genome Sequencing and Bioinformatic Analysis
Genomic DNA were extracted and purified using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China). The quality and quantity of the DNA were evaluated by electrophoresis on a 1% agarose gel and using a NanoDrop2000 (Thermo Scientific, Waltham, USA). Whole genome sequencing was performed on an Illumina Hiseq Xten platform (Illumina Inc., San Diego, USA) at Guangdong Magigene Biotechnology Co. LTD (Guangzhou, China), using the pair-end 150 bp sequencing protocol. DNA libraries were constructed using a NEBNext Ultra TM II DNA Library Prep Kit (New England BioLabs, Ipswich, USA). After sequencing, approximately 7,703,600~18,595,428 raw reads were yielded for the eight strains. Raw reads with low quality were filtered according to the following criteria: low quality base pairs at each terminal of the reads (Quality-Value < 20) were removed; reads with a short length (parameter setting at 50 bp), or > 15 bp overlap with Illumina TruSeq adapter sequences (parameter setting at 15 bp) were removed. Finally, approximately 7,363,540~17,856,944 clean reads (Q20% = 100, Q30% ≥ 95.59) were produced. High-quality reads were de novo assembled via SPAdes v3.9.0 [17] to generate contigs.
The assembled contigs were used for determining the genetic characteristics of the eight E. coli isolates. Serotypes and sequence types (ST) were determined by SerotypeFinder 2.0 [18] and Multi-Locus Sequence Typing (MLST) 2.0 [19], respectively. Acquired antimicrobial resistance genes and plasmids were determined using ResFinder 3.1 [20] and PlasmidFinder 2.0 [21], respectively. DNA identities between two sequences were calculated by ANI calculator [22]. A comparative genome analysis was performed and visualized using the BRIG package [23] and/or the EasyFig package [24]. Phylogenetic analysis was performed by the maximum likelihood method using the Tamura-Nei model [25] on MEGAX [26] with 1000 bootstrap iterations. The whole genome sequence of E. coli MG1655 (GenBank accession no. U00096.3) was downloaded from GenBank and used as the reference genome in the study.

Plasmid Conjugation
Plasmid conjugation assays between the eight carbapenem-resistant and colistin-resistant E. coli (donor) and the rifampin resistant E. coli C600 (recipient) were performed on a nitrocellulose membrane, as described previously [27]. Briefly, mid-log phase donor and recipient strains (OD 600 = 0.5~0.6) were mixed at a ratio of 1:3 (v/v). The bacterial mixture was then spotted on a nitrocellulose membrane which was pre-plated on the LB agar. After a 12 h of incubation at 37 • C, bacteria on the membrane were washed off using LB broth followed by being shaken at 37 • C for 4 h. Finally, the transconjugants were selected on LB agar plates laced with rifampin (1000 mgl −1 ) plus imipenem (20 mgl −1 ) plus colistin (2 mgl −1 ). Antimicrobial susceptibility of the transconjugants was determined using broth microdilution method as mentioned above.

Detection of the Carbapenem-Resistant Gene blaNDM-1 and Colistin-Resistant Gene mcr-1
PCR assays revealed that the eight carbapenem-resistant and colistin-resistant E. coli strains contained the carbapenem-resistant gene blaNDM-1 and colistin-resistant gene mcr-1, simultaneously. In addition, PCR assays using plasmids extracted from the eight strains as template showed positive results for the detection of the two genes, suggesting that the two genes were located on plasmids ( Figure 2).  In addition, PCR assays using plasmids extracted from the eight strains as template showed positive results for the detection of the two genes, suggesting that the two genes were located on plasmids ( Figure 2).

Plasmid Determination
Because the results of the PCR assays revealed that the blaNDM-1 gene and the mcr-1 gene were located on plasmids (Figure 2), we used the whole genome sequences to analyze the putative blaNDM-1-carring and/or mcr-1-carrying plasmids that were harbored by the eight carbapenem-resistant and colistin-resistant E. coli isolates. Bioinformatical analysis revealed the blaNDM-1 gene was located on an IncFII-type plasmid, while the mcr-1.1 gene carried by RXD007, RXD012, RXD020, RXD024, RXD033, RXD035, and RXD115 was located on an IncX4-type plasmid. However, we did not find the putative mcr-1-carrying plasmid in RXD100 showing any homologous to any plasmids with DNA sequences available in GenBank. The IncFII-type plasmid carrying the blaNDM-1 gene was homologous to pHNEC55 (GenBank accession: KT879914), and this pHNEC55-like plasmid also carried fosA3, blaTEM-1B and rmtB ( Figure 5A). An IncX4-type plasmid carrying mcr-1.1 presence in RXD007, RXD012, RXD020, RXD024, RXD033, RXD035, and RXD115 was homologous to pIBMC_mcr1 (GenBank accession: MF449287) ( Figure 5B).

Plasmid Determination
Because the results of the PCR assays revealed that the bla NDM-1 gene and the mcr-1 gene were located on plasmids (Figure 2), we used the whole genome sequences to analyze the putative bla NDM-1 -carring and/or mcr-1-carrying plasmids that were harbored by the eight carbapenem-resistant and colistin-resistant E. coli isolates. Bioinformatical analysis revealed the bla NDM-1 gene was located on an IncFII-type plasmid, while the mcr-1.1 gene carried by RXD007, RXD012, RXD020, RXD024, RXD033, RXD035, and RXD115 was located on an IncX4-type plasmid. However, we did not find the putative mcr-1-carrying plasmid in RXD100 showing any homologous to any plasmids with DNA sequences available in GenBank. The IncFII-type plasmid carrying the bla NDM-1 gene was homologous to pHNEC55 (GenBank accession: KT879914), and this pHNEC55-like plasmid also carried fosA3, blaTEM-1B and rmtB ( Figure 5A). An IncX4-type plasmid carrying mcr-1.1 presence in RXD007, RXD012, RXD020, RXD024, RXD033, RXD035, and RXD115 was homologous to pIBMC_mcr1 (GenBank accession: MF449287) ( Figure 5B).

Plasmid Conjugation
To assess the transferability of the plasmids and the resistance genes, conjugation experiments between the eight were performed with selective plates laced with rifampin (1000 mgl -1 ) plus imipenem (20 mgl -1 ) plus colistin (2 mgl -1 ). Transconjugants incubated on selective agars were selected for antimicrobial susceptibility testing. The result showed that the plasmids in seven of the eight carbapenem-resistant and colistin-resistant E. coli transferred into the recipient strain and after conjugation, the resistance phenotypes of the recipient E. coli (C600) changed remarkably (Table 1).

Plasmid Conjugation
To assess the transferability of the plasmids and the resistance genes, conjugation experiments between the eight were performed with selective plates laced with rifampin (1000 mgl −1 ) plus imipenem (20 mgl −1 ) plus colistin (2 mgl −1 ). Transconjugants incubated on selective agars were selected for antimicrobial susceptibility testing. The result showed that the plasmids in seven of the eight carbapenem-resistant and colistin-resistant E. coli transferred into the recipient strain and after conjugation, the resistance phenotypes of the recipient E. coli (C600) changed remarkably (Table 1). Table 1. Phenotypic characteristics of the transconjugants of the eight carbapenem-resistant and colistin-resistant E. coli.

Discussion
Carbapenem and colistin are the last-resort antibiotics used for treating multidrug-resistant Gram-negative pathogens [8].
It is of great public health significance to monitor the carbapenem-resistant and colistin-resistant bacteria and investigate the mechanisms for the acquisition of the resistant phenotypes. In this study, we isolated eight carbapenem-resistant and colistin-resistant E. coli from a pig farm in Henan Province, China. Antimicrobial susceptibility testing revealed the eight carbapenem-resistant and colistin-resistant isolates were also resistant to aminoglycosides, cephalosporins, β-lactam combination agents, phenicols (CHL), tetracyclines, fluoroquinolones, and sulfonamides (SXT) (Figures 1 and 2). Since most of these phenotypes in E. coli were conferred by resistance genes mostly through horizontal gene transfer [5], the presence of those strains in pigs/farms may represent a real public health concern: (1) these MDR isolates might transmit between pigs/environment and other animal species including humans through numerous pathways such as via direct/indirect contact and/or via food-chain, which might therefore lead to treatment failures in both human and veterinary medicine; (2) such strains might also act as a major reservoir of resistance genes, which might contribute to the spread of antimicrobial resistance.
Corresponding to the resistant phenotypes determined, prediction using the whole genome sequences identified many types of acquired genes conferring resistance to β-lactams (including cephalosporins and carbapenems), aminoglycosides, fluoroquinolones, polymyxins, tetracyclines, phenicols, sulfonamides, trimethoprim, and fosfomycin ( Figure 3). Of particularly note is the bla NDM-1 gene, which encodes the New Delhi metallo-β lactamase 1 (bla NDM-1 ) and confers the resistance to carbapenems [28,29], and the mcr-1 gene, which encodes the phosphoethanolamine-lipid A transferase and confers the resistance to colistin [9]. Both of these two genes have substantial importance worldwide in terms of resistance [7,[29][30][31] and they were identified in the eight carbapenem-resistant and colistin-resistant E. coli strains (Figures 2 and 3), suggesting that the co-existence of bla NDM-1 and mcr-1 mediates the resistance phenotypes to carbapenems and colistin.
Three of the eight carbapenem-resistant and colistin-resistant E. coli were isolated from fecal samples of different diarrheal pigs. Two of them, designated RDX033 and RDX035, were found to be ST746 (Figure 4). It is worth noting that MCR-1-carrying, extended-spectrum β-lactamase (ESBL)-producing E. coli ST746 has been recovered from community-acquired urinary tract infection [32]. The human sourced E. coli ST746 contained plasmid-carrying mcr-1 as well as two kinds of β-lactam-resistant genes bla CTX-M-14 and bla TEM-1B but not bla NDM-1 gene [32]. However, the two ST746 strains recovered from diarrheal pigs carried both mcr-1 and bla NDM-1 in addition to bla CTX-M- 14 and bla TEM-1B (Figure 3), and they displayed a broader spectrum of resistance compared with the human sourced ST746. The identification of such strains in pigs might represent a potential risk on public health. Another carbapenem-resistant and colistin-resistant E. coli recovered from different diarrheal pigs was RXD100, which was identified as ST695. While this isolate showed resistance to most of the antibiotics tested, it was not as resistant as the ST746 strains RDX033 and RDX035 (Figure 1). Fewer numbers of resistant genes carried by RXD100 compared to RDX033 and RDX035 might explain the difference (Figure 3).
It has been widely reported that plasmids play an important role in the dissemination of bla NDM-1 and mcr-1 [5,9,29,31]. Consistently, our PCR results using the plasmids extracted as templates from the eight strains revealed that both of the genes were located on plasmids (Figure 2). Plasmid prediction using whole genome sequences identified several putative types of plasmids harbored by the eight strains ( Figure 4). Among these putative types of plasmids, IncHI2A, IncFIB(K), IncFII, IncFI1, IncR, IncHI2, and IncFIA plasmids carrying bla NDM-1 have been reported [33][34][35][36][37], while IncX4, IncFIB(K), IncFII, IncFI1, IncY, IncHI2, IncX1, and IncFIA plasmids carrying mcr-1 have been reported [38][39][40][41][42]. Although more accurate and reliable technologies such as the third-generation sequencing technologies are necessary for determining the accurate type and sequence of the plasmids, bioinformatical analysis using whole genome sequences revealed that an IncFII-type plasmid homologous to pHNEC55 (GenBank accession: KT879914) likely carried the bla NDM-1 gene, and an IncX4-type plasmid homologous to pIBMC_mcr1 (GenBank accession: MF449287) likely carried the mcr-1 gene, as the backbones of both plasmids were found in the genomes of the eight strains ( Figure 5). Interestingly, the IncFII plasmid pHNEC55 carrying bla NDM-1 was also identified in a carbapenem-resistant E. coli strain isolated from pigs in Henan Province [43]. The multidrug resistance-encoding mobile elements of this plasmid contains several other resistant genes in addition to bla NDM-1 , including mphA, which confers resistance to macrolides; fosA3, which confers resistance to Fosfomycin; rmtB, which confers resistance to aminoglycosides; and bla TEM , which confers resistance to β-lactams [43]. These genes were also carried by the eight carbapenem-resistant and colistin-resistant bacteria and they were also likely to be carried by the bla NDM-1 -carrying plasmids ( Figure 5A). In the next step, we intend to use more accurate and reliable technologies such as third-generation sequencing or a combination of second-generation with third-generation sequencing to determine the complete genome sequences and the accurate structures of the MDR plasmids in the eight E. coli strains.
Plasmid conjugation assays revealed that the MDR plasmids harbored by the eight carbapenem-resistant and colistin-resistant bacteria were transferrable, and it seems that these MDR plasmids are capable of co-conjugation at appropriate conditions, as the conjugation of the plasmids conferred both carbapenem and colistin resistance to the recipient bacteria simultaneously ( Table 1). The same results have also been found in other articles [14]. In addition to carbapenems and colistin, the conjugation of these plasmids was also able to disseminate resistance to many types of antibiotics tested, including aminoglycosides, cephalosporins, β-lactam combination agents, phenicol, tetracyclines, fluoroquinolones, and sulfonamides ( Table 1). Many of these antibiotics are commonly used antibiotics for bacterial infections in both human and veterinary medicine [5], and many are classified as critically important antimicrobials by the WHO and their usage should be severely restricted (https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/). Therefore, the presence of these conjugative MDR plasmids might lead to the spread of the MDR plasmids and treatment failures in both human and veterinary medicine. More active actions should be taken to monitor the prevalence of such plasmids as well as their bacterial hosts. In the next step, we will evaluate the conjugative capacity and efficacy of each of the resistant plasmids after their complete genome sequences are determined by third-generation sequencing technologies.

Conclusions
In conclusion, a total of eight carbapenem-resistant and colistin-resistant E. coli from pigs/farms in Central China were characterized in the present study. The carbapenem-resistant and colistin-resistant E. coli isolates were commonly resistant to aminoglycosides, cephalosporins, β-lactam combination agents, phenicol, tetracyclines, fluoroquinolones, and sulfonamides. A coexistence of a putative IncFII-type plasmid homologous to pHNEC55 and an IncX4-type plasmid homologous to pIBMC_mcr1 is likely to be responsible for the dissemination of the carbapenem-resistant gene bla NDM-1 and colistin-resistant gene mcr-1, and both genes/plasmids are conjugative and could be co-conjugatively transferred. In the next study, we intend to use more accurate and reliable technologies like third-generation sequencing to determine the complete genome sequences and the accurate structures of these MDR plasmids.