The Role Of Staphylococcus Aureus Small Colony Variants Intraosseous Invasion And Colonization In The Pathogenesis Of Periprosthetic Joint Infection

Objective:This study aimed to explore the role and mechanism of small colony variant(SCV)of Staphylococcus aureus(S.aureus)intraosseous invasion and colonization in periprosthetic joint infection(PJI),so as to provide basis for the development of novel therapeutic targets for PJI.Methods:A PJI diagnosis was made according to The American Society for Musculoskeletal Infection(MSIS)for PJI,patients were divided into acute PJI(≤ 3moths)and chronic PJI(>3 months).(1)After approved by institutional review board,synovial fluid,periprosthetic tissues and sonication fluid were collected intraoperatively for microbial culture routinely,the morphology,growth characteristics were recorded and the expression levels of virulence factors were detected when S.aureus was isolated.(2)Bone samples were also collected intraoperatively.Bone samples collected from PJI patients who infected with S.aureus were selected for further investigation.After decalcification,transmission electron microscope(TEM)and immunofluorescence(IF)were performed to observe the intraosseous invasion and colonization of S.aureus.(3)The normal phenotype and SCVs of S.aureus were collected,then the following experiments were carried out: 1)osteoblasts were infected with the normal phenotype and SCVs of S.aureus,respectively,labeled by Green fluorescent protein(GFP),and the invasion and colonization of S.aureus in osteoblasts was determination by flow cytometry(FC)and laser confocal;2)PJI mouse models were also constructed which infected by normal phenotype and SCVs of S.aureus,respectively.Then TEM and IF were performed once again to observed the presence of S.aureus in bone samples after decalcification.(4)Chronic S.aureus PJI bone tissue,in which invasion and colonization was confirmed by TEM and IF,and acute PJI bone tissue were selected for transcriptional sequencing.The sequencing results were analyzed by cluster analysis,Principal Component Analysis(PCA),Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,and the bioinformatics analysis results were verified by polymerase chain reaction(PCR).Results:(1)SCVs could be isolated from samples collected from chronic PJIs intraoperatively,with characteristics of slow growth,smaller diameter and lower expression levels of virulence factors than normal phenotypes,while SCVs could not isolated from acute PJIs.(2)TEM and IF showed the presence of S.aureus in bone samples collected from chronic PJIs,but not in bone samples from acute PJIs,which provided a potential mechanism of PJI.(3)FC and laser confocal results showed that SCVs of S.aureus were more likely to invade osteoblasts in vitro.Furthermore,TEM and IF also demonstrated that SCV of S.aureus were more likely to invade and colonize in bone samples in vivo.(4)The cluster analysis and PCA analysis showed that there were significant differences in gene expression profiles between chronic PJI and acute PJI bone samples.GO and KEGG analysis showed that these differentially expressed genes were enriched to chemokine-related signal pathways.PCR results also showed that the expression level of chemokine in chronic PJI bone tissue was significantly lower than that of acute PJI bone samples.Conclusions:The present study showed that the S.aureus SCVs has stronger ability to invade and colonize in bone.The underlying mechanism might be that SCV could reduce the expression level of chemokines in bone tissue compared with normal phenotype,so that immune cells such as neutrophils could not be recruited quickly and effectively to clear S.aureus,lest S.aureus colonized in bone tissues long-term,thus mediating the pathogenesis of chronic PJI.