| Listeria monocytogenes(Lm)is the pathogen of zoonotic listeriosis,which is widely distributed in nature.Lm transmits grade by grade along the industry chain including farms,slaughterhouses,transportation and sale,causing human infection through contaminated food,and the main symptoms of listeriosis include meningitis,septicemia,miscarriage or stillbirth,with a fatality rate of up to 30%.In this study,the contamination characteristics ofLm in retail pork with different processing,packaging and storage methods in Yangzhou were investigated qualitatively and quantitatively.On the basis of genomic sequencing of Lm isolates,the genetic and evolutionary relationship of the isolates was studied through comparative analysis of serotype,virulence gene and drug resistance gene,as well as multi-locus sequence typing(MLST),CRISPR-Cas system typing and SNP typing.Aiming at the Lm contamination of retail pork,L-lactic acid bacteria reduction technology was developed,which provided an effective pathway for the prevention and control of Listeria monocytogenes in pig slaughtering industry chain(1)Investigation of Lm contamination in retail porkDuring the period from March 2019 to February 2020,pork samples were collected totally 12 times from three supermarkets in Yangzhou City.A total of 424 pork samples were collected and 99 Lm strains were isolated with the isolation rate of 23.3%.The separation rate of Lm in different months is variable.The Lm isolation rate of the three supermarkets was different,and the Lm isolation rate of supermarket C was significantly higher than that of A and B supermarkets.The isolated strain of Lm was composed of three serotypes,the dominant serotype was 1/2a(79/99,79.7%),the second was 1/2c(16/99,16.1%),and 4 strains was 1/2b(4/99,4.0%).Results showed that there was a certain correlation between Lm contamination rate and packaging mode,storage mode and pork type.The separation rate of packaged meat products was lower than that of bulk meat products;the separation rate of frozen meat was higher than that of chilled meat;and the separation rate of cooked food was the lowest.(2)Quantitative analysis of Lm pollution in pork in C supermarketIn October 2019,July and August 2020,the quantitative determinations of Lm were performed on 47 samples of retail pork from the C supermarket in Yangzhou City,respectively.Lm was found in 24 samples,and the isolation rate was 51.0%.Among them,there were 2 strains of serotype 1/2b(ST5 and ST87).The separation rate of Lm in bulk meat products was 72.7%,and the separation rate of Lm in boxed meat products was 40.0%.Severe contamination(MPN?110 CFU/g)samples was 14.8%(7/47);moderate contamination(110>MPN?10 CFU/g)samples was 29.7%(14/47);light contamination(10>MPN?0 CFU/g)sample was 6.3%(3/47).The separation rate of bulk meat products was high,but the contamination level was low.On the contrary,the separation rate of boxed meat products was low,but the pollution level was high.Among them,the Lm contamination in three samples was extremely serious(?1100 CFU/g).Lm was not separated from different types of cooked pork;the separation rate of cold and fresh meat was as high as 60.0%,and 83.3%samples were contaminated at medium pollution level or above(110>MPN?10 CFU/g);the separation rate of frozen meat was 14.2%,85.7%samples were contaminated at moderate level or above(110>MPN>10 CFU/g).(3)Molecular typing of LmGenome sequencing and genetic evolution analysis of 101 strains of Lm derived from retail pork in Yangzhou City showed that 101 strains of Lm were divided into 10 types of ST(Sequence Type),and the dominant ST types were ST 155(33/99,32.67%).There is one-to-one correspondence between ST and serotype except that ST9 corresponds to 1/2a and 1/2c.There were mainly type IB and type IIA CRISPR-Cas systems in 101 Lm,60.3%of Lm strains had CRISPR-Cas system,39.7%of strains had no intact CRISPR-Cas system(including ST8 and ST9),and the CRISPR-Cas system types of Lm strains were closely related to serotype and St type.The detection rate of drug resistance gene was 100%.Among them,the detection rates of lincomide antibiotic resistance gene(lin),fosfomycin resistance gene(fosX)and peptide antibiotic resistance gene(mprF)were as high as 98.0%and above.Notably,several ST9 Lm strains showed multiple drug resistance,which may be related to the use of antibiotics during feeding.99.0%of the Lm isolates carried complete listeria virulence island 1(LIPI-1)(prfA,plcA,hly,mpl,actA,plcB)and internalin gene island(inlA,inlB,inlC-inlH).2.9%of the Lm isolates carried the complete listeria virulence island 3(LIPI-3),and 1.9%of the Lm isolates carried the complete listeria virulence island 4(LIPI-4).SNP cluster analysis showed that 101strains of pork origin and 26 strains of clinical Lm were divided into five evolutionary branches(A,B,C,D and E).Most of the clade C were serotype 1/2b strains,and a total of 16 Lm strains were clustered together.The SNP difference between the 646 and 859 strains of branch C and the clinical strains in Hangzhou and Sichuan is very small,indicating that these strains have a near genetic relationship in evolution.This result suggests that Lm in retail pork has a potential risk of causing listeriosis,and the surveillance and control of Lm in retail pork should be strengthened.(4)The reduction effect of lactic acid on LmTaking the international standard strain EGD-e as the research object,the temperature,time and concentration of L-lactic acid were optimized by response surface method.The results showed that treatment for 4min at 21? by 1.1%L-lactic acid were the optimal reduction condition.Under the condition,96.0%Lm was killed,and there was no significant effect on pork pH and fat oxidation,while the Brightness Index(L*)was larger than that before treatment,and the meat color was brighter.This study provided an effective control technology for pork preservation and storage.In summary,this study investigated the molecular epidemic law of Lm in retail pork in Yangzhou for one year.The results showed that the contamination of Lm in retail pork in Yangzhou was serious,and there was certain relationship between packaging,storage and pork type and the Lm contamination degree.Lm isolates contribute potential pathogenetic risk to human health for they have multiple antibiotic resistance gene and carry intact Listeria virulence island.The Lm contamination in retail pork is serious,so the optimized L-lactic acid bacteria reduction technology can effectively control the Lm contamination in retail pork.Certainly,L-lactic acid can be employed to reduce the Lm contamination level in retail pork.The results present insight into Lm monitor,alert and risk evaluation and are significant for Lm prevention and control. |
PDF Full Text Request