Peptide Nucleic Acid Probes for Detection of Bacteria in Biofilms

Biofilms provide a reservoir of potentially infectious microorganisms, that are resistant to antimicrobial agents and, when adhered to biomaterials, can cause failure of medical devices. Fluorescent in situ hybridisation (FISH) is a technique that can be used to identify biofilm microorganisms including those encountered in the oral cavity. Objectives: To develop a FISH assay for detection and determination of the spatial distribution of specific bacteria, within microbial biofilms prepared on synthetic material (PTFE) and host tissue (reconstituted human epithelium; RHE). Methods: The study employed Peptide Nucleic Acid (PNA) probes in conjunction with confocal laser scanning microscopy (CLSM). The specificity of PNA probes for Staphylococcus aureus, Pseudomonas aeruginosa and universal bacterial detection was initially determined using planktonic bacterial preparations. Subsequently, biofilms comprising of target and non-target species (Micrococcus luteus and Streptococcus oralis) were prepared on PTFE discs in a constant depth film fermenter, and on reconstituted human epithelium (SkinEthic Laboratories). Biofilms were washed with PBS and PNA probes (100-400nM) applied for 1 h at 55°C. Sections of these stained biofilms were then viewed by CLSM. Results: The specificity of the PNA probes was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. The identification of individual target bacteria within the biofilms was also evident, confirming the suitability of the approach for biofilm analysis. Conclusions: The development of this standardised procedure makes available an assay for simultaneous identification of single or multi-species bacterial populations using distinctly labelled PNA probes. The protocol presented gives a reliable basis to design and perform PNA FISH analysis on both bacterial biofilms and infected tissue.