Clinical Biofilms – Current Concepts and Advanced Techniques
ISBN: 978-989-97478-4-5, Universidade do Minho – DEB, Braga (Portugal), 2014, pp: 173-192,
Almeida,C; Rodrigues CF; Jorge P; Costa AR.
Quorum Sensing (QS) involves the production, release, detection, and response to secondary metabolites that act as small hormones and are termed auto-inducers. These metabolites enable microorganisms to ‘sense’ cell density and alter their behavior on a population-wide scale in response to environmental factors, such as nutrient availability, toxic compounds and host–immune responses. N-acyl homoserine lactones (HSLs) are part of a basic QS circuit that has been firstly described for Vibrio fischeri, the LuxI/LuxR system, and that is very similar throughout Gram-negative bacteria. HSLs secreted by bacteria usually differ in the length of the acyl-chain moiety, saturation and substitution on the third carbon by either oxo or hydroxyl group. For instance, in P. aeruginosa the two main HSLs produced are the short-chain molecule N-butanoyl-l-homoserine lactone (C4-HSL) and the long-chain molecule N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), which are involved in coordinately regulating virulence and secondary metabolite production. While no gram-positive HSL producers have been reported yet, it is known that some HSL can interact and modulate the behavior of gram-positive species. For instance, long-chain AHLs, such as 3-oxo-C12-HSL produced by P. aeruginosa, are capable of interacting with the S. aureus, reducing exotoxin production and down-regulating cell surface colonization proteins. In this practical class we will focus on the production of HSLs on mixed biofilm populations. As a case study, a mixed biofilm of P. fluorescens and Staphyloccocus epidermidis, will be evaluated. Crude extracts of the mixed biofilm supernatants will be evaluated for the presence of HSL and compared with those of single species biofilms. First, a fractionation procedure, that will provide a partial purification of the biofilm supernatant, will be presented. Then, screening procedures for HSL and their isolation by High Pressure Liquid Chromatography (HPLC) will be addressed.