| China is a big country in pork production and consumption,accounting for more than60% of China’s total meat consumption.Chinese pork output reached 48 million tons in2020,an increase of about 5 million tons over 2019.Staphylococcus aureus(S.aureus)has a high detection rate in fresh pork at home and abroad,and it is one of the main foodborne pathogens that pollute fresh pork.The incidence of food poisoning events caused by S.aureus and its toxins ranks among the top four.S.aureus has complex drug resistance mechanism and multiple virulence genes,which pose a great threat to food safety and human health.The purpose of this study was to understand the contamination rate,drug resistance and virulence gene carriage of S.aureus in fresh pork in Yucheng District,Ya’an City.Whole-genome sequencing of S.aureus to analyze its genetic information.The test results are as follows:1.A total of 351 fresh pork samples were collected from Yucheng District in summer and winter in 2020.According to the improved first method of GB4789.10-2016,the thermostable nuclease gene(nuc)of S.aureus was amplified by PCR technique for isolation and identification.The results showed that the total contamination rate of samples was 19.94%(70/351),and 73 strains of S.aureus were isolated;the overall contamination rates of summer samples,room temperature samples and refrigerated shredded meat(minced meat)were 15.28%(35/229),16.75%(34/203)and 3.85%(1/26),respectively.The overall contamination rate of winter samples was 28.69%(35/122),and the positive rate of room temperature samples was 38.04%(35/92).No refrigerated samples were detected.2.The disk diffusion method was used to test the resistance of 73 strains of S.aureus to 16 kinds of antibiotics.The results showed that 73 strains of S.aureus had the highest tolerance rate to penicillin(93.15%).The resistance rate to tetracycline,erythromycin,clindamycin,ciprofloxacin,ofloxacin,cefotaxime and kanamycin was high(45.20%,42.46%,28.77%,27.40%,26.03%,21.92%).The tolerance rate to chloramphenicol,sulfamethoxazole,rifampicin,nitrofurantoin and teicoplanin was low(15.07%,15.07%,12.33%,12.33%,10.96%).The resistance rate to cefoxitin,amikacin and gentamicin was the lowest(6.85%,5.48%,2.74%);4.11% of the strains were non-resistant,and 61.64%were resistant to at least 2 antibiotics.5 strains of S.aureus resistant to cefoxitin carried mecA gene and were identified as MRSA strains.3.The virulence genes of S.aureus were amplified by PCR test.The results showed that the detection rate of traditional enterotoxin gene(sea～see)was 4.11%,12.33%,4.11%and 0%,respectively,and that of toxic shock syndrome toxin gene(TSST-1)was 15.07%.The detection rate of leukotoxin gene(PVL)was 1.37%.The detection rates of hemolysin genes(hla,hlb,hld)were 43.84%,76.71% and 87.67%,respectively.The detection rates of desquamate toxins(eta,etb,etd)were 1.37%,0% and 4.11%,respectively.Generally speaking,the detection rate of seb and sec in traditional enterotoxin was higher,the detection rate of hemolytic toxin was higher,but hld was the highest,and the detection rate of etd in desquamated toxin was the highest.83.56% of the strains carried 2 or more virulence genes.4.The film-forming ability of S.aureus was evaluated by qualitative,quantitative and PCR techniques.The Congo red test showed that the positive strains accounted for 84.93%and the negative strains accounted for 15.07%.The crystal violet staining test showed that the non-adhesion rate was 5.48%,the weak adhesion rate was 12.33%,the medium adhesion rate was 20.55%,and the strong adhesion rate was 61.64%.Among the film-forming genes,the highest detection rate of icaD was 93.15%,and the highest detection rates of clfA,clfB,fnbB and fnbA in adhesion-related genes were 86.30%,100.00%,95.89% and 100.00%,respectively.5.4 pairs of accessory regulatory genes(agr)and 7 pairs of housekeeping genes of S.aureus were amplified by PCR test,and the products were purified and sequenced after electrophoresis.Agr typing showed that agr Ⅰ(50.68%),agr Ⅱ(9.59%),agr Ⅲ(17.81%),agr Ⅳ was not detected,21.92% of the strains were not typed.In MLST typing,11 strains were not typed,while the other strains were divided into 26 ST types.The isolation rates of ST188(13.70%),ST88(12.33%),ST15(10.96%)and ST398(8.22%)were high,with a total of 5 clones,mainly CC1 and CC15.6.DNA was extracted from 5 strains of MRSA,second generation library was constructed and sequenced to obtain high quality cleandate.The quality of the assembled genome was controlled by GC-depth distribution and K-mer frequency distribution,and then scanned map assembly and basic genome information,gene function annotation and other data analysis.The results showed that the total genome sequence of the five isolates was 2731898～2808746 bp,and the content of GC in the gene sequence was32.689%-32.775%.strains 43-1 and 11-1 had the most drug resistance genes and virulence genes.To sum up,this paper analyzed the contamination of S.aureus in fresh pork in Ya’an,and analyzed the drug resistance,film-forming ability,virulence gene distribution,molecular typing and genome information of the isolates.The test results have certain practical significance to prevent food safety incidents caused by S.aureus and its toxins. |
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