Identification of mcr-1 positive E. coli (MCRPEC) isolates
A total of 142 E. coli isolates were isolated from pigs with diarrhea/dyspnea in Guangxi in 2018. Seventy-two (50.7%, 72/142) E. coli isolates were tested with MICs of colistin ≥3.5 mg/L. To investigate the proportion of mcr genes in E. coli, PCR amplification was performed to test mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8. A total of 33 mcr-1-positive E. coli strains were detected. While no other mcr genes were detected. The MCRPEC strains accounted for 45.8% (33/72) in colistin resistant E. coli strains and 23.2% (33/142) in all isolated E. coli strains. The thirty-three MCRPEC isolates were used for subsequent study.
The full-length 16S rRNA nucleotide sequences of the 33 MCRPEC strains were used to generate a phylogenetic tree by means of Neighbor Joining method in MEGA-X (Fig. 1). Thirty-three MCRPEC strains were classified into eight distinct STs, including ST10, ST224, ST361, ST410, ST641, ST1408, ST3345, and an unknown ST. ST10 and ST224 were the dominant STs, which accounted for 69.7% (23/33) (Fig. 1). More information about MLST is included in the supplementary materials (supplementary materials Table 2).
In order to identify incompatibility plasmid groups, multiple PCR was performed by using total plasmid genes of MCRPEC isolates. As is shown in Figure 1, a total of 6 incompatibility plasmid groups were detected, including IncFI (97.0%, 32/33), IncHI1 (12.1%, 4/33), IncY (48.5%, 16/33), IncN (15.2%, 5/33), IncI1 (3.0%, 1/33) and IncX1 (6.1%, 2/33). (Fig. 1).
Antimicrobial resistance in MCRPEC
The antimicrobial resistance proportion of thirty-three MCRPEC isolates were as follows: gentamicin (72.7%, 24/33), amikacin (48.5%, 16/33), ceftaroline (69.7%, 23/33), piperacillin-tazobactam (24.2%, 8/33), imipenem (36.4%, 12/33), meropenem (24.2%, 8/33), cefalexin (69.7%, 23/33), cefuroxime (57.6%, 19/33), cefotaxime (57.6%, 19/33), ceftriaxone (57.6%, 19/33), cefepime (39.4%, 13/33), cefoxitin (0%, 0/33), ciprofloxacin (75.8%, 25/33), sulfadiazine (24.2%, 8/33), trimethoprim-sulphamethoxazole (0%, 0/33), aztreonam (24.2%, 8/33), ampicillin (97.0%, 32/33), amoxicillin-clavulanic acid (0%, 0/33), ampicillin-sulbactam (24.2%, 8/33), chloramphenicol (84.8%, 28/33), fosfomycin (78.8%, 26/33), tetracycline (100%, 33/33), doxycycline (72.7%, 24/33), azithromycin (57.6%, 19/33), polymyxin B (100%, 33/33) and colistin (100%, 33/33) (Fig. 2a). In addition, each MCRPEC isolate showed significant antimicrobial resistance (Fig. 2b). According to the results of cephalosporin susceptibility test, nineteen strains (57.6%, 19/33) were resistant to the 3rd and 4th generation cephalosporins and twelve strains (36.4%, 12/33) were resistant to carbapenem (Fig. 2a). According to the definition criteria of MDR, XDR, and PDR bacteria, all 33 MCRPEC isolates were identified as MDR (Fig. 2b) [2]. Moreover, three of the MDR MCRPEC isolates were identified as XDR (Fig. 2c).
Coexistence of mcr-1 gene in plasmids with β-lactamase antimicrobial resistance genes and non-β-lactamase antimicrobial resistance genes in the MCRPEC isolates
To investigate whether the mcr-1 gene coexist with β-lactamase antimicrobial resistance genes and non β-lactamase genes in the plasmids of MCRPEC strains. Total plasmid DNA of MCRPEC strains were tested by using multiplex PCR. There were 22 MCRPEC isolates harbored ESBL genes including two blaOXA-1 and thirty-two blaCTX-M. The dominate blaCTX-M gene was blaCTX-M-14 (59.4%, 19/32), followed by blaCTX-M-123 (37.5%, 12/32) and blaCTX-M-24 (3.1%, 1/32) (Fig. 3a, Fig. 3b). Additionally, there were two and eight MCRPEC isolates identified to harbor blaCMY-2 and blaNDM-5 respectively. (Fig. 3a, Fig. 3c).
Furthermore, many non-β-lactamase antimicrobial resistance genes were also investigated in MCPEC isolates, including fluoroquinolone resistance gene, tetracycline resistance genes, sulfanilamide resistance genes, aminoglycoside resistance genes and chloramphenicol resistance genes. As is shown in Fig 3a, the detection rates of fluoroquinolone resistance related genes qnrA, qnrB, qnrS, and aac(6’)-Ib-cr were respectively 36.4% (12/33), 36.4% (12/33), 33.3% (11/33), and 24.2%( 8/33). Tetracycline resistance related genes tetA, tetB, and tetX accounted for 100% (33/33), 18.2% (6/33), and 0% (0/33), respectively. Sulfanilamide resistance related genes sul1 and sul2 accounted for 90.0% (30/33) and 78.8% (26/33), respectively. Aminoglycoside resistance related gene aadA (100%, 33/33) and chloramphenicol resistance related gene floR (100%, 33/33) both have 100% detection rates.