Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacteriumPhotorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression ofpbgPE,an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence inP. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process ofP. luminescensis based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.

Odilorhabdins (ODLs) constitute a novel antibiotic family with promising properties for treating problematic multidrug-resistant Gram-negative bacterial infections. ODLs are 10-mer linear cationic peptides inhibiting bacterial translation by binding to the small subunit of the ribosome.

NOSO-502 is a preclinical antibiotic candidate of the odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the colistin (CST)-resistant strains.

Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen X. nematophila . We demonstrated that selection occurring in aged infection favors lrp defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of X. nematophila , we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host.

Summary: The resistance‐nodulation‐division (RND)‐type efflux pumps AcrAB and MdtABC contribute to multidrug‐resistance (MDR) in Gram‐negative bacteria. Photorhabdus is a symbiotic bacterium of soil nematodes that also produces virulence factors killing insects by septicaemia. We previously showed that mdtA deletion in Photorhabdus laumondii TT01 resulted in no detrimental phenotypes. Here, we investigated the roles of the last two putative RND transporters in TT01 genome, AcrAB and AcrAB‐like (Plu0759‐Plu0758). Only Δ acrA and Δ mdtA Δ acrA mutants were multidrug sensitive, even to triphenyltetrazolium chloride and bromothymol blue used for Photorhabdus isolation from nematodes on the nutrient bromothymol blue‐triphenyltetrazolium chloride agar (NBTA) medium. Both mutants also displayed slightly attenuated virulence after injection into Spodoptera littoralis . Transcriptional analysis revealed intermediate levels of acrAB expression in vitro , in vivo and post‐mortem, whereas its putative transcriptional repressor acrR was weakly expressed. Yet, plasmid‐mediated acrR overexpression did not decrease acrAB transcript levels neither MDR in TT01 WT. While no pertinent mutations were detected in acrR of the same P. laumondii strain grown either on NBTA or nutrient agar, we suggest that AcrR‐mediated repression of acrAB is not physiologically required under conditions tested. Finally, we propose that AcrAB is the primary RND‐efflux pump, which is essential for MDR in Photorhabdus and may confer adaptive advantages during insect infection.