Genomic Epidemiology Study On The Correlation Of Staphylococcus Aureus And MRSA Between Students And School Environment

ObjectiveThis study aims to use whole-genome sequencing to analyze the characteristics of resistance genes and virulence genes of Staphylococcus aureus(S.aureus,SA)and Methicillin-resistant Staphylococcus aureus(MRSA)isolated from students and school environment in Guangzhou at the genomic level,which could provide a theoretical basis for the prevention and control of S.aureus and MRSA infection.Exploring the genetic mutations of S.aureus could reveal the resistance mechanism of the strains.Besides,we investigate molecular typing and compare the genes of different ST types of strains to explore the potential associations.In addition,we also explore the homology of MRSA between students and school environment and understand the genetic background of the strains,which could reveal the potential transmission risks of MRSA between students and school environment.MethodsWe selected S.aureus strains preserved in the laboratory according to the study purpose,including strains of students and school environment.All strains were performing whole-genome sequencing by Illumina Hiseq Xten platform.The online database was used to obtain MLST typing,SCCmec typing,spa typing,resistance genes,gene mutation sites and virulence genes of S.aureus strains.The Pearson χ ~2 test or Fisher’s exact test was used to compare the differences between two groups.LASSO regression was conducted to select variables of resistance gene mutation sites,then the Pearson χ ~2 test or Fisher’s exact test was applied for the comparison of gene mutation sites,and OR(Odds Ratio)and 95%CI(Confidence Interval)obtained by unconditional logistic regression.The UPGMA cluster analysis and χ ~2 test were carried out to explore the relationship between molecular typing and gene pattern.The correspondence analysis was used to explore the potential association between molecular typing and the number of resistance and virulence genes.Results1.Information about strains:A total of 221 strains of S.aureus were selected,including 173 strains in students and 48 strains in school environment.103 strains and 32 strains of MRSA were identified in students and school environment,respectively.2.Molecular typing:A total of 26 ST types of S.aureus were identified,and ST45(27.15%)and ST59(16.74%)were predominant STs.A total of46 different spa types were detected,mainly t116(22.17%)and t437(17.65%).Among the students,ST45(32.04%)and ST59(30.10%)were predominant STs in MRSA,and three predominant STs in MSSA were ST188(15.71%),ST30(14.29%)and ST45(12.86%).Two of the most predominant spa types in MRSA were t437(31.07%)and t116(24.27%),and three predominant spa types in MSSA were t189(14.29%),t116(10.00%),and t338(10.00%).The most predominant SCCmec type of MRSA was IVa(58.25%).Among the school environment,two of the most predominant STs were ST45(53.13%)and ST59(15.63%)in MRSA,and ST30(25.00%)and ST6(18.75%)in MSSA.Two predominant spa types in MRSA were t116(50.00%)and t437(15.63%),and the most predominant spa types in MSSA was t338(25.00%).The most predominant SCCmec type of MRSA was IVa(62.50%).Two of the most predominant molecular type of MRSA between students and school environment were both ST59-t437-SCCmec IVa and ST45-t116-SCCmec IVa.According to the cluster analysis,there was potential transmission between students and school environment.3.Resistant genes detection of S.aureus:A total of 23 resistance genes of S.aureus were detected.The predominant of resistance genes were beta-lactams(bla Z/mec A)(95.48%/44.80%),followed by macrolides(erm(A)/erm(B)/erm(C))(11.76%-34.39%),aminoglycosides(ant(9)-Ia/aph(3’)-III/ant(6)-Ia)(11.76%-15.84%)and tetracyclines(tet(K))(16.74%).Among the students,the detection rates of mec A,erm(B),aph(3’)-III,ant(6)-Ia,tet(K)genes in MRSA were higher than MSSA,while MSSA carried higher proportion of erm(A)and ant(9)-Ia.Among the common STs of S.aureus,the proportion of strains carrying 3 or more resistance genes was 63.02%,and ST59 was associated with carrying 5 or more resistance genes.4.Virulence genes detection of S.aureus:A total of 37 virulence genes of S.aureus were detected.The predominant of virulence genes were hemolysin(hlg A/hlg B/hlg C)(98.64%),followed by extracellular enzyme genes(aur/splA/spl B/spl E)(28.96%-98.19%),immune evasion genes(sak/scn)(85.97%-95.48%),enterotoxin(seg/sei/sem/sen/seo/seu)(56.56%-57.47%)and leukotoxin(luk D/luk E)(34.39%-34.84%).Among the students,the detection rates of sec,sec3,sek,sel,seq genes in MRSA were higher than that of MSSA with higher proportion of splA,spl B,spl E,edin A,luk D and luk E.Among the school environment,the detection rates of sec,sec3,sel genes in MRSA were higher than MSSA,while MSSA carried higher proportion of sea,splA,splB,lukD and lukE.Among the common STs of S.aureus,the proportion of strains carrying 11 or more virulence genes was75.34%,and ST1 and ST45 strains were associated with carrying 16 or more virulence genes.5.Gene mutation:The mutation rate of rpoB gene of S.aureus was39.37%,and the mutation rate of gyrA,grlA and grlB genes were more than80%,and the gene mutation sites showed polymorphism.After screening for important variables,the mutation rates of MRSA at H481N and D320N of rpoB gene,Q489P of gylA gene,and H767D and I483V of grlA gene were higher than those of MSSA,but E409D and D885N of gyrA gene were lower than those of MSSA.Except that the expression of H481N of rpoB gene in the environment was higher than that of the students,the mutation rates of the other sites between the students and the environment were not statistically significant,suggesting that the mutation rates were equivalent between students and school environment.6.Genes detection of MRSA:The same resistance gene pattern of MRSA between students and the environment was blaZ-mecA-erm(C)and blaZ-mecA-erm(B)-aph(3’)-III-ant(6)-Ia.ST59/ST45/ST1 strains mainly carried blaZ-mecA-erm(C)pattern,ST59/ST338 strains mainly carried blaZ-mecA-erm(B)-aph(3’)-III-ant(6)-Ia pattern.The correspondence analysis results showed that ST59 MRSA was associated with carrying 6 or more resistance genes.The same virulence gene pattern of MRSA between students and the environment was hlgA-hlgB-hlgC-aur-scn-sak and seg-sei-sem-sen-seo-seu.ST45/ST30/ST121/ST5 strains mainly carried seg-sei-sem-sen-seo-seu pattern,ST59/ST338 strains mainly carried seb-sek-seq pattern and ST59/ST45/ST1 strains mainly carried hlg A-hlg B-hlg C-aur-scn-sak pattern.The correspondence analysis results showed that ST45 MRSA was associated with carrying 9 or more enterotoxin genes,and ST1 MRSA was associated with carrying 12 or more other virulence genes.Conclusions1.The molecular types of S.aureus are partly the same between students and environment,indicating that there may be clonal transmission.The dominant clones,resistance genes pattern,and virulence genes pattern are the same in MRSA between students and environment,which further suggests that school environment may be source of student MRSA colonization.2.There are a large difference in the number of resistance genes and virulence genes among different types of S.aureus and MRSA.ST59 strains carry more resistance genes,while ST1 and ST45 strains carry more virulence genes.There is an association between different molecular types and gene patterns of MRSA.The resistance gene pattern and virulence gene pattern are blaZ-mec A-erm(C)and hlgA-hlgB-hlg C-aur-scn-sak in ST59/ST45/ST1 strains,blaZ-mec A-erm(B)-aph(3’)-III-ant(6)-Ia and seb-sek-seq in ST59/ST338 strains,and seg-sei-sem-sen-seo-seu in ST45/ST30/ST121/ST5 strains,revealing that specific types of MRSA may carry specific genes,and providing a direction for future research on specific molecular types of MRSA.3.The gene mutation sites of rpoB,gyrA,grlA,and grlB resistance genes in S.aureus are polymorphic and the phenomenon of multi-site combined mutation is serious,suggesting that there may be new resistance mechanism in S.aureus,so it is necessary to further combine the minimum inhibitory concentration(MIC)level of antibacterial drugs to explore the relationship between gene mutations and drug-resistant phenotypes,and to offer a reference basis for control of S.aureus infection in clinical practice.