| Food-borne pathogens could cause disease to human and animals,which is one of the most important problems for food safety.The abuse of antibiotics in animal industry could cause increase of drug resistance rate.At the same time,the antibiotic genes could horizontally tranfer to other bacteria,which is a big threat to livestock and poultry industry chain.The cold fresh chicken industry in China develops slowly and production technology is backward.In the process of production and marketing,chilled chicken is very easily contaminated by microorganism.Thus,it is of vital practical significance to evaluate the microbial contamination of chilled chicken and analyse the drug resistance situation for Escherichia coli from chilled chicken.This study used chilled chicken form market in six regions of Zhejiang province as raw material,in order to investigate microbial contamination level and analysis drug-resistance of Escherichia coli from chilled chicken.The study was divided into three parts.Firstly,microbial contamination index was detected in order to assess the microbial contamination level of chilled chicken.Secondly,Escherichia coli strains were isolated and their drug resistance type,antibiotic genes and virulence genes were tested.Lastly,13 typical Escherichia coli strains were sequenced.Based on the result of whole genome sequencing,their antibiotic genes,serotypes and plasmids were predicted and multi-locus sequence typing were conducted.This study collected 129 chilled chicken in total from six regions(NingBo,HangZhou,ShaoXing,HuZhou,JiaXing and JinHua)both in agricultural markets and supermarkets.55 chilled chicken was collected from supermarkets and 74 from agricultures.According to methods from National Food Safety Standard,aerobic plate count,coliforms,moulds amd yeasts were detected.The results show that the aerobic plate count was detected at five to eight ordersofmagnitude.The microbial pollution levels from minimum to maximum were ShaoXing,NingBo,HangZhou,HuZhou,JinHua and JiaXing.Among of that,there was no significant difference between agricultural market and supermarket in May and June.However,there was siginificant difference between two kinds of markets in July and August.The average contamination level of coliforms of all the sources was more than 8.52 ×104MPN/g.The pollution level of moulds was between three and five ordersofmagnitude,which was more than the pollution level of yeasts.There were no significant difference between agricultural market and supermarket regarding moulds and yeasts.This study isolated 133 Escherichia coli strains and their drug-resistance,antibiotic genes and virulence genes were tested.The positive rate of Escherichia coli strains from agricultural samples was JinHua(100%),NingBo(78.6%),ShaoXing(54.5%),HuZhou(54.5%),JiaXing(41.2%)and HangZhou(40%)respectively.While the positive rate of Escherichia coli strains from agricultural samples was JinHua(92.9),NingBo(75%),JiaXing(71.4%),ShaoXing(50%),HangZhou(33.3%)and HuZhou(30.8%).Regarding the results of drug resistance type,the Escherichia coli strains showed highest percent of drug resistance to ampicillin and the percent was 63.85%.The drug resistance rate of sulfamethoxazole/trimethoprim is 55.39%.The drug resistance rate of other antibiotics such as cefazolin(30%),ceftriaxone(26.16%),aztreonam(15.39%),ciprofloxacin(23.85%),levofloxacin(16.93%)and gentamicin(14.62%)was 30%,26.16%,15.39%,23.85%,16.93% and 14.62,respectively.All the isolated strains were sensitive to piperacillin,ertapenem and tigecycline.Both the strains from agricultural market and supermarket showed more than 50% resistance to ampicillin and sulfamethoxazole/trimethoprim and less than 40% to other antibiotics.As for the multi-drug resistance,36.1% of the isolated strains showed resistance to more than 3 antibiotics.Four strains showed drug resistance to 6 kinds of antibiotics,that is,penicillins,cephalosporin,aminoglycosides,quinolones,tetracyclines and sulfonamides.Regarding the detection results of antibiotic genes,the positive rate of quinolones genes,such as qnrS,aac(6')-Ib,oqxA and oqxB was 56.4%,33.8%,25.6% and 17.3%,respectively.The positive rate of cephalosporin genes,such as TEM,CTX-M-2,CTX-M-9 and CTX-M-1was41.4%,37.6%,27.8% and 13.5%,respectively.The positive rate of ?-lactam antibiotics genes,such as ompCA and ompFA was 75.2% and 57.1%,respectively.The positive rate of aminoglycosides genes,such as aphA30,aadA and aacC2 was63.2%,47.4% and 16.5%,respectively.The positive rate of tetracyclines genes,such as tetC,tetM,tetX,tetG and tetO was 9%,3.6%,4.5%,3% and 2.6%,respectively.The positive rate of sulfonamides genes,such as sul?and sul? was 77.4% and 87.2%.As for the detection results of virulence genes,the gene eae and stx were negative.The positive rate of est,elt,ipaH and aggR was about 10%.Thirteen Escherichia coli strains were sequenced and the reults of whole genome sequencing were analysed.Several kinds of antibiotic genes of 12 strains were predicted including the genes of aminoglycosides,?-lactam antibiotic,tetracyclines,trimethoprim and sulfonamides.The plasmids were predicted positively in nine strain and these plasmids could match with other plasmids from bacteria of Enterobacteriaceae.The predicted plasmids of six strains could match with the replicon of predicted genes,which verified that the plasmid containing antibiotic genes play a key role in the spread of drug resistance.Among thirteen strains,10 kinds of O serotype and 9 kinds of H serotype were predicted.The serotype O174,O9,H4 and H10 showed more than twice in the results.It showed that the serotype of 13 strains was quite different.The results of MLST based on seven housekeeping genes showed that the strains from HangZhou,HuZhou and ShaoXing had close genetic relationship.Strains from NingBo and JinHua had close genetic relationship.The strain from JiaXing was different from others. |
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