Pathogenicity Of Listeria Monocytogenes Isolated From Frozen Chicken To Chicken And Determination And Analysis Of Whole Genome Sequence

Listeria monocytogenes(Listeria monocytogenes,LM)is a food-borne intracellular pathogen that can infect humans and animals,cause meningoencephalitis,miscarriage,and sepsis.The mortality rate can be as high as 20%-30%.The annual survey results of food LM contamination in various countries show that the detection rate of LM in chicken is between 1.1%and 5.51%.The source of chicken LM may be pollution from the processing environment,or it may be carried by the chicken itself.Purpose:This experiment will study the pathogenicity of chicken source LM to chickens,so as to cause food factories and poultry farms to pay attention to pollution sources.And through whole-genome sequencing,analysis of its genome evolution and virulence characteristics,to provide a basis for the pathogenic mechanism of isolates.Methods:(1)PCR detection of virulence genes,determination of hemolysis value,modified Kou's calculation method to calculate chicken embryo LD₅₀and weighing to obtain chicken embryo liver index,and compare the virulence of 17 isolates through the results.(2)Three strains of significantly different virulence strains,LM925,LM929,and LM873,were selected to infect 9-day-old chicks by intramuscular injection.The LD₅₀of chicks was obtained by the trimmed Spearman-Karber method,liver,spleen and brain tissue were ground,and the plate count method was used to calculate the load.Bacteria count,observe the general and histopathological changes of chicks,etc.,study the pathogenicity of LM to chicks;(3)Select LM925 and LM873 with the largest virulence difference,and measure absorbance under different conditions of temperature,p H,Na Cl concentration and Et OH concentration OD₆₀₀obtained the growth curves of the two strains,and the microplate quantitative method was used to compare the biofilm formation ability in different time periods.(4)Complete genome sequencing of the isolates LM925 and LM873,and analyze their genome characteristics,virulence genes,MLST typing and genome evolution.Results:(1)41.2%(7/17)of the isolates carried the 8 virulence genes tested,and all isolates carried lmo2821,which was decisive for virulence.All 17 isolates lysed sheep red blood cells,and the hemolysis value was in the range of Between 1:4-1:16,17 isolates can kill chicken embryos,58.8%of isolates have LD₅₀between 1.5 and 2,and 41.2%of isolates have LD₅₀between 2 and 5.The LD₅₀of the chicken embryo of the virulent strain was 1.533,and the LD₅₀of the chicken embryo of the weakest strain was4.733.The liver index of the chicken embryo inoculated with LM was significantly increased.(2)The chick infection experiment showed that the three strains are pathogenic to the chicks.The LD₅₀of the chicks of the virulent strain LM925 and LM929 were 7.733 and 8.133,respectively,while the LD₅₀of the chick of the relatively weak strain LM873 was 8.733.LM925 had the highest bacterial load in each tissue,and LM873 was the lowest.The anatomy of the chicks revealed the most obvious liver disease.The liver of chicks infected with LM929 can see obvious white necrotic foci,while LM925 is obviously in the majority,and the liver of chicks infected with LM873 has no visible lesions.Histopathological observation showed that the lesions were mainly liver cell necrosis,inflammatory cell infiltration,congestion and purulent foci.The most lesions were LM925,and the least was LM873.Only local congestion was observed.(3)Under different conditions,37?,p H 7.0,and 5.0%Na Cl are the optimal conditions for the growth of the three LM plants,and 4%Et OH is the minimum growth inhibitory concentration.The growth trend of LM925 is always higher than that of LM873,and the growth trend of LM925 is always higher than that of LM873.The growth trend of the two strains under the conditions of,4.5%Na Cl and 4%Et OH was significantly different(P<0.05);the microplate quantitative method was used to determine the biofilm formation of the bacteria at different time periods,and it was found that there was a small amount of bacteria after 6-24 hours of cultivation.LM begins to attach and the formation of microcolonies.Compared with LM873,there are significant differences in the formation of biofilm between LM925 and LM873 at 6h,8h and 12h(P<0.05).At 10h and 24h,the difference between LM925 and LM873 is extremely significant.(P<0.01);At 36h,the microcolonies are connected into a network structure,which can form a mature and dense biofilm,and the difference between LM925 and LM873 is extremely significant(P<0.01),the biofilm network structure gradually dissolves at 48h,LM925 and LM873 There was no significant difference in comparison(P>0.05).It was found that LM925 has stronger environmental adaptability and biofilm formation ability.(4)The results of genome-wide annotation analysis show that the LM925 genome is 2,965,010bp in length,with a GC content of 37.83%,encoding 2928 protein predictive genes,and the coding region accounts for 91.03%of the entire genome.The average protein length is 872.38 amino acids and contains56 t RNAs and 6 r RNAs.The LM873 genome is 2,972,618 bp in length,with a GC content of 37.83%,and it encodes 2928 protein predictive genes.The coding region accounts for 91.03%of the entire genome.The average length of the protein is 862.57 amino acids,containing 55 t RNAs and 6 r RNAs.In the analysis and comparison between LM925 and LM873,in addition to the virulence genes shared by the two bacteria,LM925 also contains gene clusters lls B,lls D,lls H,lls X,lls Y,and lls G that encode LIPI-3.These genes encode hemolysin S(LLS),LM873 has gene clusters esx B and gsp G encoding LIPI-4,which encode secreted proteins.The sequencing results of the two strains were compared with the database information,and the sequence typing of LM873 was ST87,belonging to the CC87 clone group,and LM925 was ST386,belonging to the CC224 clone group.In the evolutionary analysis,LM873 is closely related to the Chinese clinical isolate SHL012 and the American FSLJ1-194 that caused the outbreak of human listeriosis;LM925 is closely related to FSLJ1-194,SHL012,the American food isolate FSLN1-017 and the American clinical isolate The isolate FSLR2-503 is in the same evolutionary position,showing that they evolved from the same ancestor.The comprehensive analysis of the two results showed that both LM873 and LM925 have the potential to cause the outbreak of human listeriosis.Conclusion:Frozen chicken Listeria monocytogenes isolates are lethal to chicken embryos and chicks,but the virulence is not the same.Infecting chicken embryos and chicken livers are the more obvious organs.The environmental adaptability of LM frozen chicken isolates is positively correlated with the biofilm formation ability and virulence.Both LM873 and LM925 have the potential to cause an outbreak of human listeriosis.