Study On The Drug-resistant Gene Bla_CMY-2 Of Escherichia Coli Isolates Of Chicken Origin And Salmonella Isolates Of Pig Origin In Shandong Province

Cephalosporins are one of the most efficacious antibiotics which are usually used to treat infections caused by Escherichia coli and Salmonella. Unfortunately, drug-resistant strains are emergencing consistently in accordance with the illogical use or even abuse of antibiotics clinically. The appearance of drug-resistant strains, especial the appearance of cephalosporin-resistant strains has caused a huge threat against the safety of animal food and the health of human beings. Correlative studies demonstrated that the predominant mechanism of bacterial resistance to cephalosporins is the production ofβ-lactamases, which can effectively hydrolyze cephalosporins. CMY-2-type AmpCβ-lactamase encoded by the blaCMY-2-gene harbouring in plasmid is the most prevalent and damagingβ-lactamase all over the world. In the present study, 15 E.coli isolates of chicken origin and 6 Salmonella isolates of pig origin recovered from Shandong province were demonstrated to be positive for blaCMY-2 by PCR detection. Then Antimicrobial susceptibility testing were carried out to determine the susceptibility of the blaCMY-2-positive isolates to the selected antibiotics, especially to the 1st-, 2nd-, 3rd-, 4th-generation cephalosporins. Besides, certain characterizations of these isolates were also studied in order to provide theory basis and research foundation for controlling colibacillosis of chickens and salmonellosis of pigs caused by cephalosporin-resistant isolates. This study was divided into three parts:PartⅠPCR detection of blaCMY-2 gene and DNA sequence analysis With the specific primers for blaCMY-2 gene, PCR analysis was performed on plasmid DNA of E.coli and Salmonella isolates to detect all the isolates. The results of PCR demonstrated that 15 (11.9%) of 126 E.coli and 6 (3.8%) of 156 Salmonella isolates were positive for blaCMY-2 gene. Besides, another pair of primers was designed to amplify the entire blaCMY-2 gene. DNA sequence analysis showed that all the DNA sequences were identical to the published blaCMY-2 sequences, except for isolates JN10 and S103 which harboured a substitution of thymine at position 1026 by adenine and a substitution of thymine at position 482 by cytosine, respectively.PartⅡAntibiotic resistance phenotype testing of the blaCMY-2 gene-positive isolatesThe Kirby-Bauer disk diffusion method and microdilution method were carried out to determine susceptibilities of the 15 and 6 blaCMY-2 gene-positive E.coli and Salmonella isolates to selected antibiotics according to Clinical and Laboratory Standards Institute (CLSI) recommendations, respectively. Susceptibility results showed that all the blaCMY-2 gene-positive isolates were completely resistant to first-generation cephalosporins cefalexin, cefradine, cefalothin, cefazolin; second-generation cephalosporin cefaclor; third-generation cephalosporin cefixime. All isolates were intermediate to two second-generation cephalosporins cefamandole and cefuroxime except for several isolates which were resistant to them. Besides, the blaCMY-2 gene-positive isolates were also resistant to ampicillin, streptomycin, tetracycline, chloramphenicol, amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole. Three (JN5, JN9 and HZ9) E.coli isolates were resistant to two third-generation cephalosporins ceftriaxone and ceftazidime. Three (S90, S103 and S121) and four (S90, S103, S121 and S138) Salmonella isolates were resistant to two third-generation cephalosporins ceftriaxone and ceftazidime, respectively. However, all isolates were sensitive to fourth-generation cephalosporin cefepime. It was demonstrated in the present study that all the blaCMY-2 gene-positive isolates were multidrug-resistant strains, and their antibiotic resistance phenotypes were alike. PartⅢDetection of enzyme-type of blaCMY-2 gene-positive isolates and study on transfer of the blaCMY-2 geneIn the present study, three dimension enzyme-inhibition test was carried out to determine the enzyme-type of these blaCMY-2 gene-positive isolates. Besides, plasmid conjugation test and transformation experiment were carried out to determine whether the blaCMY-2 gene could be transferred between same or different species. The results showed that the blaCMY-2 gene could be successfully transferred not only between same species, but also between different species, but the latter one was more difficult to happen. The transfer of plasmid harbouring drug-resistant gene greatly accelerated the dissemination of bacterial resistance, which brought great threat to the health of human beings.