| Listeria monocytogenes is an important food-borne zoonotic pathogen,which is widely distributed in nature and can cause diseases in humans and animals.It mainly manifests as meningitis,encephalitis,septicemia and monocytosis after infection and the mortality rate is as high as 20%-30%.This strain has been listed as one of the four foodborne pathogens by WHO as early as 2000.According to literature reports,the annual incidence of listeriosis in clinic is approximately 2-15/100,000 in some developed countries.For example,in the United States,about 2600 people are infected with Listeria monocytogenes and nearly 260 people die from this each year.Listeria monocytogenes is mainly spread through the food chain and can survive in many aspects of meat manufacture,resulting in meat contamination and food poisoning.Listeria monocytogenes has many serotypes,a wide range of survival adaptation,strong tolerance to external unfavorable environments,and a long incubation period.These factors make it difficult to detect,trace and control the transmission of the bacteria.At present,there are few relevant data on the investigation of Listeria monocytogenes contamination in meat in Chongqing.The study aims to provide technical guidance and useful reference for the healthy development of pigs and poultry breeding industry and the protection of animal material food safety in Chongqing via understanding the contamination of Listeria monocytogenes in pork and chicken and their machining process and sales in Chongqing and surrounding areas.The samples in this experiment were collected from chicken,pork and processing and sales related environments in some slaughterhouses,supermarkets and farmers markets in Chongqing,Shaanxi and Yunnan.Then identified Listeria monocytogenes isolated from the samples and performed serotyping testing,drug resistance testing and genotyping.A total of 1,445 samples were collected and 79 Listeria monocytogenes strains were detected,and the total detection rate was 5.5%.Among them,the detection rate of chicken samples was the highest,15.7%,which was statistically significant compared with pork and environmental samples.Through analyzing the contamination of Listeria monocytogenes in environmental swabs at several production stages,it was found that the slaughterhouse was the most seriously contaminated with Listeria monocytogenes and the supermarkets had the lowest detection rate,which was statistically significant compared with slaughterhouses and farmers markets.Among environmental swabs in farmers markets,the scales and countertops were more seriously contaminated by Listeria monocytogenes,and the detection rates were 20.0% and 14.3%,respectively.Listeria monocytogenes resistance rates to oxacillin,cefotaxime and polymyxin B were as high as 89.9%,40.5% and 21.5%,respectively.Among the isolated strains,14 strains were resistant to 3 or more antibiotics in the isolates,and the multi-drug resistance rate was 17.7%.Molecular serotyping results show that Listeria monocytogenes isolates were divided into four major types,1/2b(1/2b-3b-7)and 4b(4b-4d-4e)belong to lineage I.1/2a(1/2a-3a)and 1/2c(1/2c-3c)belong to lineage II.Among them,the number of isolates of type 1/2b,1/2c,1/2a and 4b respectively accounted for 38.0%,26.6%,24.0% and 11.4% of the total number of isolates.And 4b isolates were distributed in the meat and environment of sales.By analyzed the MLST genotyping of 79 Listeria monocytogenes isolates,a total of 18 ST types and 14 clonal complexes were obtained,and the Simpson's index was 0.8880.ST9 was the most widely distributed,with a total of 18 strains,and in which 16 strains were isolated from the meat and environment of sales.There were 15 strains belong to ST87,in which 14 were isolated from the meat and environment of sales.There were 9 strains belong to ST5,which were all isolated from the slaughter environment.There were 5 strains belong to ST8,which were all isolated from the meat and environment of sales.A total of 39 MTs were obtained by MLVA genotyping,and the Simpson's index was 0.9656.The traceability results of genotyping indicated that the Listeria monocytogenes isolated from the slaughterhouses in Wanzhou district of Chongqing and other districts of Chongqing had the same ST type and were located on the same branch of the MLST phylogenetic tree.In the MLVA phylogenetic tree,MT27,MT28 and MT32 were all isolated from different environments in the same slaughterhouse,with 4 strains,3 strains and 4 strains,respectively.A total of 9 strains belong to MT18 were isolated from sales in Chongqing and Shaanxi.There are 8 strains belong to MT23 were isolated from sales in Chongqing and Yunnan.The phylogenetic tree structure of MLVA and MLST genotyping were basically similar,but the same clonal complex and ST type strain could be further subdivided by MLVA genotyping.In addition,we selected eight optimal loci combinations(LMV2,LMV9,LMV1,Lm10,Lm11,Lm15,Lm23 and LMTR6)from 14 MLVA loci.Listeria monocytogenes contamination was found in some slaughterhouses,supermarkets,and farmers markets in Chongqing,Shaanxi and Yunnan provinces.The chicken in the sales was mainly frozen/chilled meat,which was the most seriously contaminated by the bacteria.And the cleaning and disinfection of the environment in slaughterhouses and farmers markets should be strengthened because of the higher contamination rate of Listeria monocytogenes in these area.Listeria monocytogenes isolated from Chongqing and its surrounding areas resistance to antibiotics is constantly expanding,and the phenomenon of multi-drug resistance is becoming more and more serious.It is necessary to strengthen the management of commonly used antibiotics in the clinic and advocate that drug susceptibility tests should be conducted before the use of antibiotics.There are various serotypes associated with human listeriosis and various ST types that cause food-borne listeriosis in sales of pork and chicken.These high-risk strains breed for a long time in slaughtering and processing of animals,and spread through the food chain to meat sales.There was cross-contamination of the isolates in the slaughter and processing of the same area,and there were also close relatives in a few of the isolates in the sales in different regions.The Simpson's index of the MLVA genotyping is higher than MLST genotyping,indicating that MLVA has stronger typing ability than MLST.The 8-loci and 14-loci combination screened from all detection sites have the same typing ability,indicating that this combination scheme is the optimal loci combination scheme for Listeria monocytogenes MLVA genotyping in Chongqing. |
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