| Staphylococcus aureus is a common gram-positive bacteria in nature,also a common public health problem caused by pollution in food.Food poisoning caused by Staphylococcus aureus and its toxins ranks third in all bacterial food poisoning every year in China,second only to Salmonella and Vibrio parahaemolyticus,which is a common foodborne pathogenic bacteria.The consumption of food contaminated by Staphylococcus aureus or its enterotoxin by consumers can cause gastrointestinal symptoms such as nausea,vomiting,diarrhea,and even collapse or shock in severe cases.As the most ingested animal food in the diet of Chinese,the food safety of chicken is closely related to consumers and must be paid special attention to.In recent years,the detection of methicillin-resistant Staphylococcus aureus(MRSA)in food has become more and more common in China.In addition,with the food trade and population movements,the contamination of MRS A in food become more and more widespread.In different foods,the rates of MRSA vary greatly and the main types are complex.The traceability survey of foodborne diseases in China has also found food poisoning incidents caused by MRSA,even reports that the serious infection caused by MRSA from food.Therefore,MRSA pollution in food can cause greater safety risks to consumers.In this study,we investigated the contamination of Staphylococcus aureus in commercially raw chicken in Jinan,evaluated its risk of causing foodborne disease outbreaks,and elucidated the genotype,drug resistance,and virulence gene prevalence characteristics of the strains by complete gene sequence analysis to understand the genomic epidemiological characteristics of stains;screened MRSA strains and analyzed their predominant types,pathogenicity,and biofilm-forming ability.The results of this study illustrated the characteristics and pathogenic risks of S.aureus contamination in commercially raw chicken in Jinan and provided basic data support for food-borne pathogenic bacteria contamination control and reducing the risk of foodborne disease outbreaks in food.Objective1.To investigate the contamination of Staphylococcus aureus in raw chicken in Jinan,and to evaluate the risk of foodborne disease outbreaks caused by Staphylococcus aureus.Use genome-wide sequence analysis.to detect the drug resistance characteristics of the strains,analyze the main epidemic types of the strains,epidemic characteristics of drug resistance genes and virulence genes,and genetic evolution relationships of various types of strains.2.To screen MRS A strains and explore the source of contamination,analyze the pathogenic characteristics of MRSA strains,and evaluate the potential harm of MRSA contamination in food.Methods1.Sample collection and detection:We collected raw chicken in major supermarkets,farmers’ markets,and other places in all districts of Jinan from March 2021 to November 2021,with 20 samples per month.Then the contamination rate and the level of Staphylococcus aureus in the samples were tested by GB 4789.10-2016 "Food Microbiological Examination of Staphylococcus aureus",and the suspected strains were isolated,purified,and frozen.2.Strains review and enterotoxin gene identification:5 types of classical enterotoxin gene carriage among strains were tested using PCR assays;meantime identified the species of strains by PCR and time-of-flight mass spectrometry.3.Data sorting and risk assessment:Samples were grouped according to the sample type and sampling location,and calculated the contamination rate and level of Staphylococcus aureus of samples in each group.The test results of each group were statistically analyzed by SPSS25.0,Assessing the risk of foodborne disease outbreak caused by Staphylococcus aureus in the samples of each group by Risk Ranger semi-quantitative risk assessment software.4.Antibacterial drug sensitivity test:19 kinds of antimicrobial drugs from 13 categories were used to detect the minimum inhibitory concentration of the strains by the methods of micro broth method and agar dilution,and the drug resistance of the strains was determined by the CLSI antimicrobial drug sensitivity test execution standard.5.Strain sequence analysis:The genetic sequence of the strain was extracted to secondgeneration sequencing analysis,splice the whole genome sequence of the strain,and upload to the line comparison platform Center for Genomic Epidemiology(CGE)to obtain the strain type,and drug resistance gene and virulence gene carrying and genetic evolution relationship.6.MRSA strain screening and pathogenic characteristics analysis:MRSA strains were screened according to the drug resistance phenotype and genotype of the strains,the pathogenicity of the strains was verified by the toxicity test of the wax borer,the biofilmforming ability of the strains was determined by Congo red agar,and the pathogenic characteristics of MRSA strains were analyzed in combination with the drug resistance phenotype.Results1.A total of 55 positive samples were detected from 180 raw chicken samples,with a total detection rate of 30.56%,the rate of frozen chicken products from supermarkets was 26.23%,the rate of frozen chicken products from farmers’ markets and meat retail stores was 50.00%,the rate of fresh chicken products was 15.25%.In 55 samples with positive qualitative results,the overall geometric mean of pollution level is 21.9 MPN/g.All strains were identified as Staphylococcus aureus by various methods.2.Risk assessment results indicated that the risk level of food poisoning caused by Staphylococcus aureus in raw chicken in Jinan was high,and the highest risk value was the group of frozen chicken products from farmers’ markets and meat retail stores,which the risk value was 53;the second was the group of fresh whole chicken,with a risk value of 52;frozen chicken products from supermarkets and e-commerce platforms had the lowest risk value of 49.3.There are 14 types of ST and 20 types of spa were detected among the 55 strains of Staphylococcus aureus.A total of 26 strains was ST7,the most detected type,which was the dominant type in the region.Among them,22 strains were ST7-t91 Staphylococcus aureus,3 strains were ST7-t976 Staphylococcus aureus,and 1 strain was ST7-t605 Staphylococcus aureus.Different types of strains showed different distribution characteristics of drug resistance genes and virulence genes.4.The drug resistance rate of the strains was 98.18%;the rate of multidrug resistance was 54.55%.Among the 13 classes of 19 antimicrobial drugs selected in this study,the resistance rate of the strains to penicillin was the highest,reaching 92.73%(51/55),the resistance rates of erythromycin and tetracycline were 56.36%(31/55),and the resistance rate to clindamycin was 54.55%(30/55).All strains were sensitive to ceftaroline,vancomycin,teicoplanin,daptomycin,linezolid,tigecycline,and furantoin,and 5 strains showed rifampicin intermediary.5.A total of 8 strains out of 55 strains were MRS A,and 7 strains were multidrug resistant.All strains show resistance to oxacillin,cefoxitin,and penicillin;At the same time,some strains were resistant to erythromycin,clindamycin,tetracycline,chloramphenicol,gentamicin,ciprofloxacin,levofloxacin,moxifloxacin,and other antibacterial drugs.All MRSA have the biofilm-forming ability.6.There were 4 ST types of 8 MRSA strains.In addition to the common types of LAMRSA in China,there were also common types of LA-MRSA and CA-MRSA in foreign countries.The results of the toxicity test of the giant wax borer showed that the number of dead larvae was positively correlated with the number of virulence genes carried by the strain,and the strain carrying the leucocidin gene might have higher pathogenicity.Conclusions1.There is a certain degree of Staphylococcus aureus contamination in raw chicken in Jinan,especially in frozen chicken products.The results of the semi-quantitative risk assessment suggest that the risk of foodborne disease outbreaks is high.The departments should strengthen supervision and take measures to control the contamination of pathogenic microorganisms in food to reduce the risk of foodborne disease outbreaks.2.There are various types of contaminated strains,and ST7 is the dominant type of local pollution.The strains have a high resistance rate to commonly used antibacterial drugs in animal husbandry and are sensitive to commonly used clinical bacterial infection treatment drugs.However,it is found that some strains carry rifampicin resistance gene mutations,which are intermediated by rifampicin,which should be concern.The carrying rate of virulence genes of the strains is high and the types are wide.In addition to enterotoxin genes,some strains also carry leucocytocide genes and toxic shock syndrome genes,that has a risk of serious bacterial infection in humans.3.The rate of MRSA in the strains is gradually increasing,the types of pollution are complex and the sources are diverse.The strain has strong drug resistance and pathogenicity,as also the ability to biofilm forming,which has great health risks.It is necessary to strengthen the monitoring and traceability of MRSA pollution in food to reduce the pollution and spread of MRSA in food. |
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