Antimicrobial Susceptibility Analysis And ESBLs And Enterotoxin Gene Detection Of Staphylococcus Aureus Isolated From Animal Farms In Chongqing

Staphylococcus aureus belongs to the genus Staphylococcus,and it is an important zoonotic gram-positive bacterium.It exists in the respiratory tract,digestive tract and body surface of animals and humans widely.S.aureus has variety ways of infection,different routes of infection lead to clinical symptoms are also different.The gastrointestinal infections can cause food poisoning and gastroenteritis.By body surface infection can cause suppurative diseases,and can worsen as sepsis.Respiratory tract infections can cause bronchitis and pneumonia.It often cause diseases such as mastitis,arthritis,omphalitis,local abscess and so onin veterinary clinics.It also can cause human skin suppuration and sepsis,and the production of enterotoxins is one of culprits which can cause human food poisoning.According to statistics,food poisoning caused by S.aureus enterotoxin in the United States accounts for 33%and Canada accounts for 45%,causing serious public health problems.Since the first methicillin-resistant S.aureus?MRSA?was detected by Eriksen in 1961,more and more reports on the resistance of S.aureus to various antimicrobial agents have been reported so far.It has been appeared resistant strains that resistance to ciprofloxacin,erythromycin,gentamicin,tetracycline,vancomycin.Especially the multi-drug resistance is more and more serious.So that leading to a series of public health problems that have become one of the focuses of the world attention.In order to understand the resistance,the basic information of carrying Extended-spectrum?-lactamases?ESBLs?and enterotoxin genes of animal-derived S.aureus in Chongqing.A total of 1371 samples were collected from some pig farms,chicken farms,dairy farms,goat farms and rabbit farms in Chongqing from 2014.03 to 2017.12.The samples were pretreated and then added to Mueller-Hinton high salt medium for enrichment,streaked in mannitol sodium chloride plate,pick up the suspected colonies in LB liquid medium for expansion culture,followed extraction the genome of bacterial by boiled as the PCR template.The S.aureus specific gene nuc was amplified from the suspected colonies.According to the American Society of Clinical and Laboratory Standards?CLSI?Antimicrobial Susceptibility Test Standard,a drug sensitive test was conducted by K-B disk diffusion test for clinical isolates identified as S.aureus to detect the resistance of S.aureus in animal origin in Chongqing.And extracted plasmid by kit,then detected whether the strains carrying ESBL genes of bla_TEM?bla _CTX-M?bla_SHV?bla_OXA-2?bla_OXA-10?bla_VEB?bla_PERER and bla_GESby PCR.Sequenced the production of PCR and analyzed with DNAstar software to determine the subtype of the ESBL gene.To detect 20 enterotoxigenic of isolates by multiple-PCR,to understand the genotypes of enterotoxins in animal origin of S.aureus in Chongqing.Eighty-nine S.aureus clinical strains were isolated and identified from 1371 samples.The total isolation rate is about 6.5%.The highest isolation rate is in rabbit farm about 15.2%.And the lowest isolation rate is in cattle farm about 1.7%.The 89 isolates including 25pig-associated strains,32 chicken-associated strains,6 dairy cattle-associated strains,10goat-associated strains,and 16 rabbit-associated strains.The isolation rates were 7.3%,6.7%,1.7%,11.4%,and 15.2%.The highest isolation rate of S.aureus in the sample was 22.6%in Dazu district,followed by Beibei district,Changshou,Dianjiang,Shapingba,Rongchang,Bishan,Pengshui,and Yubei.The isolation rates respectively were:11.4%,7.3%,6.7%,6.7%,6%,5.2%,4.6%,and 2%,and the lowest isolation rate is in Hechuan about 1%.All isolates were resistant to two or more tested antimicrobials.The isolates were most resistant to penicillins,and also more resistant to tetracyclines,macrolides,and quinolone.The resistance of swine and chicken strains was more severe than that of sheep,cattle,and rabbit strains.Bacterial resistance spectrum in Beibei,Rongchang and Dianjiang regions was wider than other regions.All isolates were multi-drug resistant strains and resistant to 1 to 21antimicrobials.The most prevalent multi-drug resistant strains were pig-associated.On the contrary,rabbit-associated strains were the most sensitive isolates.The multi-drug resistance of the strains in Beibei,Bishan,Dazu,Pengshui,Yubei,and Changshou areas was mainly below 10,while the strains in Rongchang and Shapingba areas were mainly resistant to more than 10 drugs.And the distribution of multi-drug resistance is extensive in strains from Pengshui and Hechuan areas,and the multi-drug resistance in Pengshui area varies from 3-18.ESBL gene detection results showed theat only TEM-type ESBL gene detected,and didn't detect other 7 ESBL genotypes.Sequencing analysis revealed that all the TEM were bla_TEM-1aEM-1a subtype,and 86 strains carried the gene with a carrying rate of 96.6%.A total of 88 strains of enterotoxigenic genes were detected by multiplex PCR in 89 S.aureus,the positive rate was 98.9%.In this study,30 strains detected SEA-SEE traditional enterotoxin gene,mainly sec and sed.88 strains detected a new type of enterotoxin.The main circulating enterotoxin genes were seg,sei,tst-1.This test hasn't detected selq gene.The gene of sea and seb were only detected in chicken strains and bovine strains,and serl gene was detected only in bovine strains and rabbit strains.The gene of seg,sei,selj,selk,selo,tst-1,and sell were all detected in five source strains.Detection of S.aureus enterotoxin genes in different regions shows a certain concentration trend.There is a certain difference between different animal strains of carry enterotoxin gene.The chicken-associated strains carry enterotoxigenic up to 17 kinds of specie,and rabbit-associated strains carry 11 kinds of specie as the fewest.From each strain carrying enterotoxin gene situation,only one rabbit-associated strain did not detect any enterotoxin gene,and a pig-associated strains carried enterotoxigenic up to 10.The other strains carrying enterotoxin gene range from 1-9.The results of this experiment showed that the overall drug resistance of isolates was serious,and the resistance rate to penicillins was more than 90%.And the multi-drug resistance was serious.Among them,the resistance of swine-asociated strains were more serious than other animal-derived strains,and the multi-drug resistance in Rongchang and Shapingba areas was serious.ESBL gene test results showed that S.aureus widely carried bla_TEM-1aEM-1a ESBL gene in Chongqing.Enterotoxin gene carrying was more serious.The main epidemic toxins gene were seg,sei which were new enterotoxin gene.And the detection rate of traditional enterotoxin genes was relatively small.