| Staphylococcus aureus is the important food-borne pathogen that threatens food safety and exists widely in nature.As the largest pork producer and consumer in the world,the detection of Staphylococcus aureus in the process of pork production and processing is particularly important in China.In this study,Staphylococcus aureus was detected in slaughter chains of swine slaughterhouses in different areas,and its epidemic characteristics were analyzed.Whole genome sequencing was performed on representative isolates from different slaughtering stages in the same slaughterhouse,and the homologous evolutionary relationship of isolates in different slaughtering stages in the slaughterhouse was analyzed to provide scientific data support for the risk assessment and prevention and control of Staphylococcus aureus.1.Epidemic analysis of Staphylococcus aureus from swine slaughter.From May 2019 to October 2020,sampling was conducted 7 times in different slaughtering links in part of slaughterhouses in Henan,Hebei and Shanxi provinces,and a total of 1680 samples were collected.A total of 212 strains(12.6%)of Staphylococcus aureus were isolated using conventional detection methods and molecular biological techniques,and MRSA strains accounted for 21.2%(45/212)of the isolates.The detection rates of Staphylococcus aureus in Henan,C1 nd Hebei provinces were 11.1%,12.8% and 14.4%,respectively.The detection rates of Staphylococcus aureus in slaughterhouses in A,B2,C1,D,and E were 13.8%,12.4%,15.6%,13.8%,and 15.0%,respectively.The contamination rate of Staphylococcus aureus was relatively close(P>0.05).The detection rates of B1 and C2 slaughterhouses were lower,7.1% and 10.4%,respectively.There were also some differences among different batches.For example,the detection rates of B1 and B2 isolates were 7.1% and12.4%,respectively,and the detection rates of C1 and C2 isolates were 15.6% and 10.4%,respectively.From the sampling site,the average detection rate of Staphylococcus aureus in the nasal cavity was 21.3%,which was significantly higher than the average detection rate of 3.3% in surface swab samples,which indicated that the swine nasal cavity was the main contaminated site of Staphylococcus aureus;In addition,Staphylococcus aureus was also detected in the knife shank and environmental samples,suggesting that Staphylococcus aureus contamination exists in slaughter processing tools and slaughterhouse environments,and there is a risk of secondary contamination of pork.2.Epidemic distribution and drug resistance analysis of Staphylococcus aureus isolates with different spa types.All isolates were identified by spa typing,and the results showed that there were mainly 4 types of spa types in the 212 isolates,t571(99/212),t1456(47/212),t011(21/212)and t1451(21/212)respectively.Among them,the t571 type is the main spa type,which widely exists in various slaughterhouses and is distributed in each slaughtering link.Next,the t1456 type mainly comes from the A and Luohe slaughterhouses,and the t011 type is distributed in the E slaughterhouse and all are MRSA strains,the t1451 type strains are mainly distributed in D slaughterhouse.A total of 15 kinds of antibacterial drugs were selected in this experiment,and 212 strains of Staphylococcus aureus isolated by the method of agar dilution were tested for drug resistance phenotype.The test results showed that there were 202 strains resistant to more than one kind of antibiotics,accounting for 95.3% of all tested strains,and the isolation rate of strains resistant to more than 3 antimicrobial drugs was 68.9%.The most common isolates were resistant to Erythromycin,with a resistance rate of 84.9%;Lincomycin(64.2%),Tetracycline(69.3%),Florfenicol(67.0%)and Doxycycline(62.3%)showed a high resistance rate.All strains were sensitive to Vancomycin,Cefotaxime and Amikacin.Resistance phenotypes of all Staphylococcus aureus isolates present 34 resistance profiles,of which the dominant drug resistance profile was ERY(20.8%,44/212),the second is LCM/OFL/CIP/ERY/TET/NOR/FFC/DOX(17.5%,37/212).The results of drug resistance gene detection showed that drug resistance genes erm(T),tet(M),bla Z,fex A,erm(C)and lnu(B)were commonly carried in Staphylococcus aureus isolates.Notably,44 of the 45 MRSA isolates in this study carried the mec A gene,but only 30 Staphylococcus aureus isolates were resistant to Oxacillin.Systematic analysis of drug resistance among major spa-typed isolates.Type t571 and type t1451 have the same dominant drug resistance spectrum,which is LCM/OFL/CIP/ERY/TET/NOR/FFC/DOX,and both generally carry drug resistance genes erm(C),tet(M),fex A,bla Z,The resistance genes such as lnu(B),erm(T),tet(L)and las(E)were more common in t571 isolates.The 21 t011 isolates were all resistant to Oxacillin,Tetracycline,Florfenicol and Doxycycline,and all carried the resistance genes mec A,tet(M),fex A,and bla Z.Among them,the dominant drug resistance spectrum OXA/LCM/KAN/CHL/OFL/CIP/ERY/GEN/TET/NOR/FFC/DOX(12/21)all carry drug resistance genes mec A,tet(M),fex A,bla Z,lsa(E),lun(B)and dfr G.Most of the t1456 Staphylococcus aureus isolates were only resistant to erythromycin,and the results of drug resistance gene detection showed that they all carried the drug resistance gene erm(T).All isolates were tested for 17 enterotoxin genes,and the results showed that only 9enterotoxin genes(seb,seg,sei,sek,sem,sen,seo,seq,seu)were detected.41 of the 212 Staphylococcus aureus isolates carried enterotoxin genes and were spa-specific,and only t899,t571,t441 and t011 strains carried enterotoxin genes,among which the complete egc gene cluster gene(seg-sei-sem-sen-seo-seu and seb-sek-seq)are only carried in t899,t571 and t441 isolates.3.Genomic characteristics and genetic evolution analysis of Staphylococcus aureus isolates based on a slaughterhouse.In order to explore the epidemic spread and genetic evolution of isolates at different slaughtering stages in the slaughterhouse,22 Staphylococcus aureus strains were selected in different slaughtering stages in D slaughterhouse for whole genome sequencing.The results of the phylogenetic tree analysis of the genome sequence constructed by SNP showed that,the Staphylococcus aureus isolates were able to spread along the swine slaughter chain,and cross-contaminated in different slaughter links through the environment.Most of the strains contained drug resistance genes bla Z,tet(M),fex A and erm(C),among which 17 isolates carried mobile transposon(Tn6009,Tn558),and the Tn558 transposon carried the drug resistance gene fex A.In the genome of the MRSA strain isolate numbered XBZ1,it carries the mobile transposon Tn917,which carries the drug resistance gene erm(B),and Untyped plasma coagulase-negative MRSA strains carry mec B,aac(6’)-aph(2’’)and lnu(A)and other drug resistance genes,and carry the quaternary ammonium compound resistance gene qac Z.In this study,Staphylococcus aureus in swine slaughterhouses in different regions was studied by means of spa typing,genetic testing,drug susceptibility testing,and genome sequence analysis,in order to explore the epidemic and transmission rules,gene carrying and drug resistance of Staphylococcus aureus,and tracing it back to its source.Establishing the key risk control points of Staphylococcus aureus during the slaughtering process of live swines,formulate reasonable clinical medication,so as to achieve effective prevention and control and monitoring of Staphylococcus aureus. |
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