Methicillin-resistant (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of-generated TO-resistant strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the gene in SCVs or introducing the mutation found in the gene of one of the high-level TO-resistant mutants into the gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10-fold for FC04-100.

Staphylococcus aureus and Pseudomonas aeruginosa are prevalent lung pathogens in cystic fibrosis (CF). Whereas co-infection worsens the clinical outcome, prototypical strains are usually antagonistic in vitro. We sought to resolve the discrepancy between these in vitro and in vivo observations. In vitro, growth kinetics for co-cultures of coisolates from CF patients showed that not all P. aeruginosa strains affected S. aureus viability. On solid media, S. aureus slow-growing colonies were visualized around some P. aeruginosa strains whether or not S. aureus viability was reduced in liquid co-cultures. The S. aureus-P. aeruginosa interactions were then characterized in a mouse lung infection model. Lung homogenates were plated on selective media allowing colony counts of either bacterium. Overall, 35 P. aeruginosa and 10 S. aureus strains (clinical, reference, and mutant strains), for a total of 200 co-infections, were evaluated. We observed that S. aureus colonization of lung tissues was promoted by P. aeruginosa and even by strains showing antagonism in vitro. Promotion was proportional to the extent of P. aeruginosa colonization, but no correlation was found with the degree of myeloperoxidase quantification (as marker of inflammation) or with specific virulenceassociated factors using known mutant strains of S. aureus and P. aeruginosa. On the other hand, P. aeruginosa significantly increased the expression of two possible cell receptors for S. aureus, i.e., ICAM-1 and ITGA-5 (marker for integrin α 5 β 1) in lung tissue, while mono-infections by S. aureus did not. This study provides insights on polymicrobial interactions that may influence the progression of CF-associated pulmonary infections.

Tomatidine (TO), a steroid alkaloid, exerts a strong bactericidal activity on the infectionpersistent phenotype of Staphylococcus aureus, the small-colony variant (SCV), with a minimal inhibitory concentration (MIC) of 0.06 µg/ml. Also, the combination of TO to an aminoglycoside (AMG) shows a strong synergistic effect against prototypical (WT) S. aureus (MIC 0.06 µg/ml), which is otherwise unaffected by TO alone (MIC > 128 µg/ml). We have recently established that the ATP synthase (subunit AtpE) was the molecular target of TO and that TO reduces the production of ATP in S. aureus. The purpose of this study was to understand how TO and the TO-AMG combination exert bactericidal activities against S. aureus SCV and WT strains, respectively. The impact of TO and of the TO-gentamicin (GEN) combination on the membrane potential and generation of reactive oxygen species (ROS) were determined using florescent probes. GEN uptake in WT was assessed in the presence of TO. Virulence of SCV and WT strains as well as of in vitro-selected mutants showing resistance to TO or the TO-GEN combination was evaluated in a murine thigh infection model. TO causes a reduction in membrane potential in both WT and SCV, but significant amounts of ROS are only produced in SCVs. Besides, the presence of TO improves the uptake of GEN by the WT strain and the combination TO-GEN generated 2.5-folds more ROS in WT, compared to that induced by GEN alone. Under anaerobic conditions, WT adopts a fermentative slow-growth phenotype and becomes susceptible to TO even if used alone. In vivo, TO-or TO-GEN-resistant strains were significantly altered in their ability to colonize tissues. These results shed light on the mechanism of action of TO and its synergy with AMGs against S. aureus WT. TO bactericidal activity against SCVs