Molecular Typing And Potential Pathogenicity Of Staphylococci Sourced By Raw Chicken Meat

There are severe microbial direct exposure risk and cross-contamination risk for raw chicken meat products in feeding,slaughtering,processing,transporting,storing and retailing stages.Staphylococci as potential pathogen which is widely spread within animal and human skins,the potential pathogenicity of staphylococci in raw chicken meat is closely related with the health of consumers.Therefore,it is meaningful to investigate the prevalence of staphylococci,study the potential pathogenicity in raw chicken meat,thus to constitute and develop the targeting control strategies.Our study isolated staphylococci strains based on the selection of different retailing settings,storing temperature,packaging types and product types of raw chicken meat,employing multi molecular typing methods to identify the isolates,and evaluating the potential pathogenicity by antibiotic resistance assay,virulence gene determination and biofilm forming ability.Subsequently,regarding coagulase-negative staphylococci served as reserviors for methicillin resistance gene,the origin of methicillin resistance gene mecA was analyzed based on codon usage bias comparison between staphylococci genome and mecA.Regarding the typical characteristic of producing superantigenic enterotoxins of Staphylococcus aureus,the the influence of potential stimulative nutrients of chicken meat on enterotoxin genes expression were studied,thus to explore the key regulatory factors which modulate enterotoxin genes expression with the presence of stimulative factor of chicken meat.The main research were introducd as follow:1.Contamination and pathogenicity of Staphylococcus aureus isolated from raw chicken meatThis study aimed to determine the prevalence,antibiotic resistance(routine animal used and nosocomial treatment antibiotics)and biofilm forming abilityof S.aureus.In total,464 raw chicken meat products were collected in a variety of conditions:from local supermarkets and wet markets;between summer and winter;of cut and whole carcass product types;stored at both low temperature and room temperature;and in both packed and unpacked states.The overall prevalence of S.aureus was 11.5%,contamination level of S.aureus among different products types maintained 102-104 CFU/g.Whereas,penicillin-and tetracycline-resistant isolates were common in products with skin,cuts,particularly,extremely strong biofilm formation isolates were found in products with skin.Our investigation indicates that cutting process and packing were the key factors for the contamination of S.aureus,suggesting a possible critical control points in processing and marketing stages.Routine antibiotic resistance and strong biofilm forming ability raise the need for concern over possibility of human infection.2.Molecular typing and virulence gene determination of Staphylococcus aureus isolated from raw chicken meatThe genetic characterization of Staphylococcus aureus in raw chicken meat products were determined in this study.Multi molecular typing methods(including MLST,spa typing and agr typing)were employed for classification of S.aureus isolates,the virulence gene existence was also determined for molecular determination,thus,comprehensive analysis of epidemiology and potential virulence characteristics of S.aureus was obtained in various raw chicken products.The results showed that CC5 had common presence in all of products,and spa typing did not appear further classification on the basis of MLST,comparison among different products revealed that CC5 might be sourced from processing environments,CC12 was from retailing environments,products with skin showed more variously molecular types,centralized distribution of agr Ⅱ and agr Ⅲ suggested that different genetic types could utilize the common signal factor,thus synchronized the regulation of virulence genes expression.The virulence gene determination showed that typical virulence genes such as pvl,tst and enterotoxin genes had relatively low prevalence in S.aureus isolates,it revealed that toxic S.aureus has low presence in raw chicken meat products,whereas,common prevalence of atypical enterotoxin genes need to be concerned regarding the potential unclearly pathogenicity of S.aureus.Comparison among different product types showed that cuts and products with skin in supermarkets has higher prevalence of enterotoxin genes,and whole carcass in wet markets had highest prevalence of enterotoxin genes,it suggested that cutting and retailing are main factors that causing the contamination of pathogenic S.aureus.3.Pathogenicity of coagulase-negative staphylococci isolated from retailing raw chicken meatDiverse coagulase-negative staphylococci(CNS)species isolated from meat are previously reported and considered as potential pathogenic staphylococci through obtain horizontal transferred elements(e.g.toxic,antibiotic resistance factors)from other bacteria.In this study,prevalence,homology and pathogenicity of CNS isolated from 180 raw chicken meat samples in retail markets were studied.The overall prevalence of CNS was 11.7%and centralized in 69.2%of Staphylococcus epidermidis and 30.8%of Staphylococcus warneri among diverse sources(including different retail settings,suppliers and products).Molecular typing showed highly homogenous patterns in the same species.Antibiotic susceptibility test revealed a common penicillin and erythromycin resistance among all of CNS,while 17 S.epidermidis isolates displayed multidrug resistance of gentamycin(GM)-kanamycin(K)-penicillin(P)-erythromycin(E),except one methicillin-resistant Sepidermidis isolate which was typed in staphylococcal cassette chromosome mec(SCCmec)V.Conclusively,diverse sourced raw chicken meat products exhibited a centralized prevalence of S.epidermidis and S.warneri,and a highly homogenous of genome inthe same CNS specie.Though the negative toxigenicity of CNS was determined,constant multidrug resistance in S.epidermidis and P-E resistance in CNS need to be highly concerned regarding human health.4.Analysis of methicillin-resistant gene mecA’s origin based on codon usage biasAs the central element of SCCmec,Gene mecA gave SCCmec elements the characteristic of methicillin resistance,showing widely dissemination in staphylococci and critical mortality in infections.Whereas,the origin of mecA is still remain unclear,according to the previous studies demonstrated that codon usage bias could influence the translation efficiency for target gene,our study hypothesized that compatible codon usage between mecA and staphylococcal genome could be employed to track the origin of mecA,Here,the mecA was extracted from staphylococcal whole genome,codon usage bias between mecA and staphylococcal genome and compatibility of codon within mecA and anticodon of tRNA within staphylococcal genome were compared.Our results indicated that point mutation of mecA has a negative effect on codon usage bias,RSCU between mecA and staphylococcal genome suggested mecA has apparent characteristics to spread within staphylococci,codon and anticodon compatibility between mecA and tRNA showed that S.haemolyticus is highly possible serve as ancestral carrier of mecA,and whole-genome phylogenetic tree exhibited closely genetic relationship in methicillin-resistant staphylococci,especially for the common methicillin-resistant species like S.aureus and S.haemolyticus.In summary,according to the codon comparative results of between mecA and staphylococcal genome,we believed that S.haemolyticus served as original staphylococci for mecA.5.Effect of chicken meat,mass protein and vitamin B2 on enterotoxin gene expression of Staphylococcus aureusThis study compared the growth and enterotoxin gene expression of S.aureus under different culturing matrix(including meat matrix and TSB),it revealed that chicken meat matrix had significant stimulating effect on enterotoxin gene expression with exception of affecting cell growth,especially for the enterotoxin b,c and d,which exhibit closely relationship with cell growth.The enterotoxin expression difference within various nutrients of meat were also determined,using Short Time-series Expression Miner(STEM)to analyze the time-series expression of regulators and enterotoxins under different conditions.The results indicated that,though experimental strains possess all of regulatory genes,different strains showed a diverse expression preference,it suggested that the regulatory network is more complex than we previously acknowledged,according to the putative regulatory network summarized by amounts of previously reports,we can still obtain a common conclusion,when cell sensing abundant energy or nitrogen sources,they will prior choose to unregulate the energy metabolic pathway,so as to provide sufficient energy for replication and metabolism,instead of improving virulence for competition,when the environment contain stimulative factors like vitamin that could contribute to the energy metabolism via improving nutrient availability,they will choose to upregulate the virulence gene expression for improving environmental competition.