Shoulder
Neer Award 2017: A rapid method for detecting Propionibacterium acnes in surgical biopsy specimens from the shoulder

https://doi.org/10.1016/j.jse.2016.10.001Get rights and content

Background

Propionibacterium (P) acnes infection of the shoulder after arthroplasty is a common and serious complication. Current detection methods for P acnes involve anaerobic cultures that require prolonged incubation periods (typically 7-14 days). We have developed a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) approach that sensitively and specifically identifies P acnes in tissue specimens within a 24-hour period.

Methods

Primers were designed to amplify a unique region of the 16S rRNA gene in P acnes that contained a unique HaeIII restriction enzyme site. PCR and RFLP analyses were optimized to detect P acnes DNA in in vitro cultures and in arthroscopic surgical biopsy specimens from patients with P acnes infections.

Results

A 564 base-pair PCR amplicon was derived from all of the known P acnes strains. HaeIII digests of the amplicon yielded a restriction fragment pattern that was unique to P acnes. P acnes-specific amplicons were detected in as few as 10 bacterial cells and in clinical biopsy specimens of infected shoulder tissues.

Conclusion

This PCR-RFLP assay combines the sensitivity of PCR with the specificity of RFLP mapping to identify P acnes in surgical isolates. The assay is robust and rapid, and a P acnes-positive tissue specimen can be confirmed within 24 hours of sampling, facilitating treatment decision making, targeted antibiotic therapy, and monitoring to minimize implant failure and revision surgery.

Section snippets

Primer design and enzyme digestion site identification

Genomic sequences of the 16S ribosomal RNA genes for a broad range of Propionibacterium spp, including P acnes, P avidum, P propionicus, P acidipropionici, P theonii, P granulosum, and P lymphophilum were obtained from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). GenBank, the annotated collection of publicly available DNA sequences provided by NCBI, was used to obtain these Propionibacterium sequences in FASTA format, and they were entered into the European

PCR design, optimization, and validation

As described in the “Materials and methods,” comparative in silico analyses of the 16S rRNA gene sequences of 7 different Propionibacterium spp were performed to identify P acnes-specific targets for PCR amplification. One region of the 16S rRNA gene in P acnes that contained regions that differed from other closely related species and all other bacterial species is shown in Fig. 1. Some of these regions had been identified previously as targets for P acnes-specific PCR analyses in published

Discussion

There is an identified need for more rapid and accurate technologies to diagnose P acnes infections in patients after shoulder arthroplasty.11 The current standard for P acnes identification is a tissue swab for anaerobic culture,17 an approach that carries substantial risk of contamination from the adjacent skin and other sites where P acnes is a commensal organism. The length of time required for anaerobic culture and subsequent confirmatory analyses averages 6 or more days.17 In contrast,

Conclusion

The PCR-RFLP assay reported here is simple, robust, distinguishes between P acnes and other Propionibacterium spp, and is (theoretically) cost effective. In its current form, the assay provides proof of concept for using a PCR-RFLP approach to rapidly and consistently identify P acnes in tissues from shoulder biopsies within 24 hours. A potential additional advantage of this approach is that the minute sample size required for analysis may decrease the false-positive rate in cultures caused by

Disclaimer

Scott Holmes received financial support from the Schulich Research Opportunities Program in the Schulich School of Medicine and Dentistry at the University of Western Ontario. Kenneth J. Faber and David B. O'Gorman received support from the Department of Surgery at the University of Western Ontario through an Internal Research Fund award. George S. Athwal and David B. O'Gorman received a Canadian Orthopedics Research Legacy Award.

The authors, their immediate families, and any research

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