Transmission Of Listeria Monocytogenes In The Pork Production Chain

Listeria monocytogenes(Lm)is a foodborne pathogen that can infect humans and more than 40 species of animals.Listeriosis is often caused by eating food contaminated with Lm.The incubation period of listeriosis is very long,with an average of 2 to 3 weeks,and the longest can exceed 70 days.Listeriosis can cause symptoms such as meningitis,septicemia,miscarriage or stillbirth.The bacterium is widely distributed in the environment,and can cause listeriosis in animals through the pollution of farm and animal forage,causing economic losses.Lm carried by animal spreads along the slaughtering,processing,transportation and sales links,eventually infecting humans.In recent years,foodbome listeriosis has been frequent all around the world.In 2019,listeriosis broke out in many European countries,39 people were infected by eating sausage,and 2 people died.At present,listeriosis caused by Lm has been reported regularly in pregnant women,neonates and immunocompromised patients.Lm poses a global economic and health burden,which may have implications for public.This study investigated the epidemiological status on listeriosis,analyzed and summarized the clinical characteristics and prevalence of listeriosis to provide a theoretical basis for the understanding of characteristics of pathogens,transmission routes,and sources of infection.In addition,we explored the transmission of Lm in the pork industry chain.Genome sequencing was performed to analyze their molecular characteristics and genetic evolution relationship,revealing the distribution of virulence and pathogenicity,providing important guidance for effectively controlling the pollution and spread of Lm.1.Epidemiology of human listeriosis in China during 2008-2017Both electronic and manual retrieval systems of relevant literature about listeriosis were employed to collect and analyze the clinical manifestations,basic data and outcomes in China between 2008 and 2017.A total of 759 cases were reported from 22 provinces,including 245 nonperinatal patients(32%)and 514 perinatal patients(68%).Among the clinical cases,the most common symptom was septicemia(49%),followed by CNS infection(25%).The overall mortality rate was 18%,of which neonatal mortality rate up to 73%.Recently,sporadic listeriosis has been reported annually,with a peak in 2014,frequently in March to May.The median age of the nonperinatal patients was 36(range,0-102)years and the predominance of the male sex among them(52%).Further in-depth research and collaboration are needed to improve the capacity of clinical diagnosis and treatment to prevent and control the spread of listeriosis.Human listeriosis has occurred in China,and listeriosis cases caused by ingesting animal products have also been reported,which prompts to strengthen the monitoring of Lm in animal foods to prevent and control the transmission of foodborne listeriosis.2.Isolation and identification of Listeria monocytogenes in the slaughterhouse,transport and supermarketsA total of 905 samples were obtained from the slaughterhouse,transport and pork in supermarkets,including 663 samples from 9 links,63 samples from the transport and 179 samples of the sales link,with a total separation rate of 10,6%.None of the samples from all steps except the chilling link of the slaughterhouse were positive for Lm.12 Lm strains were isolated from the chiling link,with a isolation rate of 10.9%.Among the pork refrigerated transport,11 samples(17.5%)were identified as positive for Lm.73(40.8%)samples were positive for Lm from the supermarkets.All isolates were serotyped into two serotypes,61 isolates(63.5%)of 1/2a and 35 isolates(36.5%)of 1/2c.The most common serotype from different links was 1/2a.According to the detection results of Lm in the pork industry chain,with the progress of slaughtering,transportation and sales,the pollution of Lm presents a trend of progressive enlargement.The antibiotic susceptibility tests were performed on 96 Lm strains from different steps,with a resistance rate of 90.6%.Among them,the clinical first-line drugs ampicillin(35 strains)and clindamycin(81 strains)have higher resistance rates.Moreover,57 strains were resistant to meropenem,18 strains were resistant to trimethoprim-sulfa methoxazole;and 18 strains are resistant to tetracycline.It is worth mentioning that 19.5%of the strains are multidrug-resistant.The emergence of a large number of food-borne drug-resistant strains suggests that the relevant departments should strengthen the monitoring of Lm drug resistance and regulate the use of drugs in animals.3.Molecular typing of Listeria monocytogenes in the pig industry chainAll Lm strains comprised of 4 ST types,ST9(n=40),ST121(n=27),ST8(n=13),and ST155(n=15).The slaughtering and marketing links have the same ST type(ST155 and ST9),and the dominant ST types is ST9 and ST121,which suggests that Lm can transmit along the pig industry chain and occur cross-contaminationMolecular typing methods core genome multilocus sequence typing(cg-MLST)based on whole-genome sequencing classified the strains into4 sublineages,which were highly concordant with STs.Different clusters included the same batch of strains from different stages,indicating that the pollution of Lm in the sales link originated from the slaughterhouse.It suggests that cg-MLST shows a higher discrimination power compared to traditional typing methods MLST.According to the clustered regularly interspaced short palindromic repeats(CRISPR)typing method,only ST121 and ST155 strains carry an intact CRISRP-Cas system.Then CRISPR was used to cluster strains into 11 CRISPR genotypes and 2 groups.The spacer sequences and numbers were highly consistent between the same ST strains from different steps,suggesting the spread law of Lm in the pig industry chain,and also reflecting the microevolution between the strains,which has a good application prospect in the traceability of Lm.Considering the resistance gene clustering results,fos(X)gene was widely distributed in all strains,strains carrying dfrG gene were detected frequently.Only one multidrug-resistant strain carried genes fos(X),dfrG,tet(S),erm(B),lnu(B),lsa(E),cat,etc.,while multidrug-resistant genes was rarely found in food isolates.Based on whole-genome sequencing,we detected 82 virulence genes of 67 Lm strains from different sources.28.4%isolates carried complete LIPI-1.None of the isolates harboured LIPI-2,LIPI-3 and LIPI-4.In addition,7 internalin genes existed in 76.1%strains and actA was absent in 71.6%isolates.All the strains belonged to lineage ?,not hypevirulent strain,which have the potential to cause sporadic listeriosis.The supervision and control of Lm in the pig industry chain should be strengthenedSNP(Single-nucleotide polymorphism)distributed Lm strains from slaughtering,transportation,marketing,and clinical into 4 major clusters.The cluster A with the most strains was the main contamination categorie.The strains isolated from different batches of chilling links were in the same branch with a close genetic and evolutionary relationship,and the SNPs differences was 3-6.The strains isolated from different batches of transport links were in the same cluster with high affinity,suggesting that there was cross contamination between the carcass,environment and tools in the chilling and transport links.Strains from different sources on the same branch appeared to have a close evolutionary relationship,indicating that Lm is spreading along the pig industry chain.The strains isolated in the pig industry chain and clinical isolates were divided into the same branch,suggesting that Lm in the pig industry chain has potential pathogenic risks to consumers.Taken together,we have investigated the epidemic law of listeriosis in mainland China over a ten-year period.Listeriosis in China is mainly sporadic,and septicemia was the most common presentation.Many cases are related to the consumption of pork products.Thus,the study explored the epidemiological characteristics of Lm from three stages of the slaughterhouse,transport and supermarkets,found that with the process of each link,the pollution presents a progressive enlargement characteristic.The strains exhibited extensively drug-resistant and multi-drug resistant,indicating that the resistance spectrum of Lm is broadening.Based on typing methods such as cg-MLST,CRISPR and SNP,suggesting that isolates from different sources have closely genetic relationship,with risks of food contamination and human infection.This study would be useful for provided new insight to Lm and prevention and control of listeriosis.