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 colistin (MIC 3.5mg/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. 33 mcr-1-positive E. coli strains detected. The percentage of MCRPEC strains accounted for 45.8% (33/72) of colistin resistant strains and 23.2% (33/142) of all isolated strains. The thirty-three MCRPEC isolates were used for subsequent study.
The full-length 16S rRNA gene 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 was included in the supplementary materials (supplementary materials Table 2).
Multiple PCR was used to identify incompatibility plasmid groups in MCRPEC by using plasmid DNA of MCRPEC isolates. As is shown in Figure 1, six incompatibility plasmid groups were detected, including IncFI (97.0%, 32/33), IncHI (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). Ten (30.3%, 10/33) MCRPEC isolates were detected to carry one incompatibility plasmid group (IncFI). Sixteen (16/33, 48.5%) MCRPEC isolates were detected to carry two incompatibility plasmid groups, among which eleven (11/16, 68.8%) isolates were the combination of IncFI and IncY, four (4/16, 25%) isolates displayed the combination of IncHI and IncFI, and one (1/16, 6.2%) isolate exhibited the combination of IncFI and IncX1. Six (6/33, 18.2%) MCRPEC isolates were detected to carry three incompatibility plasmid groups, among which four (4/6, 66.6%) isolates were the combination of IncFI, IncN and IncY, one (1/6, 16.7%) isolate was the combination of IncFI, IncI1 and IncY, and one (1/16, 16.7) isolate was the combination of IncFI, IncN and IncX1. In addition, one (1/33, 3.0%) MCRPEC isolate was not detected to carry incompatibility plasmid group.
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 of MDR, XDR, and PDR bacteria, all 33 MCRPEC isolates were identified as MDR (Fig. 2b). Among them, 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
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 with blaCMY-2 and blaNDM-5 respectively. (Fig. 3a, Fig. 3c).
Furthermore, many non-β-lactamase antimicrobial resistance genes were also detected 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 rates of fluoroquinolone resistance related genes qnrA, qnrB, qnrS, and aac(6’)-Ib-cr were 36.4% (12/33), 36.4% (12/33), 33.3% (11/33), and 24.2%( 8/33) individually. Tetracycline resistance related genes tetA, tetB, and tetX accounted for 100% (33/33), 18.2% (6/33), and 0% (0/33), respectively. Sulfanilamide resistance determinants 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.
Besides, virulence genes (Enterotoxigenic E. coli and Shigatoxin-pruducing E. coli) of thirty-three MCRPEC isolates from clinical diagnostic were detected. The results were shown in Table 3 of supplementary materials. Twenty-six (26/33, 78.8%) MCRPEC isolates were identified as pathogenic E. coli, of which twenty-two (22/26, 84.6%) isolates were identified as Enteroxigenic E. coli (ETEC), three (3/26, 11.5%) isolates were identified as Shigatoxin-pruducing E. coli (STEC), and one (1/26, 3.9%) isolate was both ETEC and STEC. Fifteen (15/22, 68.2%) ETEC isolates only carried STb gene, the rest of ETEC isolates (7/22, 31.8%) carried STb and LT genes. Three STEC isolates only carried stx2e gene. The ETEC/STEC isolate carried STb, LT and stx2e genes.