Characterization Of Staphylococcus Aureus Strains With An Incomplete Hemolytic Phenotype

ObjectiveStaphylococcus aureus（S.aureus）is one of the important pathogenic bacteria,causing acute suppurative infection and can spread to tissues or transfer to other parts by bacteremia metastasis.S.aureus is a common pathogen causing hospital and community acquired infection.Hemolysin is an important virulence factor of S.aureus,which can cause typical beta hemolysis and the phenotype is called complete hemolytic phenotype.Since 2013,S.aureus strainswith an incomplete hemolytic phenotype were separated from clinical samples in this hospital,which has not been reported before.The phenotype of incompelete hemolysis was significantly different from complete hemolysis.This article focused on characterization of the hemolysis,transcription and expression of genes encoding hemolysin,drug resistance and virulence genes of S.aureus strainswith an incomplete hemolytic phenotype.The epidemic trend of the strains was further analyzed by molecular typing.Our results provided theoretical and experimental basis for the clinical effective treatment,prevention and control of infection caused by the special S.aureus strains.Methods1.Hemolytic characteristics of Staphylococcus aureus strains with an incomplete hemolytic phenotype（1）Staphylococcus aureus strains with an incomplete hemolytic phenotype were inoculated on different commercial blood platesBacterial cultures were inoculated in plates produced by different companies（bioMerieux,Antu,kemajia）and the self-made Columbia sheep blood agar plates.The inoculated plates were incubated at 35℃under different oxygen concentrations for 24h.After continuous passage,we observed the hemolysis phenomenon.At the same time,the standard strain ATCC25923 which with an complete haemolytic phenotype of Staphylococcus aureus was used as a control.（2）The dectecion of the mRNA expression of hemolysin by qRT-PCRHemolysin is an exotoxin.The standard strain,complete hemolytic strain and incomplete hemolytic strain were digested by lysostaphin,then total RNA were extracted,purified and reverse treanscripted to cDNA.The expression of hla,hlb,hlc and hld was detected by RT-PCR.The relative expression level of these genes in the standard strain,complete hemolytic strain and incomplete hemolytic strain was calculated by 2^{-ΔΔC t} and statistics was analyzed by Student’s t test.（3）The detection of the expression of Hla by Western blotTo verify whether the expression of hemolysin proteins consistent with the expression of the genes encoding the proteins,we detected the expreesion of Hla by Western blot.Briefly,we extracted the bacterial proteins,separated the proteins by PAGE electrophoresis,and then transferred the proteins to a nitrocellulose membrane.After blocked by 5%skim milk,we add Hla polyclonal antibody to the membrane and cultured overnight.After washed the membrane with HRP labled anti-IgG antibody,we added chromogenic substrate.The result was observed by chemiluminescence imager,a positive band was33KD.2.Drug resistance and virulence genes of Staphylococcus aureus strains with an incomplete hemolytic phenotype（1）Detection of drug resistance of Staphylococcus aureus stains with an incomplete hemolytic phenotypeThe Staphylococcus aureus stains with an incomplete hemolytic phenotype and the strains after 10 consecutive passage were tested for drug resistance by BD Phoenix-100System.The results were judged according to the clinical and Laboratory Standards Institute（CLSI）standard.The methicillin-resistant Staphylococcus aureus（MRSA）was defined as minimum inhibitory concentration（MIC）value of cefoxitin≥8μg/ml or the MIC value of oxacillin≥2μg/ml according to CLSI standard.（2）Detection of mecA,pvl and tst-1 genes in Staphylococcus aureus strains with an incomplete hemolytic phenotype by PCRThe mecA,pvl and tst-1 genes in Staphylococcus aureus strains with an incomplete hemolytic phenotype were amplified by PCR and sequenced.The results were compared by GenBank.3.Multilocus sequence typingWe obtained the sequences of the 7 house keeping genes of Staphylococcus aureus by MLST database and designed the primers for MLST.We amplified the house keeping genes,sequenced them and analyzed the sequence by MLST database.We got the clone types of 50 Staphylococcus aureus strains with an an incomplete hemolytic phenotype and analyzed the epidemiology.Resluts1.Hemolytic characteristics of Staphylococcus aureus strains with an incomplete hemolytic phenotype（1）All the 50 Staphylococcus aureus strains with an incomplete hemolytic phenotype collected in the clinical samples showed the phenomenon of incomplete hemolysis after 24h incubation.So as the strains after 10 continuous passages.（2）Took the standard strain ATCC25923 as a reference,the mRNA expression of hla,hlb,hlc and hld of Staphylococcus aureus strains with an incomplete hemolytic phenotype was statistically significant.The expression of genes encoding alpha,gamma,delta hemolysin were down-regulated,the expression of the gene encoding beta hemolysin was up-regulated.（3）Western blot showed that the expression of incomplete haemolytic phenotype of Staphylococcus aureus hemolysin Hla protein level is lower than the standard strain and complete hemolysis strains,the results were consistent with the mRNA gene expression level.2.Drug resistance and virulence genes of Staphylococcus aureus strains with an incomplete hemolytic phenotype（1）The drug resistance to cefoxitin and oxacillin of Staphylococcus aureus strains with an incomplete hemolytic phenotype and the strains after passaging were identical,all the tested strains were MRSA.（2）All the 50 Staphylococcus aureus strains with an incomplete hemolytic phenotype carried drug resistance gene mecA and virulence gene pvl and tst gene.3.Multilocus sequence typingWe identified the allotypes of Staphylococcus aureus strains with an incomplete hemolytic phenotype,all the 50 tested strains in this study were MRSA ST5.ConclusionsExcluded external factor,the incompleted phenomenon of the Staphylococcus aureus strains with an incomplete hemolytic phenotype in this study was stable.The expression of genes encoding alpha,gamma,delta hemolysin were down-regulated,the expression of the gene encoding beta hemolysin was up-regulated.The 50 Staphylococcus aureus strains with an incomplete hemolytic phenotype in this study all carried mecA,pvl and tst genes,which implied that these are high virulent MRSA and all of them are ST5 clones,which is a common clone type of MRSA in the world...