Sonication of orthopedic implants has been used to increase biofilm detection by culture, presumably by causing the detachment of firmly adhered biofilm bacteria into

the sonicate, which may then be cultured (Trampuz et al., 2007; Esteban et al., 2008). It has been estimated that up to 13 million Americans per year suffer from microbial infections with a biofilm buy EPZ-6438 involvement (Wolcott et al., 2010). We have used modern molecular techniques for the detection and direct identification of bacteria in chronic biofilm infections of the middle ear (Post et al., 1996), and we have confirmed these data by direct observations of the bacteria in the infected tissues using rRNA-specific probes (Hall-Stoodley et al., 2006). These FISH probes consist of oligonucleotides that match variable regions of

the 16S rRNA gene of bacteria, and they provide both visualization of the cells and unequivocal identification at the genus or the species level (Moter & Gobel, 2000). In other surgical areas, we have examined culture-negative infections of sutures (Kathju et al., 2010) and of orthopedic hardware (Stoodley et al., 2008), and have detected and identified bacteria using PCR-based methods (Stoodley et al., 2008) and visualized the infecting organisms using FISH probes (Kathju et al., 2009). Because PCR-based methods for bacterial detection and identification and the FISH probes operate independently, bacteria can be detected and identified by the former (with their high Ponatinib mouse sensitivity), and this detection and identification can be confirmed (and the cells visualized) by the latter. We use confocal microscopy

of the tissues and prosthetic surfaces themselves as our definitive evidence of infecting bacteria and biofilm formation, because (1) cells must be firmly adhered to withstand the multiple rinsing steps and (2) the presence of aggregates of bacteria in a biofilm is strong evidence of a ‘growth in place process’ (Hall-Stoodley & Stoodley, 2009). To further maximize our confidence that we have detected an active infection, we use reverse transcriptase (RT)-PCR to identify bacterial mRNA, which is highly labile. The half-life of the housekeeping genes we use, hut and gap, is <5 and <15 min, respectively (Roberts crotamiton et al., 2006); thus, evidence of these mRNA species may be taken as evidence of bacterial viability, because in the absence of cell integrity, they would be rapidly degraded and lost. In the present study, we have added the new Ibis universal biosensor technology to PCR-based molecular methods for the detection and identification of bacteria, because of the potential of this technique to provide rapid and accurate data to support clinical decisions without the need for a priori supposition of the causative agents involved.