China Animal Husbandry and Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (3): 1286-1297.doi: 10.16431/j.cnki.1671-7236.2024.03.041

• Basic Veterinary Medicine • Previous Articles    

Co-transmission Characteristics of blaCTX-M-65 and mcr-1 Genes in Escherichia coli Isolated from Diarrhea Piglets in Guangdong Province

ZHANG Yan1, CAO Mengtao1, LU Yuewei1, JIANG Hongxia1, WANG Ling2, LI Bin3   

  1. 1. Guangdong Key Laboratory of Veterinary Drug Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
    2. Guangzhou Animal Health Supervision Institution, Guangzhou 510440, China;
    3. Equine Disease Free Zone Management Center of Conghua District, Guangzhou City, Guangzhou 510900, China
  • Received:2023-08-14 Published:2024-02-27

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Abstract: 【Objective】 This study was aimed to investigate and analyze the drug resistance of diarrheal piglets in a pig farm and the molecular characteristics and the co-transmission mechanism of blaCTX-M-65 and mcr-1 drug resistance genes positive Escherichia coli (E. coli) in Guangdong province,so as to provide scientific basis for drug resistance monitoring and risk prevention and control.【Method】 E. coli strains were isolated and identified from 90 intestinal samples of diarrhea pigs in a pig farm in Zhaoqing,Guangdong province.ESBLs and mcr-1 genes were detected by PCR,and subtyping of CTX-M ESBLs was determined by sequencing.The minimum inhibitory concentration (MIC) of strains to 12 antibiotics was performed using agar double dilution method and broth microdilution method.Genetic relatedness of blaCTX-M-65 and mcr-1 genes co-carrying strains was analyzed by pulsed field gel electrophoresis (PFGE) and multiple locus sequence typing (MLST).The ability of plasmid horizontal transfer was determined by conjugation transfer,and plasmid replicon types were detected by PCR-based replicon typing.The localization of blaCTX-M-65 and mcr-1 genes and the size of plasmids were determined by S1-PFGE and Southern blotting.The recombination event of plasmid was determined by whole-genome sequencing and bioinformatics analyses.【Result】 A total of 86 strains of E. coli were isolated and identified.44 strains carried CTX-M ESBLs genes,blaCTX-M-55 gene was the predominant subtype (n=16,36.4%),followed by blaCTX-M-65 (n=10,22.7%),blaCTX-M-27 (n=8,18.2%),blaCTX-M-15 (n=5,11.4%),blaCTX-M-79 (n=2,4.5%),blaCTX-M-14 (n=2,4.5%) and blaCTX-M-24 (n=1,2.3%).Of 10 blaCTX-M-65 positive strains,8 were confirmed co-carrying mcr-1 gene,presenting multidrug-resistance phenotypes,including resistance to gentamicin,florfenicol,aztreonam and other antibiotics.The 8 strains were divided into 6 ST types,contained 7 PFGE profiles.A total of 6 transconjugates co-carrying blaCTX-M-65 and mcr-1 genes were obtained.The blaCTX-M-65 and mcr-1 genes in all 6 conjugates were located on the IncHI2 (253 kb) plasmids.The resulting 323 kb plasmid was a fusion of a 253 kb IncHI2 plasmid and a 69 kb IncFⅡ plasmid,which was formed during conjugation transfer.Sequence analysis of plasmids revealed that IS26 mediated the recombination of the IncHI2 plasmid and the IncFⅡ plasmid.【Conclusion】 IncHI2 plasmid mediated the widely spread of blaCTX-M-65 and mcr-1 genes.IS26 led to the fusion of IncHI2 plasmid and IncFⅡ plasmid through integration with the target site GTTTCACT.Such recombination events of plasmids played an important role in expanding the antibiotic resistance spectrum,accelerating the spread of resistance genes,making multidrug resistance more serious and posing a threat to public health security.This study provided a basis for elucidating the transmission mechanism of multi-drug resistant E. coli.

Key words: Escherichia coli; blaCTX-M-65; mcr-1; IS26; plasmid fusion

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