The Analysis Of Antimicrobial Resistance And The Plasmid Mediated Resistant Genes In Salmonella Typhimurium Isolates

Salmonella enterica serovar Typhimurium(S.Typhimurium)is one of the most significant foodborne zoonotic pathogens.It can result in sporadic outbreaks almost every year especially in developed countries such as Europe and America.The origin of S.Typhimurium is widely and it can cause infection between animals and human through the food chain.Antimicrobial resistance and multi-drug resistance of S.Typhimurium has become an increasing problem worldwide,favoured by overuse or misuse of antibiotics.In addition,the spread of resistance genes is one of the reasons for the emergence of resistant strains.The resistance genes is usually located on the moble element.Plasmid carrying resistant determinant factor provides an important way to spread drug resistance.This may take huge hidden dangers to public health.However,there are few studies on antibiotic resistance and plasmid mediated resistance genes of S.Typhimurium in China.In order to analyze the resistance of S.Typhimurium isolates,antimicrobial susceptibility testing to 18 antimicrobial agents of 330 strains was performed using the broth microdilution method.Investigation was carried out to analysis the prevalence of PMQR in these 330 S.Typhimurium isolates.A mcr-1 positive isolate was identified from 330 strains and further studies were implemented such as whole genome sequencing and the conjugation experiments.1 Antimicrobial resistance of S.Typhimurium isolates of different originsAntibiotic susceptibility of the isolates was tested by the broth microdilution method,using parameter of minimum inhibitory concentration(MIC).The results of antibiotic susceptibility to 18 antimicrobial agents revealed that the antimicrobial agents with higher occurrence of resistance was to amoxicillin/clavulanic acid(75.8%),tetracycline(73.9%)and ampicillin(73.6%).The isolates displayed resistance to streptomycin,chloramphenicol,cephazolin,nalidixic,trimethoprim-sulfamethoxazole and colistin with rate all over 50%.They showed lower resistance to aztreonam,kanamycin,gentamicin,enrofloxacin and olaquindox.About 90%of the isolates showed sensitive to ciprofloxacin and nitrofurantoin.Almost all of the strains showed sensitive to amikacin and meropenem.S.Typhimurium isolates in different origins of breeding,slaughterhouse,market and human beings showed similar resistance to amoxicillin/clavulanic acid,tetracycline,ampicillin,chloramphenicol,kanamycin,ciprofloxacin,meropenem,amikacin and nitrofurantoin.But for the remaining 8 antimicrobial agents,the resistance performance was different.In each origin,the S.Typhimurium isoaltes share the similar antimicrobial resistance to 18 antimicrobial agents with the total strains.The resistance of S.Typhimurium isolates in breeding process were higher than in the other three processes.The second was the market processs,and the resistance rates of strains in slaughter and human beings were similar.260 of 310 isolates showed resistance to at least 3 antibiotics and up to 83.9%,implied that multiple antimicrobial resistance in S.Typhimurium isolates was a common phenomenon.The highest multi-resistance rate was observed in market process(100%),whereas the lower level of multi-resistance rate were in breeding process(99.0%)and human beings(73.9%),and the lowest level was in slaughter process(70.1%).The monitoring of S.Typhimurium isolates in the desired pig farm showed that the MICmin and MICso value of isolates were generally higher than those of total isolates.Therefore,the resistance of S.Typhimurium isolates in the desired pig farm was very serious during that period.2 Prevalence of PMQR genes in S.Typhimurium isolatesThe presence of PMQR genes was tested by PCR.The positive rate of PMQR genes was 51.2%.Among all the PMQR genes,the highest rates were 29.7%of oqxAB,followed by aac(6')-Ib-cr and qnrS(19.4%and 10.9%respectively).qnrA,qnrC and qnrD genes were not been detected.The positive rate of oqxAB was significantly higher than that of other PMQR genes,especially in isolates from pigs.The qnr gene subtypes detection showed that both qnrB and qnrS had 2 subtypes.The occurrence of qnrS along with aac(6')-Ib-cr in a same strain was the most common phenomenon and oqxAB along with aac(6,)-Ib-cr was also common.Correlation analysis of genotype and phenotype of PMQR positive strains showed that the strains carrying PMQR gene were accounted for 73.6%among quinolone resistance S.Typhimurium isoaltes.We also examined the level of decreased ciprofloxacin susceptibility(DCS,defined as a ciprofloxacin MIC of 0.12-1?g/mL).Almost all the PMQR positive strains showed decreased sensitivity to ciprofloxacin.3 Identification of the plasmid-mediated colistin resistance gene mcr-1 in S.Typhimurium isolatesThe prevalence of mcr-1 was investigated by PCR and only one strain carried with mcr-1 was identified from 330 isolates.Futher study was carried out by whole genome sequencing.This strain was completely sequenced,which revealed it 5,232,290 bp in size with an average GC content of 51.4%.Futher study showed that this strain possessed five backbones:IncHI2,IncX1,IncI2,IncHI2A and IncQ1.This revealed that the strain may carry more than one plasmids.The plasmid carrying mcr-1 was transfered to an E coli recipient by conjugation.Transconjugant showed the resistance to colistin(8 ?g/mL)and streptomycin(64 ?g/mL).No other antimicrobial resistance was co-transferred with the donor strain to the recipient strain.The resistance gene of streptomycin along with mcr-1 gene transfered together to the recipient.Due to the presence of this plasmid,the isolate showed decreased sensitivity to ciprofloxacin,enrofloxacin and nalidixic acid,compared with the recipient strain with the value of MIC increased 8-16 times respectively.