With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin a macrolide antibiotic devoid of bactericidal activity on (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type but not of the and results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed. ClinicalTrials.gov “type”:”clinical-trial” attrs :”text”:”NCT00610623″ term_id :”NCT00610623″NCT00610623 Author Summary With the rising development of antibiotic resistance and rapid spread of nosocomial pathogens the search for new treatments beyond conventional antibiotics becomes a key aim of public health research. As such AC220 anti-virulence therapies might be alternative antimicrobial strategies. However consideration must be given to the potential evolutionary and environmental consequences of such interventions. Here we demonstrate a significant evolutionary impact of an anti-virulence intervention. Virulence and cooperative social behaviour of rely on the quorum-sensing (QS) controlled production of extracellular products. In the absence AC220 of a specific intervention non-cooperating (and hence less virulent) QS-mutants exploit and benefit from products provided by wild type isolates. As a consequence these less virulent AC220 mutants increase in frequency and provide a relative protection against infection to a colonized patient. In contrast when QS-gene expression is reduced by the QS-inhibiting drug azithromycin this advantage of QS-mutants is lost and virulent isolates predominate both in colonized patients and during experiments. These results suggest that QS-blockade may increase the prevalence of more virulent QS-responders among colonizing isolates in the hospital environment. More generally the impact of anti-virulence interventions on the ecology and evolution of virulence of pathogenic bacteria needs to be assessed. Introduction Anti-virulence therapies have been recently suggested as alternative strategies to circumvent the growing problem of antibiotic resistance  . In inhibition of Quorum-Sensing (QS) seems particularly attractive as QS regulates many virulence determinants of this pathogen . Azithromycin is a widely used macrolide antibiotic without significant bactericidal activity on . Recent studies suggest azithromycin might be of benefit against this bacterium because it interferes with the QS-circuit and thereby inhibits the expression of a wide range of extracellular virulence factors . Inhibition of QS is likely to have important evolutionary consequences for and studies suggest that mutants (QS-cheats) that don’t respond to QS (specifically mutants that are defective in one of the QS-receptors LasR) can have a selective advantage in the presence of QS-wildtypes  . This has been recently demonstrated during colonization of untreated colonized patients in whom QS-cheats accumulated over time . The most likely explanation for this advantage is that the mutants exploit the AC220 wild type public goods without paying the metabolic cost of their AC220 production CREBBP -; although other AC220 direct costs of QS in clinical contexts can’t be ruled out -. Regardless of the reasons why QS-mutants have a fitness advantage this advantage is unlikely to be realised if QS is blocked in wild type bacteria. Azithromycin (or any QS-blocker) will therefore reduce or remove selection for less virulent QS-cheats and maintain the predominance of more virulent QS-wild type bacteria. We tested this hypothesis by following the evolutionary dynamics of QS (mutants rapidly increased in the untreated control patients the proportion did not change in the azithromycin-treated patients. This.