| The safety of animal derived foods is a major public health issue that is high concern to the international community,pathogenic microorganisms are the key factors to food safety.Many factors cause food safety problems,and more than 70%of diseases is caused by pathogenic microorganisms such as Listeria monocytogenes(Lm,Salmonella,Escherichia coli,Streptococcus,etc.Listeria monocytogenes is an important zoonotic foodborme pathogen that can cause meningitis,gastroenteritis,sepsis,miscarriage and other symptoms,the mortality rate is as high as 20%-30%.Lm has the characteristics of tolerance to low temperature and high salt,etc.,it is widely distributed in environment,animals and food processing environment,and causes great treat to food safety.Listeriosis outbreaks have been reported in Europe and United States,most cases are caused by consumers eating contaminated pork products.Pork is the main meat product in China,with the improvement of people's living standards,people are paying more attention to food safety issues.During the processing and sales of pork,pathogenic microorganisms are usually contaminated and pose a potential treat to human health.Therefore,to clarify the current situation of listeria contamination during the pig slaughter,the transmission characterization along the slaughter chain was investigated.Our research work is beneficial to effectively solve the problem related with animal derived foods safety from the farm to the industrial chain.This study investigated the characterization of Lm in a pig slaughterhouse in Jiangsu Province and investigated the transmission patterns and key control points of Lm in the slaughter chain.Genome sequencing was performed to analyze their evolutionary relationships such as population structure,antibiotics sensitivity and virulence from the genome level.1 Isolation and identification of Lm in pig slaughter chainA total of 936 samples were collected from the slaughterhouse,the positive rate in slaughterhouse was 4.9%,Lm was transmitted in the process of dehairing,singeing,eviscerating,washing,trimming,precooling and,chilling,as well as the ground and cutting tools,the highest separation rate is in the chilling link(11.6%).Among the 78 samples of pork refrigerated transport,the positive rate is 20.5%.All isolates of Lm were divided into 4 serotypes,including 29 isolates(46.8%)of serotype 1/2a,4 isolates(6.5%)of 1/2b,17 isolates(27.4%)of 1/2c,and 12 isolates of 4b/4e(19.4%).In summary,with the process of slaughtering,the pollution of Lm presents a trend of progressive enlargement,Lm pollution rate in the chilling link is the highest,and the chilling link is the key control point of Lm pollution in the slaughterhouse.The antibiotic susceptibility results of Lm isolates revealed that 6 strains were resistant to two anitibiotics,of which 5 strains were resistant to ampicillin and another antibiotic(clindamycin,ciprofloxacin or penicillin);20 strains were resistant to ampicillin;3 strains were resistant to clindamycin;and 5 strains were resistant to penicillin,ciprofloxacin,tetracycline,gentamicin and compound sulfamethoxazole respectively.Among them,the high resistance rate to antibiotics was ampicillin(32.8%),followed by clindamycin(4.9%).All strains were sensitive to chloramphenicol,imipenem,erythromycin,streptomycin,vancomycin and rifampin.In a word,the antibiotic resistance of Lm isolates appeared the trends of increasing resistance rate and broadening of resistance spectrum.2 Molecular subtyping based on genome sequencing61 Lm isolates from the slaughter house were genome resequencing,then the genetic evolution,multi-virulence-locus sequence typing(MLST),Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR),virulence genes analyze,plasmid analyze and single nucleotide polymorphism(SNP)analysis were performed.The MLST classification results showed that all Lm isolates were divided into 6 ST types(3,8,9,121,155 and 626),the predominant ST types were ST9(n=29),ST121(n=12),and ST626(n=12),other ST types including ST3(n=4),ST8(n=4),and ST155(n=1).The results of cluster analysis showed that there was a high correlation between ST type and serotype except ST9 consisting of serotype 1/2a and 1/2c strains,and other five ST types corresponded to one specific serotype respectively.The same ST strains were isolated from different slaughter stages and transportation process,suggesting that Lm transmitted along the slaughtering and refrigerated transport processes.Cluster analysis based on CRISPR spacers showed that all strains were divided into two evolutionary Clusters,Cluster 1 was consisted of ST 121,ST3 and ST 155 strain,Cluster II was composed of ST9,ST8,ST626,and ST121 strains.The ST types in each Cluster were highly cosistent and the spacer sequences were identical.It is worth noting that the strains ST626 and ST155 isolated from the slaughterhouse formed a unique evolutionary sub-branch respectively.The above results showed that the CRISPR clustering is highly polymorphic,and can distinguish between subtypes based on the difference in interval sequences,suggesting that this molecular subtyping method plays important role in study of micro-evolution among strains.Comparing the virulence genes of all genome resequencing strains,we found that the strains of the same ST type contained the same virulence genes,and some virulence genes were missed in specific ST strains.In all ST3 strains,inlF and vip genes were missed,strains belonging to ST3 have the tendency to cause listeriosis in humans,this suggested that ST3 strains possibly have strong virulence;and all ST8 strains were absent of htrA and vip genes;ST9 strains were lost csA gene,and 99 percent of isolates were lost Ami gene;all ST121 strains were not harbored sipX,inlF,inlJ,actA,and inlC genes.The results suggested that the distribution of virulence genes in the same lineage strains are samilar,and the virulence genes among different lineage strains are somewhat different.The above results indicated that different ST strains carrying different virulent genes have different potential pathogenic risks.Lm isolates of genome resequencing were analyzed with single-nucleotide polymorphisms(SNPs),the results showed that all strains were divided into two Lineages(Lineage I and Lineage ?),Lineage I consisted of all ST3 isolates,and other isolates were belonged to Lineage?,importantly,each sub-branche of Lineages was consisted of unique ST type.The research work related with SNP anlysis suggested that isolates from different stages of pork production chain had a closer genetic evolution relationship,heretofore,it confirme*d the law of Lm carried by pigs and further transmitted along the slaughter chain in the slaughterhouse. |
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