| "Hunger breeds discontentment, food safety first", the food safety problem has been thefocus attention. Prevention of food contamination and foodborne disease control is thepriority domain of global public health. Based on the statistical data, foodborne pathogensis the main factor caused food borne illness. Epidemiological monitoring datashowa continued trend towardsfood borne infections, andvariable pathogenpool. Besides commonintestinal bactieria, an emerging pathogen witht high toxicity and lethality,has spreaded inalmost all kinds of food, such as meat, milk, cereal, vegetable, fruit,et al, and bringhuge safety challenge.Effective and improved prevention of foodborne infections will require a thoroughunderstanding of the evolutionand ecology of pathogens, e.g., in order to allow forappropriate source tracking and source attribution. For example, development and applicationof molecular subtyping methods, which can differentiate strains,have been critical for thedetection of foodborne disease outbreaks and their sources.Furthermore,Molecular mechanism of foodborne pathogen to adapt to the complex food processingenvironment and evade the host immune system has not been very clear, it isimperative that we do in-depth study in order to find a suitable control or remove means.This study focused on one of the leading death caused foodborne pathogen-Listeriamonocytogene, and aimed to investigate the occurrence in food of Chinaandthe potentiallyvirulence, a rapid detection and tracing method was developed and the structure and potentialfunction of a new important defense system--CRISPR/Cas system was anylzed anddemostrated, in order to pre-propose and eliminate various potentialListeria-related food-borne safety issues during food production and sugarproduction. The main content of this study were as follows:1) Statistical analysis almost13years food contaminants monitoring results of15provinces,and found that the detection rate L. monocytogenes (6.37%) weresignificantly higher than those of common salmonella (5.29%) and Vibrioparahaemolyticus (5.91%), become the leading foodborne pathogen.Raw meat andquick-frozen rice flour food are the first two polluted sources,bring food cold-chain transport and food preservation major security risk.2) Through investigating serotype, drug resistance and resistance genes of90isolatesfrom6types of food in Hebei province, we found the main serotype of the isolates is1/2a, resistant11kinds of antibiotics, and the highest resistant rate were detected forthree second-line antibiotics, ciprofloxacin (15.56%), tetracycline (12.22%)andstreptomycin (11.11%); For the first time detected a first-line antibiotics-penicillinresistant strains. Tetracycline resistance mainly by tet (M) gene in transposons Tn916,an important mobile elements spreading antibiotic resistance.3) Based on two kinds of bio-marker-genome and fatty acids, we used PFGE geneticfingerprint technology and fatty acid methyl ester fingerprint technology to analysisthe homology between the90isolates from food. We found12large clones popular inHebei province, C3(1/2c, T, Fa3) and raw meat and quick-frozen rice flour food werecross contamination by different popular clones. C3(1/2c, T, Fa3) cloning is thebiggest epidemic clone, were detected in five city. Meat and frozen food rice havebeen cross-contaminated by of several epidemic clones. Acquiring resistance genes isan important factor to divergent evolution. In practical application, the combination ofautomated high-throughp FAME along with accuracy PFGE enabled us better toidentify the differences among L. monocytogenes isolates.4) A total of three relatively fixed CRISPR loci (LMa, LMb, LMc) were inserted threedistinct CRISPR system types: CRISPR RliB (Listeria specific), the CRISPR I-B, theCRISPR II-A. The activity of three loci from strong to weak was followed by LMc>LMb> LMa, as well as the inserted orderaccidentally in the opposite, CRISPR arraysshowed significant lineage specific, lineages II and I strains showed several identityspacers at the far end of leader, indicating that they may divergent from the sameancestor. In addition to the major function of defence against viruses, CRISPR-Cassystem might also be related to virulence and durg resistance, as well as involve inregulating genetic recombination and physiological activities such as the formation ofbiofilm in L. monocytogenes, since we found two independent self-targeting spacersin only a subset of LMb loci （53.66%）targeted two endogenous regulating geneaadB (ATP-dependent nuclease subunit B) and dgc (diguanylate cyclase). The positive correlation between the evolution trend of the three CRISPR-Cas system andthe virulence as well as the environment adaptive ability, suggested that the epidemicstrains should be high virulence with better environment adaptability in the future,thus needed more attention. |
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