Antibiotic Resistance Protocols

Manuel Alcalde-Rico, José Luis Martínez

One relevant aspect for understanding the bottlenecks that modulate the spread of resistance among bacterial pathogens consists in the effect that the acquisition of resistance may have on the microbial physiology . Whereas studies on the effect of acquiring resistance of bacterial growth are frequently performed, more detailed analyses aiming to understand in depth the cross talk between resistance and virulence, including bacterial communication are less frequent. The bacterial quorum sensing system, is an important intraspecific and interspecific communication system highly relevant for many physiological processes, including virulence and bacterial/host interactions. Some works have shown that the acquisition of antibiotic resistance may impair the quorum sensing response. In addition, some antibiotics as antimicrobial peptides can affect the production and accumulation of the quorum sensing signal molecules. Given the relevance that this system has in the bacterial behavior in the human host, it is important to study the effect that the acquisition of antibiotic resistance may have on the production of quorum sensing signals. In this chapter we present a set of methods for measuring quorum sensing signals based on the use of biosensor strains, either coupled to Thin Layer Chromatography or for performing automated luminometry/spectrophotometry assays. We use Pseudomonas aeruginosa as bacterial model because it has a complex quorum system than encloses different signals. Namely, P. aeruginosa quorum sensing system consists in three different interconnected regulatory networks, each one presenting a specific autoinducer molecule: the las system, which signal is N-(3-oxo-dodecanoyl)-L-homoserine lactone, the rhl system, which signal is N-butanoyl-homoserine lactone and the pqs system, which signals are 2-heptyl-3-hydroxy-4(1H)-quinolone together with its immediate precursor 2-heptyl-4-hydroxy-quinoline.