Analysis of mixed microbial species in clinically relevant biofilms using denaturing HPLC

Abstract number: 1733_909

Jury F., Bueid A., Ollier W., Fox A., Upton M.

Objective: Many infections have a multi-species aetiology with causative agents existing in bacterial biofilms. Differentiation of these organisms usually is possible using culture based approaches. However, in an age when molecular detection and typing methods are being heralded as the future of diagnostic microbiology, they are not optimal for analysis of mixed species populations. We have previously shown that denaturing HPLC (DHPLC) has utility for rapid identification and typing of Staphylococcus aureus strains. Here we investigate the application of DHPLC to analysis of mixed bacterial species from culture and clinical swabs.

Methods: DNA recovered from cultured cells and wound or screening swabs was used as template in PCR using universal primers. The primers target the 16S rRNA genes and the forward primer carried a 40bp GC clamp to allow optimal separation during DHPLC. Conditions for DHPLC were based on those shown to be useful for separation of amplicons from environmental samples. DHPLC separation was carried out using fragments amplified from swabs known to carry single and mixed species.

Results: Amplification of 16S rDNA from strains and swabs was successful following some minor modifications. Separation of multiple peaks representing individual rDNA species was possible.

Conclusions: DHPLC has potential to be a powerful tool for separation of mixed amplification products for species identification and for analysing population dynamics in bacterial biofilms. The method could have a significant impact on direct detection of clinically relevant organisms and rapid identification for targeted patient management and antimicrobial therapy selection.