Bacteria commonly form dense biofilms encased in extracellular polymeric substances (EPS). Biofilms are often extremely tolerant to antimicrobials but their reliance on shared EPS may also be a weakness as social evolution theory predicts that inhibiting shared traits can select against resistance. Here we show that EPS of Salmonella biofilms is a cooperative trait whose benefit is shared among cells, and that EPS inhibition reduces both cell attachment and antimicrobial tolerance. We then compare an EPS inhibitor to conventional antimicrobials in an evolutionary experiment. While resistance against conventional antimicrobials rapidly evolves, we see no evolution of resistance to EPS inhibition. We further show that a resistant strain is outcompeted by a susceptible strain under EPS inhibitor treatment, explaining why resistance does not evolve. Our work suggests that targeting cooperative traits is a viable solution to the problem of antimicrobial resistance.

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21The Pseudomonas quinolone signal (PQS) is a multifunctional quorum sensing molecule of key 22 importance to the P. aeruginosa metabolism. We here describe that the lytic Pseudomonas bacterial 23 virus LUZ19 targets this population-density-dependent signaling system by expressing quorum 24 sensing-associated acyltransferase (Qst) during early infection. Qst interacts with a key biosynthesis 25 pathway enzyme PqsD, resulting in decreased metabolites levels of PQS and its precursor 2-heptyl-26 4(1H)-quinolone. The lack of a functional PqsD enzyme impairs the normal LUZ19 infection but is 27 restored by external supplementation of 2-heptyl-4(1H)-quinolone, showing that LUZ19 exploits PQS 28 to successfully achieve its infection. A functional interaction network, which includes enzymes of the 29 central carbon metabolism (CoaC/ThiD) and a novel non-ribosomal peptide synthetase pathway 30 (PA1217), suggests a broader functional context for Qst, which blocks P. aeruginosa cell division. Qst 31 represents an exquisite example of intricate reprogramming of the bacterium, which may be exploited 32 towards antibiotic target discovery for this bacterial pathogen. 33

Interference with public good cooperation provides a promising novel antimicrobial strategy since social evolution theory predicts that resistant mutants will be counter-selected if they share the public benefits of their resistance with sensitive cells in the population. Although this hypothesis is supported by a limited number of pioneering studies, an extensive body of more fundamental work on social evolution describes a multitude of mechanisms and conditions that can stabilize public behaviour, thus potentially allowing resistant mutants to thrive. In this paper we theorize on how these different mechanisms can influence the evolution of resistance against public good inhibitors. Based hereon, we propose an innovative 5-step screening strategy to identify novel evolution-proof public good inhibitors, which involves a systematic evaluation of the exploitability of public goods under the most relevant experimental conditions, as well as a careful assessment of the most optimal way to interfere with their action. Overall, this opinion paper is aimed to contribute to long-term solutions to fight bacterial infections.