Abstract
Propionibacterium acnes infection of the shoulder after arthroplasty is a common complication. Current detection methodologies for P. acnes involve prolonged anaerobic cultures that can take up to three weeks before findings can be reported. Our aim was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach that is both sensitive and specific to P. acnes that would enable a 24-hour turnaround between biopsy and results.
Comparisons between the 16S ribosomal sequences of P. acnes and closely related bacteria identified two unique regions in P.acnes to which PCR primers were designed. Additionally, two unique restriction enzyme cut sites for HaeIII were identified within this amplicon. To test the PCR method, arthroscopic surgical biopsies were mechanically homogenised and boiled for 20 minutes to lyse the cellular membranes. PCR was performed using standard conditions followed by a one hour HaeIII enzymatic digest of the PCR product. Resultant fragments were visualised on polyacrylamide gels stained with ethidium bromide. All experiments included no-template controls to rule out reagent contamination and independently confirmed P. acnes DNA as a positive control. Serial dilutions of P. acnes cultures in Robertson's cooked-meat broth and spectrophotometric analysis of cellular concentration were used to assess the sensitivity of the PCR reaction.
A unique 564 base-pair PCR amplicon was derived from different strains of P. acnes. This amplicon was confirmed as P. acnes DNA by gel excision and DNA sequencing. HaeIII digests of the amplicon yielded 3 restriction fragments at the sizes predicted by in silico analyses. Sensitivity testing confirmed that as few as 10 P. acnes cells in a 50µl reaction volume could be detected using this assay. P. acnes was also detected in surgical biopsy samples.
P. acnes infections following shoulder arthroplasty are a serious complication placing a burden on the healthcare system and the patient due to the lengthy surgical revision process that follows. The infections are also difficult to diagnose. This unique assay combines the sensitivity of PCR with the specificity of RFLP mapping to specifically identify P. acnes in surgical isolates. We anticipate that this assay will allow us to determine if a biopsy is P. acnes positive within 24-hours of sampling, allowing for more aggressive antibiotic therapy and monitoring to avoid implant failure and revision surgery. Additionally, this PCR-RFLP method may decrease the false positive rate of extended length cultures due to P. acnes contamination.
