Specific Phenotypic And Molecular Characteristics Of MRSA In Swine And Human

Obejectives:The whole genome sequencing was used to comprehensively and systematically detect the variable genes in the core genome,the mobile components of the attached genome and the related encoded genes of methicillin-resistant Staphylococcus aureus(MRSA).It could elucidate systematically the genetic background of MRSA from different sources and reveal the specific molecular characteristics.Exploring the relationship between MRSA gene mutations and resistance phenotypes in order to clarify and reveal the resistance mechanism of MRSA.The comparison of the resistance phenotype and molecular characteristics of MRSA in swine and human was to explore the specific phenotypic and molecular markers of MRSA in swine and human.The specific markers were used to establish a discriminant model and predict the source category of MRSA in unknown origin occupational workers,so as to further clarify the risk and route of cross-species transmission of LA-MRSA in swine and human,as well as provide scientific basis for the effective control of the spread of LA-MRSA in human.Methods:According to the study purpose,63 strains MRSA in human,48 strains MRSA in swine and 73 strains MRSA in unknown origin occupational workers MRSA were selected.Whole genome sequencing analysis is performed on all MRSA.The sequence alignment was performed using network database to obtain MLST typing,spa typing,SCCmec typing,resistance genes,gene mutation sites,and virulence geneof MRSA.The difference between the two groups was compared using the Pearson x2 test or Fisher's exact probability method.The MEGA-X software and iTOL online tools were used to construct and beautify the phylogenetic tree.To explore the relationship between gene mutation sites and resistance phenotypes,the importance variables were screened by random forest model,and then the correlation analysis was carried out by x2 test.In the risk prediction of MRSA cross-species transmission,the important characteristic variables of strains from different sources(swine and human)were screened by the combination of parameter method(x2test)and machine learning method(random forest model).The distance discrimination model and the logistic discrimination model with and without prior probability were established for discriminant analysis,and then the optimal model was used to predict the source category of MRSA of unknown origin occupational workers.Results:1.Molecular characteristics of MRSA from different sources: swine MRSA was mainly cloned as ST9-t899-SCCmec XII(15/48,31.3%),occupational workers MRSA as ST9-t899-SCCmec XII(23/73,31.5%),and human MRSA as ST188-t189-SCCmec V(11/63,17.5%).2.Relationship between gene mutation and resistance phenotype:grl A gene mutation(536Ser?Cys,410Tyr?Phe,80Ser?Phe,223Ile?Val),grlB gene mutation(139Asn?Ser)and gyrA gene mutation(84Ser?Ala)were positively correlated with ciprofloxacin resistance.RopB gene mutation(481His?Asn)was positively correlated with rifampin resistance.3.Comparison of phenotypic and molecular characteristics of MRSA in swine and human: The resistance rate of swine MRSA is significantly higher than those of human MRSA(ciprofloxacin,gentamicin,clindamycin,erythromycin,tetracycline,etc).In the resistance genes,the tet(K)gene positive rate of swine MRSA was higher than that of humanMRSA,but the gene positive rate of aac(6')-aph(2 "),lnu(B),and tet(L)was lower than those of human MRSA.In the grlA gene,ciprofloxacin resistant swine MRSA has multiple mutation sites(V694M,S80 F,S536C,Y410 F,and I223V),while human MRSA is mostly a single mutation site(V694M).In the grlB gene,there are two mutation sites(N139S and D530G)in ciprofloxacin resistant swine MRSA,while most human MRSA has only one mutation site(D530G).In the gyr A gene,ciprofloxacin resistant swine MRSA has a mutation site(S84A).For rifampicin resistant strains,the mutation level of H481 N in ropB gene of swine and human MRSA was similar.For cefoxitin resistant strains,there is a mutation site(E315A)in pbp2 gene of both swine and human MRSA.In the pbp4 gene,cefoxitin resistant swine MRSA has one mutation site(T189S),while human MRSA mostly has three mutations sites(T25A,T189 S,and E398A).The bacteriostatic circle value of swine MRSA mutation to cefoxitin was generally smaller than that of human MRSA mutation.In the virulence genes,the genes positive rate of swine MRSA were higher than that of human MRSA(sea,seh,sep,splA,splB,splE,lukD,and lukE),but these genes positive rate were lower than human MRSA(seg,sei,sem,sen,seo,and seu).4.Screening for specific phenotypic and molecular markers:combination of parameter method and machine learning method was used to screen for specific phenotypic and molecular markers,including resistance phenotypes(ceftoxitin,clindamycin,ciprofloxacin,gentamycin,rifampicin,chloramphenicol,trimethoprim-sulfamethoxazole,linezolid),resistance gene aac(6 ')-aph(2")and virulence gene splE.5.Establishment of discriminant function for discriminant prediction:the discriminant model of swine and human MRSA was established by using the selected specific phenotyic and molecular markers,and the source category of MRSA was predicted for the occupational workers.It was predicted that 53.4% of the occupational workers were human strain and 46.6% of those were swine strain.Conclusions:1.The main clones of MRSA from both occupational workers and swine are ST9-t899-SCCmec XII,suggesting that there may be a risk of MRSA cross-species transmission between the swine and occupational workers.2.Mutations in grlA gene,grlB gene and gyrA gene were positively correlated with ciprofloxacin resistance,suggesting that mutations in grlA gene,grlB gene and gyrA gene could mediate resistance to ciprofloxacin in MRSA.There was a positive correlation between ropB gene mutation and rifampicin resistance,suggesting that ropB gene mutation could mediate resistance to rifampicin in MRSA.3.The specific phenotypic characteristics of swine MRSA were resistance to cefoxitin,clindamycin,ciprofloxacin,gentamicin,rifampicin,chloramphenicol,trimethoprim-sulfamethoxazole,linezolid,and the specific molecular characteristics were resistance gene aac(6')-aph(2")and virulence gene splE.4.The discriminant model of swine and human MRSA was established and the source category of occupational workers MRSA was predicted.It was predicted that 53.4% of occupational workers MRSA were human strains and 46.6% of those were swine strains.The above results revealed that the risk of transmission of LA-MRSA across species from animal to human in the occupational workers cannot be ignored.