| Bovine mastitis is the most serious disease in the dairy industry,which affects the most widely,is the most difficult to cure,and has a huge impact on the agricultural economy all over the world.Escherichia coli is one of the most important pathogens causing mastitis in dairy cows.With the widespread and abuse use of the third generation cephalosporin in clinical practice,the rapid spread of cephalosporin-resistant Escherichia coli between humans and animals,leading to the chronic mastitis in dairy cows,which brings huge social and economic loss to the breeding farms.The production of ESBLs(broad spectrum ?-lactamase)are one of the most important enzyme for E.coli with the resistance to the third cephalosporin antibiotics,and blaCTX-M genes are the highest prevalence and the heaviest proportion of ESBLs.The purpose of this study is to explore the molecular mechanism of the epidemic spread of the E.coli resistance to cephalosporin from bovine mastitis in Henan province.Monitoring the detection rate of blaCTX-M strains,the resistance phenotypes and subtypes of blaCTX-M genes.Through conjugation experiments,plasmids incompatibility analysis,pulsed-field gel electrophoresis,to provide theoretical basis for rational use of drugs in clinic.From March to September 2019,a total of 180 clinical mastitis milk samples were taken from 8 dairy farms in total from 7 regions in Henan.70 Escherichia coli from bovine mastitis were isolated,purified and identified,with a rate of 38.8%.All of the E.coli isolates were tested for their antimicrobial susceptibility by using the microbroth dilution methods,based on the criteria of the Clinical and Laboratory Standards Institute(CLSI2017).The following fourteen antimicrobial agents were used:Ciprofloxacin,enrofloxacin,florfenicol,doxycycline,ticacycline,amikacin,gentamicin,colistin,ampicillin sodium,ceftiofur,cefotaxime,ceftazidime,cefoxitin,meropenem.The drug resistance rate of ?-lactam antibiotics was the highest,and the drug resistance rate of ampicillin sodium,ceftiofure and cefotaxime were all more than 60%,while the drug resistance rate of cefoxetine was only 20%,followed by doxycycline(27.14%)and flufenicol(11.43%).The isolates of bovine mastitis were all sensitive to quinolones,polymyxin,aminoglycosides amikacin and gentamicin relatively.The resistance genes and their subtypes were determined by PCR and sequencing.A total of 34 strains of blaCTX-M positive bacterias were obtained,and the detection rate was as high as 48.57%among the 70 isolates.Six subtypes of CTX-M were identified by sequencing,among which CTX-M-55 was at most(14/34),followed by CTX-M-15(8/34),CTX-M-97(6/34),CTX-M-65(2/34),CTX-M-14(2/34)and CTX-M-3(2/34).Other resistance determines were also detected among the cephalosporin-resistant isolates,two blaNDM-1 and one blaCMY-2 located on different isolates respectively.Among the blaCTX-M strains,four strains also carried floR which resistant to florfenicol,and one carried rmtB resistant to aminoglycosides.All isolates with blaCTX-M were conjugated with rifampicin-resistant E.coli C600,and 22 strains were successfully conjugated,with a conjugation rate of 64.7%.Twenty-two transconjugants and their donor strains were tested from plasmid incompatibility.The results showed that most of the donor strains contained multiple plasmid types,and the transconjugants contained only one plasmid respectively.IncFII and Incll were carried on the most of the donors,and IncI1 was most in transconjugants.The results of pulsed field electrophoresis gel typing showed that 28 of the 34 blaCTX-M positive strains were successfully typed,and 18 different electrophoresis types were obtained,indicating that ESBLs strains were mostly horizontal transmission and had a lot of genetic diversity.Further S1-PFGE-typing and plasmids sequence analysis of Escherichia coli HM03 carrying blaCTX-M-14 and rmtB genes showed that blaCTX-M-14 was located on the IncI1/ST80 plasmid,forming the classic structure I SEcp1-blaCTX-M-14-IS903 which is co-transferred with blaLAP-2-qnrS1.rmtB was located on the IncFII plasmid and constitutes a common gene module with qepA.This plasmid carried a large multidrug-resistant region containing multiple drug-resistant genes,promoted the formation and spread of multi-drug resistant bacterias.In conclusion,E.coli producing ESBLs in this study was widely prevalence in bovine mastitis,subtypes of CTX-M were prevalence diversity,and most of them could be transmitted horizontally by conjugating plasmids.PFGE was conducted to classify the blaCTX-M producing E.coli from bovine mastitis,and it was found that there was clonal transmission among the strains,especially those in the same area.Multidrug resistance plasmids promote the rapidly spread of blaCTX-M E.coli in mastitis cows poses a threat to both dairy farms and humans,so timely surveillance is imperative to control and prevent the spread of the pathogenic bacterias. |
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