| Listeria monocytogenes is an important intracellular facultive foodborne pathogencausing meningitis, meningoencephalitis and abortion with the mortality rate of20%~30%,considered as one of the fourth foodborne pathogens by WHO.L. monocytogenes widely spread in natural environment, can be isolated from food andits associated environment. In addition, L. monocytogenes biofilms can tolerate highconcentrations of disinfectants and sanitizers, thereby hampering the decontamination ofsurfaces of food-procucing machinery, it is potential risk to food microbial safety. So, thisthesis was to investigate the contamination level of retail foods in South China and analyzedthe phenotype and genotype of foodborne L. monocytogenes. In addition, we also explored theprevalence and contamination patterns of L. monocytogenes in Flammulina velutipes plants.With the aim of controlling biofilm formation effectively, the relationship of between quorumsensing and biofilm formation was studied for exploring a new way for controlling biofilmformation. The specific contents are as follows:1. Ten (6.33%) of158retail RTE food samples were positive for L. monocytogenes and thecontamination levels were less than10MPN/g, while none of65dairy products was positivefor L. monocytogenes. The37strains were grouped into five clusters and two singletons, fiveclusters and two singletons, and three clusters and one singleton by enterobacterial repetitiveintergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplifiedpolymorphic DNA(RAPD) fingerprint respectively, at similarity coefficient of80%. Thesusceptibility test showed that83.8%were susceptible to15antimicrobials; virulent L.monocytogenes that possess partial antimicrobial resistance, and serotypes frequentlyassociated with listeriosis were recovered from RTE foods. Consumers may, therefore, beexposed to potential risks of L. monocytogenes infection in South China. This studycontributed to the prevalence and contamination levels of L. monocytogenes in RTE foods inSouth China for the first time, providing baseline information for Chinese regulatoryauthorities to formulate a regulatory framework for controlling L. monocytogenes to improvethe microbiological safety of RTE foods.2. About21.7%(123/567) of raw foods collected from South China were positive for L.monocytogenes,86.0%of the MPN values of positive samples did not exceed100MPN/g, wefound that84.7%(150/177) of isolates harbored the nine virulence-related genes. Among177isolates, lineage I.1(1/2a-3a) and II.2(1/2b-3b-7) were prevalent in the raw food samples,accounted for42.4%and26.0%, respectively; lineage I.2, II.1and III were18.1%,7.3%and 6.2%, respectively. Surprisingly, isolates recovered from Flammulina velutipes samples onlybelonged to lineage I.1and II.2, lineage III were mainly isolated from fresh fishery products,which may be associated with the specific environmental niche. Fourty-two antimicrobialprofiles were found among177isolates, including59.9%(106/177) were susceptible to the15testing antimicrobial,28.2%were considered as multidrug-resistant isolates, this emphasizesthe need to conduct the surveillance of antibacterial susceptibility of foodborne L.monocytogenes in South China continuously. ERIC-PCR and RAPD were applied for thediversity analysis of177foodborne L. monocytogenes isolates, the discrimination power ofthe two method were0.960and0.972, respectively.3. Four mushroom (Flammulina velutipes) production plants were sampled to investigate theprevalence and contamination source of L. monocytogenes. Among295samples, theprevalence of L. monocytogenes was18.6%; the contamination appeared to originate from themycelium-scraping machinery, contaminating both the product and upstream packagingequipment. Of55L. monocytogenes isolates, lineages I.1(1/2a-3a) and II.2(1/2b-3b-7)accounted for65.5%and34.5%, respectively. In addition, lineage I.1formed significantlythicker biofilms than those within lineage II.2, as determined by crystal violet staining andscanning electron microscopy. Genotype analyses of L. monocytogenes isolates usingenterobacteria repetitive intergenic consensus-polymerase chain reaction, and randomamplified polymorphic DNA revealed that the surfaces of mycelium-scraping machinery mayserve as the main source of L. monocytogenes contamination in three of the four plants. Thisstudy was the first report to explore the potential contamination sources of L. monocytogenesin the mushroom production chain, thereby providing baseline information for adoptingprophylactic measures for critical control points during production in mushroom plants toavoid L. monocytogenes contamination.4. Whole-genome sequencing of L. monocytogenes798-1WT. Whole-genome sequencing of798-1WT was carried out by Solexa sequencing to produce1311Mb filtered sequences,representing a435-fold coverage of the genome. The reads were assembled into62contigsand26scaffolds using the velvet denovo package, then the scaffolds make up of the wholegenome sequence. The draft genomic feature of L. monocytogenes798-1WT contained onesingle circular chromosome of3,013,905bp with an average GC content of37.5%. A total of3055open reading frame (ORF),65tRNA genes, and18rRNA operons were identified.Using L. monocytogenes EGD-e strain as a reference genome, comparative genome analysisrevealed that an approximately40kb DNA fragment of prophage A118inserted in comK genewas substituted by partly homologous sequence of L. innocua and L. welshimeri genome, this may indicate the inserted DNA sequence in comK gene asscociated with the evolution ofListeria spp; the downstream of luxS (lmo1288) gene in798-WT was not homologoussequence compared with EGD-e strains, this data paved the foundation for construction ofΔluxS mutant using homologous recombination.5. The relationship of between quorum sensing and biofilm formation of L. monocytogeneswas studied. The growth curve of ΔagrD, ΔluxS and ΔagrDΔluxS mutants were as similar as798-1WT; the colony morphology and flagellum formation were not affected by deletion ofagrD and luxS of L. monocytogenes under25°C or37°C cultivation. Compared to798-1WT,the biofilm formation of ΔagrD and ΔagrDΔluxS mutants were highly decreased (p<0.01), butΔluxS mutant was highly increased (p<0.01) after120h cultivation, indicated that agrD maypositively regulate the process of biofilm formation and luxS may negatively regulate thebiofilm formation. Compared to798-1WT, both ΔagrD and ΔluxS mutant showed increasedsensitivity to oxidative reagent(treated with12.5μmol H2O2), may imply their role in theregulation of oxidative stress tolerance. These data may explore a new way for prevention andcontrolling the biofilm formation of L. monocytogenes.To summarize, we investigated the contamination level of L. monocytogenes in foodproducts collected from South China for the first time, comprehensive analyze the phenotypeand genotype of L. monocytogenes isolates, which providing the data for the risk assessmentof L. monocytogenes in retail foods in South China. We also first explored the prevalence andcomtamination source of L. monocytogenes during Flammulina velutipes production worldwi,and analyzed the relationship of between quorum sensing and biofilm formation. These datamay be utmost of importance for prevention and controlling the biofilm formation of L.monocytogenes. |
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