Distribution,Gnetic Diversity And Toxicological Mechanism Of Foodborne Listeria Monocytogenes

Listeria monocytogenes is an important food-borne pathogen due to its widespread distribution. Consumption of food contaminated with this pathogen can lead to listeriosis. This condition has high rates of morbidity and mortality(25%~30% overall). The organism can endure adverse conditions such as freezing, drying, mild heat and anaerobic conditions. It can also grow at temperatures used in refrigeration(from-1.5 to 4 oC). Due to these characteristics, Listeria monocytogenes is a great concern for food safety.In order to know the contamination status of Listeria monocytogenes and the distribution of virulence genes in Listeria monocytogenes, to reveal the variation law of Listeria monocytogenes vary with time and region, to reveal the toxicity levels of Listeria monocytogenes in different kinds of meat products and in different culture time. In this study, there were 308 wild strains of Listeria monocytogenes isolated from 1288 samples of seven kinds of foods which were obtained from Baoding and Shijiazhuang areas of Hebei province, china. Meanwhile, the distribution of 27 virulence genes in 308 strains of Listeria monocytogenes was detected by PCR. The randomly amplified polymorphic DNA-PCR(RAPD-PCR) was employed to investigate the genetic diversity of 308 wild isolates of Listeria monocytogenes. Genetic distant coefficient analysis of the band profiles was carried out using unweighted pair-group method analysis(UPGMA) and cluster analysis. The growth curves for the pathogen were measured at OD600, and quantitative real-time PCR(q RT-PCR) was used to analyze 12 batches of 10 virulence genes: iap, prf A, plc B, act A, ami, fbp, hpt, hfq, mpr F and hly A. The expression level of 27 virulence genes in four kinds of broth was carried out by using RT-PCR. The mouse virulence test was carried out to detect the pathogenicity of Listeria monocytogenes. The conclusions of this study are as follows:(1) The total isolation rate of Listeria monocytogenes in seven kinds of foods was 23.91%. The contamination level of raw meat was the most serious in all foods, the isolation rate of Listeria monocytogenes was up to 40.60%, followed by cooked meat products(25.42%) and aquatic products(23.46%). In comparison, the contamination level of Listeria monocytogenes was higher in the animal food than in garden stuff. (2) The RAPD typing method of Listeria monocytogenes was optimizated. RAPD-PCR fingerprinting patterns were obtained. The genetic relationship dendrogram among the 308 isolates of Listeria monocytogenes was established.(3) The clustering analysis showed that the 308 isolates of Listeria monocytogenes could be grouped into six genetic clusters. Among them, Clusters Ⅰand Ⅱwere the dominant species from this region. The foodborne Listeria monocytogenes has a preference for a particular host and the spread of foodborne Listeria monocytogenes has characteristics of being regional and temporary.(4) The result of the detection of 27 virulence genes indicated that those genes which related to cell infection and intercellular spread were distributed in animal food isolates more than in garden stuff isolates. Those genes which related to cell adhesion and regulation were distributed no significant difference both in animal food isolates and in garden stuff isolates. The average number of virulence genes was more in Clusters Ⅰthan in Clusters Ⅱ. The detection rate of hfq、dlt C and iap was up to 94.68%、93.36%、91.03% respectively in 308 wild isolates, which suggested that the three gene above could be used as a reference gene in identification of Listeria monocytogenes in this area.(5) Quantification by q RT-PCR indicated that 10 virulence genes(iap, prf A, plc B, act A, ami, fbp, hpt, hfq, mpr F and hly A) had similar expression patterns in a 24 h period, with peak expression levels at about 16h~18h in late stationary phase, which thereafter declined but increased again in the death phase. The virulence gene expression of Listeria monocytogenes in four kinds of broth indicated there was a big difference in the expression level of virulence gene when the Listeria monocytogenes grew in different environment.(6) The experimental results of the mouse virulence test shown that hly A played a decisive role in pathogenicity. The virulence genes of inl A, prf A, vir R, plc A, act A, plc B, dlt A and dlt D had a great effect on the pathogenicity of Listeria monocytogenes.This study provides a theoretical basis for Listeria monocytogenes detection and prevention.