Selection And Application Of Rapid Salmonella Detection Method,and Spreading Features Investigation Of Chicken And Pig-origin Salmonellae By MLST Typing And Antibiotic Resistance Detection

Salmonella is an important zoonotic agent and a vehicle for antibiotic resistance genes.It can infect animals and humans,causing salmonellosis.Animal-derived food,especially from birds and pigs,is the main source of salmonellosis.It is of great significance to learn the status of salmonella contamination in animal-origin food for controlling the spread of salmonella,which could reduce the potential safety hazard to public health.In the present study,we developed a PCR method for rapid detection of salmonella,and make it a standard for clinical diagnosis of salmonella.With the new method,we isolate and identify several salmonella strains from clinical samples.Features including serotypes,antibiotic resistance,and multilocus sequence typing(MLST)were further investigated to all isolates.Based on these epidemiological studies,we could give a guide for Salmonellae prevention and control measures.1.A rapid detection method of salmonellaAlthough the national standard GB4789.4-2010 gives a conventional method for the isolation and identification of salmonella,we establish a new standard,which normalized clinical samples collection,medium selection and enrichment methods,in order to facilitate the quick detection of salmonella from the animal-origin samples.PCR method were used in the standard for shortening the time of clinical diagnosis,which the enrichment time in conventional method was shortened to 4-6h,more than that,the total 2-3 days originally was compressed to within 1 day by eliminating the conventional biochemical identification steps.2.Antibiotic phenotypic and genetic resistance and MLST molecular typing of salmonella isolates from chickens and pigsTotal 134 isolates from chicken and swine samples in Jiangsu area were tested with serotypes,antibiotic resistance to 14 kinds of antibiotics commonly used in clinical(including ampicillin,amoxicillin,cefotaxime and cefepime,cefoperazone,ceftriaxone,gentamicin,kanamycin and nalidixic acid,amikacin,ciprofloxacin,tetracycline).The resistant genes?-lactamase gene,sull,aacC4,aac(6')-1b and floR,etc.,which locate on the gene island-1(SGI),were also tested.With MLST,we try to analyze the Salmonella epidemic and resistant characteristics in the food chains including chicken industry and swine industry.Results showed that 134 isolates were identified to 4 serotypes:S.Enteritidis,S.Indiana,S.Derby and S.Pullorum.The isolates from healthy chicken farms were S.Enteritidis(36 strains)and S.Indiana(40 strains),and from healthy pig farms were S.Derby(14 strains)and S.Indiana(2 strains).The isolates from the ill chicken in Jiangsu and its surrounding area,which were collected from Yangzhou University veterinary hospital,included S.Enteritidis(31 strains),S.Indiana(6 strains)and S.Pullorum(5 strains).With MLST,the 134 isolates could be divided into 6 types ST:ST11,ST17,ST92,ST40,ST64 and ST155.All S.Enteritidis isolates belonged to ST11.S.Indiana isolates,ST17,S.Pullorum isolates,ST92.S.Derby isolates could be divided into three STs,ST40,ST64 and ST155.The results showed that the isolates were highly resistant to nalidixic acid,ampicillin,tetracycline and cefoperazone,accounting for 92.5%,80.7%,72.5%,and 65.1%,respectively.Isolates of S.Enteritidis were highly resistant to nalidixic acid,ampicillin,tetracycline and cephalosporin cefoperazone,accounting for 94.2%,89.8%.62.3%,and 57.9%,respectively.The swine-origin isolates,S.Derby mainly,were highly resistant to tetracycline,nalidixic acid,sulfamethoxazole,and chloramphenicol,accounting for 100%,94.7%,68.4%,and 57.8%,respectively.The isolates of S.Indiana showed the highest resistant,of which about more than 75%isolates showed resistant to 11 antibiotics at least,and about 50%isolates were resistant to all 14 kinds of antibiotics.48 strains of S.Indiana produced a total of 27 kinds of drug resistant profile,including 41 strains were resistant to more than 9 antibiotics,the other 7 strains were resistant to all antibiotics;67 strains of S.Enteritidis produced a total of 20 kinds of drug resistant profile,2 strains were resistant to more than 9 drugs,and some strains formed the fixed spectrum,like AMP-NA-TE,AMP-CFP-NA,AMP-CFP-NA-TE;14 strains of S.Derby produced 13 kinds of drug resistance profile,with high resistance to tetracycline,nalidixic acid,sulfamethoxazole and chloramphenicol,2 strains were resistant to more than 9 antibiotics.Between 134 strains of Salmonella,detection rates of 11 resistance genes,blaTEM-1-like,bla-CTX-M,blaOXA-1-like,sull,aacC4,aac6-1b,floR,tet(G),were 73.1%,37.3%,29.1%,34.3%,32.8%,32.1%,36.6%,24.6%respectively;blaCMY-2,blaPSE,and blaSHV were not detected.S.Indiana isolates mainly carried ?-iaciamase gene,blaOXA-1-like,blaCTX-M,blaTEM-1-like and floR,sull,aadA2,and aac(6')-1b,which is the main reason to become super drug-resistant clone showing highly resistant to at least 9 kinds of antibiotics.S.Enteritidis isolates mainly carried blaTEM-1-like genes,which corresponded with its moderately-resistance to ampicillin,nalidixic acid,tetracycline and cefoperazone.The antibiotics abuse with chicken and swine industry was the main sources of resistance genes.The super resistant bacteria spread through animal food and food chains,which resulted in salmonella infections in humans becoming more and more serious,and causing food security risks.So,control the abuse application in animal food industry is of great significance for preventing multidrug-resistant bacteria,which could reduce the security risks in food industry.