Molecular detection of transcriptionally active bacteria from failed prosthetic hip joints removed during revision arthroplasty

Abstract

The purpose of this study was to use microbiological culture and bacterial 16S rRNA gene sequencing methods to detect transcriptionally active bacteria present on the surface of failed prosthetic hip joints removed during revision arthroplasty. Five failed prosthetic hip joints were sonicated to dislodge adherent bacteria and subjected to microbiological culture. Bacterial RNA was extracted from each sonicate, cDNA prepared by reverse transcription and the 16S rRNA gene amplified using universal primers. Polymerase chain reaction (PCR) products were cloned, assigned to distinct groups by restriction fragment length polymorphism (RFLP) analysis and one representative clone from each group was sequenced. Bacteria were identified by comparison of the obtained 16S rRNA gene sequences with those deposited in public access sequence databases. All five specimens were positive for the presence of bacteria by both culture and PCR. Culture methods identified species from eight genera. Molecular detection of transcriptionally active bacteria identified a wider range of species. A total of 42 phylotypes were identified, of which Lysobacter gummosus was the most abundant (31.6%). Thirty-four clones (14.5%) represented uncultivable phylotypes. No potentially novel species were identified. It is concluded that a diverse range of transcriptionally active bacterial species are present within biofilms on the surface of failed prosthetic hip joints.

PCR-hybridization after sonication improves diagnosis of implant-related infection

“…Previous use of antibiotics has been implicated as one of the main causes of this problem (Trampuz et al 2007), but other causes are also possible. To solve the problem, molecular biological techniques have been proposed in order to obtain faster and more accurate results than conventional culture (Tunney et al 1999, Sauer et al 2005, Dempsey et al 2007, Fihman et al 2007, Moojen et al 2007, Gallo et al 2008, Kobayashi et al 2008, Vandercam et al 2008, De Man et al 2009, Piper et al 2009, Achermann et al 2010, Riggio et al 2010, Marin et al 2012). Most of these reports were based on protocols that were developed in-house, which are difficult to integrate into clinical microbiology routines, even though they may give good results.…”

Biofilm-degrading enzymes fromLysobacter gummosus

“…For example, L. gummosus has been isolated from the skin of redback salamanders and was identified by culture-independent methods as the most abundant bacterial species on the surface of failed prosthetic hip joints removed during arthroplasty. 20,21 After purifying the biofilm-degrading activity using three different types of strong anion-exchange columns we identified by peptide mass fingerprinting an α-lytic protease, a β-lytic metalloendopeptidase, two isoforms of a lysyl endopeptidase (Lep1 and Lep2), a metalloprotease homologous to the hemagglutinin/proteinase from V. cholera, and the C-terminal fragments of two OmpA-like proteins.…”