Combining antibiotics with antivirulence compounds can have synergistic effects and reverse selection for antibiotic resistance in Pseudomonas aeruginosa.

Antibiotics are losing efficacy due to the rapid evolution and spread of resistance. Treatments targeting bacterial virulence factors have been considered as alternatives because they target virulence instead of pathogen viability, and should therefore exert weaker selection for resistance than conventional antibiotics. However, antivirulence treatments rarely clear infections, which compromises their clinical applications. Here, we explore the potential of combining antivirulence drugs with antibiotics against the opportunistic human pathogen Pseudomonas aeruginosa. We combined two antivirulence compounds (gallium, a siderophore quencher, and furanone C-30, a quorum sensing [QS] inhibitor) together with four clinically relevant antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) in 9×9 drug concentration matrices. We found that drug-interaction patterns were concentration dependent, with promising levels of synergies occurring at intermediate drug concentrations for certain drug pairs. We then tested whether antivirulence compounds are potent adjuvants, especially when treating antibiotic resistant (AtbR) clones. We found that the addition of antivirulence compounds to antibiotics could restore growth inhibition for most AtbR clones, and even abrogate or reverse selection for resistance in five drug combination cases. Molecular analyses suggest that selection against resistant clones occurs when resistance mechanisms involve restoration of protein synthesis, but not when efflux pumps are up-regulated. Altogether, our work provides a first systematic analysis of antivirulence-antibiotic combinatorial treatments and suggests that such combinations have the potential to be both effective in treating infections and in limiting the spread of antibiotic resistance.

Harnessing bacterial interactions to manage infections: a review on the opportunistic pathogen Pseudomonas aeruginosa as a case example.

During infections, bacterial pathogens can engage in a variety of interactions with each other, ranging from the cooperative sharing of resources to deadly warfare. This is especially relevant in opportunistic infections, where different strains and species often co-infect the same patient and interact in the host. Here, we review the relevance of these social interactions during opportunistic infections using the human pathogen Pseudomonas aeruginosa as a case example. In particular, we discuss different types of pathogen-pathogen interactions, involving both cooperation and competition, and elaborate on how they impact virulence in multi-strain and multi-species infections. We then review evolutionary dynamics within pathogen populations during chronic infections. We particuarly discuss how local adaptation through niche separation, evolutionary successions and antagonistic co-evolution between pathogens can alter virulence and the damage inflicted on the host. Finally, we outline how studying bacterial social dynamics could be used to manage infections. We show that a deeper appreciation of bacterial evolution and ecology in the clinical context is important for understanding microbial infections and can inspire novel treatment strategies.

In-vivo microscopy reveals the impact of Pseudomonas aeruginosa social interactions on host colonization.

Pathogenic bacteria engage in social interactions to colonize hosts, which include quorum-sensing-mediated communication and the secretion of virulence factors that can be shared as "public goods" between individuals. While in-vitro studies demonstrated that cooperative individuals can be displaced by "cheating" mutants freeriding on social acts, we know less about social interactions in infections. Here, we developed a live imaging system to track virulence factor expression and social strain interactions in the human pathogen Pseudomonas aeruginosa colonizing the gut of Caenorhabditis elegans. We found that shareable siderophores and quorum-sensing systems are expressed during infections, affect host gut colonization, and benefit non-producers. However, non-producers were unable to successfully cheat and outcompete producers. Our results indicate that the limited success of cheats is due to a combination of the down-regulation of virulence factors over the course of the infection, the fact that each virulence factor examined contributed to but was not essential for host colonization, and the potential for negative frequency-dependent selection. Our findings shed new light on bacterial social interactions in infections and reveal potential limits of therapeutic approaches that aim to capitalize on social dynamics between strains for infection control.

Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in Pseudomonas aeruginosa.

LAY SUMMARY: We probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa. Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance. BACKGROUND AND OBJECTIVES: Treatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa. METHODOLOGY: We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance. RESULTS: We found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike. CONCLUSIONS AND IMPLICATIONS: Our work highlights that antivirulence treatments are not evolutionarily robust per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.