Characterization of microbes in prosthetic joint specimens by culture-independent molecular methods

Prosthetic joint infection (PJI) is one of the most challenging complications of joint alloplasty. Formation of biofilm is a prominent feature of PJIs and constitutes a challenge to current sampling procedures and culture practices to obtain a reliable diagnosis. The aim of the study was to investigate the microbialdiversity in surgical samples (eg. synovial fluid, periprosthetic tissue, removed prosthesis) from 22 prosthetic patients using a range of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH).Concomitant samples were cultured by standard methods. Overall, the results of culture-based and molecular methods showed concordant results for 13 patients and discrepant results for 6 patients. In the remaining cases, culture methods identified one species or a group of bacteria (e.g., coryneform rods),while molecular methods detected several distinct species including the species identified by culture. Broad range 16S rRNA gene PCR and cloning detected 39 bacterial species from 11 patients, including known pathogens and species not previously reported in PJIs, and polymicrobial communities were found in 9patients. Culture-based methods detected bacteria in 7 patients, and multiple species were only isolated from one of them. Additionally, quantification of Propionibacterium and Staphylococcus aureus using specific qPCR assays confirmed the findings by clone library approach, and FISH and confocal scanning laser microscopy visualized the presence of both single cells and microcolonies. In conclusion, cultureindependent methods identified more species and more polymicrobial infections than standard culturebasedmethods. Little is presently known about the pathogenesis of these microorganisms, their interactions and their roles in PJIs.