Characterization Of Extended-spectrum Beta-lactamase(ESBL)-Producing Escherichia Coli Isolated From Bovine Mastitis

Bovine mastitis is the most prevalent disease in dairy herds,causing huge economic losses to the dairy industry by deteriorating quality and quantity of milk.It is one of the most common reasons to use antimicrobials in dairy animals.However,microbes have self-developed or acquired tools to protect themselves from the deleterious effects of antimicrobial agents by making them inactive by various means such as inactivation of ?-lactam drugs by extended-spectrum beta-lactamases(ESBL)enzymes.The prevalence of pathogenic multi-drug resistant ESBL-producing Escherichia coli is rapidly increasing,thus becoming a global concern.Furthermore,the current increasing prevalence of colistin resistance mcr-1 in E.coli isolated from food-producing animals like pigs and poultry is quite worrisome.In veterinary context,there is paucity of data regarding the occurrence of ESBL-,carbapenemase-producing,and plasmid-mediated colistin-resistant(mcr-1)E.coli in dairy herds suffering mastitis,especially from swiftly developed huge daily herds of China.Therefore,this study was designed with the objectives to determine:1)the prevalence of ESBL-producing E.coli in cattle suffering clinical mastitis and determination of various types of ESBL encoding genes using phenotypic viz.,double-disc synergy test and PCR assays,2)the occurrence of colistin-resistant E.coli and detection of mcr-1 genes among the isolated E.coli strains,3)the antimicrobial susceptibility profiles of the ESBL-and mcr-1-positive E.coli by disc diffusion and broth microdilution(minimum inhibitory concentration)assays,4)detection of phylogenetic and integrons groups,gene cassettes and insertion sequence(IS)by PCR,restriction fragment length polymorphism(RFLP)and sequence analysis,and 5)molecular epidemiological study by PCR-based plasmid replicon(PBRT),multilocus sequence typing(MLST),and multilocus sequence analysis(MLSA),and recombination,eBURST and geoBURST analysis of the isolated ESBL-and mcr-1-positive E.coli from dairy cows suffering bovine mastitis.In the current study,a total of 49(20%)ESBL-producing E.coli out of 249 E.coli isolates(12%)were recovered from milk samples(n = 2038)of dairy cows with clinical mastitis from 73 dairy herds located in 16 different provinces of China during 2015 to 2017.Nucleotide sequence analysis of PCR amplicons revealed that blaCTX-M was the predominant ESBL gene detected in 44(90%)isolates,with blaCTX-M-15 being the major(77%)allele encoding for ESBL enzymes,followed by baCTX-M-55(9%),blaCTX-M-14(9%),blaCTX-M-3(5%),blaCTX-M-1(2%).Also,22(45%)and 8(16%)of the ESBL-producing isolates were carrying b/aTEM and blaSHV genes,respectively,in singlet or in combination.However,no carbapenemase-producing E.coli was detected in this cohort study.Furthermore,the prevalence of colistin-resistant mcr-1-carying E.coli was observed to be 2%(n = 5)among the 249 E.coli recovered mastitic-milk samples.The results of phylogenetic analysis showed that majority of these isolates belonged to phylogroup-A(74%),followed by-D(20%),-B1(6%)and-B2(4%).Strikingly,all the ESBL-and mcr-1-positive isolates were found to be multidrug resistant phenotypes showing high resistance to cephalosporins including the 4th generation cefepime and other common non-?-lactams such as aminoglycosides,fluoroquinolones,tetracyclines and sulphonamides,etc.Additionally,class-1 integrons were detected in majority of recovered ESBL-and mcr-1-positive isolates(83%).Molecular analysis of class-1 integrons variable regions indicated that they were carrying up to five different gene cassettes with a predominant combination of dfrA17-aadA5 genes in tandem,conferring resistance to aminoglycosides and trimethoprim.Interestingly,66%of the ESBL isolates were also carrying insertion sequence common region 1(ISCR1)which was found to be associated with most of the CTX-M genes.PBRT results revealed that the resistant determinants were carried on conjugative plasmid of diverse incompatibility groups(IncF was the most dominant(n = 46),while IncL/M was the least(n = 1)common).In addition,transconjugation experiments with randomly selected twelve ESBL-producing and five mcr-1-positive E.coli indicated successful transconjugation,which was verified phenotypically and genotypically.MLST of ESBL-producing E.coli showed a total of 28 sequence types(STs)with ST410 representing 10%of the isolates and nine STs(32%),that were not assigned to any STs,were identified reflecting the extent of diversity.MLSA revealed two lineages in the population,comprised of a dominant lineage containing most of the isolates.Split-decomposition analysis revealed intraspecies recombination clearly contributed in genetic recombination generating genotypic diversity among the isolates,and lack of interspecies recombination.Additionally,MLST of mcr-1 isolates indicated a total of four STs with two isolates of ST 1290,while three isolates were not assigned any ST,which indicates novel STs.MLSA showed evolutionary connection of mcr-1 carrying isolates with our ESBL-producing E.coli raising concerns of fast dissemination between similar species.Population structure analysis indicated that only ST1290 was found linked to other global STs.In conclusion,the current study reports on the high occurrence of ESBL-producing E.coli,particularly blaCTX-M-15,carrying clinical class-1 integrons and ISCR1 elements are likely indicative of their rapid and wider dissemination,posing threats to veterinary and public health.This study also indicates that dairy cattle might act as an important source of ESBL-producing and mcr-1-carrying.coli to the human beings via food chain or environment.In addition,this study will make a baseline data and may contribute to increase understanding of the molecular epidemiology of ESBL-producing and colistin-resistant E.coli in dairy cattle of China and their susceptibilities to both ?-lactam and non-?-lactam antimicrobial agents.Moreover,this study provides deep insights into the various mechanisms which help in the wide spread of antimicrobial resistance genes via horizontal gene transfer like plasmids,integrons,gene cassettes and IS elements.This coherent analysis on the drug resistance profile and genetic structure of pathogenic E.coli population isolated from mastitic-milk weaponised with resistance elements from a big and dynamically developing country will be invaluable contribution in the epidemiology,surveillance and understanding global diversity of this world wide distributed pathogen.