| Listeria monocytogenes is a pathogen of zoonoses.Listeria monocytogenes has very strong tolerance to the environment,and the distribution is very wide in the natural environment.It can cause various diseases.Although the incidence rate is low,but the mortality rate is high(20%~30%).In order to understand the distribution of the bacteria in the city of Beijing,isolation and identification of Listeria monocytogenes were done on retail food in Beijing.On this basis,the the virulence related factors of the isolates were studied,and the isolates were analyzed by genotyping and comparative genomics analysis.1.According to the national standard method,the Listeria monocytogenes were isolated and identified from retail food in Beijing,in order to study the distribution of Listeria monocytogenes in food.The multiple PCR method was used to identify the serotype of the strains.71 Listeria monocytogenes strains were isolated from 470 food samples,the positive rate was 4%(19/470).Among them,the positive rate of raw meat products was 13.4%(9/67),the positive rate of instant processed foods was 3.8%(9/237),the positive rate of cereal products was 2.0%(1/49),and no strain was detected from vegetables and fruits(0/117).The raw meat products were more susceptible to be contaminated by Listeria monocytogenes in the four categories of foods detected in Beijing.The serotypes of the isolates were 1/2a,1/2b and 1/2c.Serotype 1/2a is the dominant serotype.Serotype distribution is not necessarily related to the types of food samples2.In the study of the factors that affect the virulence of Listeria monocytogenes,the biofilm formation ability,antibiotic sensitivity,drug resistance genes and virulence genes of the isolates were detected respectively.(1)96-well plate crystal violet staining method was applied to detect the biofilm formation ability of isolates.Isolates from raw meat products had strong biofilm formation ability.So,they were more likely to persist for a long time and cause long-term pollution.(2)The sensitivity of ampicillin,ciprofloxacin,erythromycin,gentamicin,nitrofurantoin,penicillin G,rifampin,tetracycline,trimethoprim-sulfamethoxazole and vancomycin weredetected by the broth microdilution method.The isolates were resistant to ciprofloxacin universally;the resistance rate to trimethoprim-sulfamethoxazole was 68.4%;1 strain was resistant to trimethoprim-sulfamethoxazole,ciprofloxacin and penicillin G;34 strains were resistant to trimethoprim-sulfamethoxazole and ciprofloxacin;22 strains were resistant to ciprofloxacin;and all isolates were sensitive or intermediate sensitive to ampicillin,penicillin G,rifampicin,tetracycline and vancomycin.(3)The PCR method was used to detect 20 antibiotic resistance related genes,including tetracycline resistance gene tet A,tet S,tet M,macrolide resistant gene erm B,erm C,aminoglycoside resistance gene aac(6’)-Ib,β-lactam resistance gene mec A,vancomycin resistance gene van A,van B,Tn916 transposon excision enzyme Xis-Tn,polymyxin resistance gene mcr-I,quinolone resistance-determining region gene gyr A,gyr B,par C,par E,plasmid mediated quinolone resistance determining region qnr A,qnr B,qnr S,trimethoprim resistance gene dfr D.Most of the results of drug resistance genes were consistent with the results of drug sensitivity test.Some of the drug-resistant genes positive strains were not resistant to antibiotics.(4)The PCR method was used to detect 9 virulence genes of 2 virulence islands,including adhesion and infection related genes inl A,inl B,inl C,inl J,intracellular growth gene mpl,plc A,act A,hly,and regulatory gene prf A.The test results of virulence genes were all positive.Indicating that the gene deletion of isolates from Beijing was not serious,and all isolates had the potential for infection.3.The genetic diversity of isolates was analyzed by pulsed field gel electrophoresis.The cluster similarity degree of isolates from smoked salmon,which were the different batches of products from the same manufacturer,was very high(95.7%~100.0%).It indicated that the food production enterprises had the same pollution source for a long time.Some of the isolates,which were isolated from the same sample,were low in clustering similarity.It indicated that the contamination source of the food sample were complex,or the contaminated strains came from different pollution sources.4.The Hiseq Xten platform of Illumina was used for the complete genome sequencing,and60 disease-causing genes were forecasted.Among all the 21 strains,the positive rate of 31 geneswas 100%,3 genes were deleted in some isolates,and 26 genes were not detected in the tested isolates.The main virulence genes deletion of the isolates were not serious,indicating that the isolates had certain pathogenicity.Phylogenetic tree was obtained by evolution analysis using16 S r RNA sequence.To sum up,the results of this study provide evidence for Beijing’s food safety risk assessment,control and prevention of related diseases,and guide clinical treatment.The results can also provide reference for traceability of listeriosis outbreaks,and data support for the study of the biological characteristics of Listeria monocytogenes. |
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