Rationally Designed Pyrimidine Compounds: Promising Novel Antibiotics for the Treatment of Staphylococcus aureus-Associated Bovine Mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis, and antibiotic treatment is most often inefficient due to its virulence and antibiotic-resistance attributes. The development of new antibiotics for veterinary use should account for the One Health concept, in which humans, animals, and environmental wellbeing are all interconnected. S. aureus can infect cattle and humans alike and antibiotic resistance can impact both if the same classes of antibiotics are used. New effective antibiotic classes against S. aureus are thus needed in dairy farms. We previously described PC1 as a novel antibiotic, which binds the S. aureus guanine riboswitch and interrupts transcription of essential GMP synthesis genes. However, chemical instability of PC1 hindered its development, evaluation, and commercialization. Novel PC1 analogs with improved stability have now been rationally designed and synthesized, and their in vitro and in vivo activities have been evaluated. One of these novel compounds, PC206, remains stable in solution and demonstrates specific narrow-spectrum activity against S. aureus. It is active against biofilm-embedded S. aureus, its cytotoxicity profile is adequate, and in vivo tests in mice and cows show that it is effective and well tolerated. PC206 and structural analogs represent a promising new antibiotic class to treat S. aureus-induced bovine mastitis.

Staphylococcus aureus Small-Colony Variants from Airways of Adult Cystic Fibrosis Patients as Precursors of Adaptive Antibiotic-Resistant Mutations.

Prototypic Staphylococcus aureus and their small-colony variants (SCVs) are predominant in cystic fibrosis (CF), but the interdependence of these phenotypes is poorly understood. We characterized S. aureus isolates from adult CF patients over several years. Of 18 S. aureus-positive patients (58%), 13 (72%) were positive for SCVs. Characterization included genotyping, SCCmec types, auxotrophy, biofilm production, antibiotic susceptibilities and tolerance, and resistance acquisition rates. Whole-genome sequencing revealed that several patients were colonized with prototypical and SCV-related clones. Some clonal pairs showed acquisition of aminoglycoside resistance that was not explained by aminoglycoside-modifying enzymes, suggesting a mutation-based process. The characteristics of SCVs that could play a role in resistance acquisition were thus investigated further. For instance, SCV isolates produced more biofilm (p < 0.05) and showed a higher survival rate upon exposure to ciprofloxacin and vancomycin compared to their prototypic associated clones. SCVs also developed spontaneous rifampicin resistance mutations at a higher frequency. Accordingly, a laboratory-derived SCV (ΔhemB) acquired resistance to ciprofloxacin and gentamicin faster than its parent counterpart after serial passages in the presence of sub-inhibitory concentrations of antibiotics. These results suggest a role for SCVs in the establishment of persistent antibiotic-resistant clones in adult CF patients.

Effective Treatment of Staphylococcus aureus Intramammary Infection in a Murine Model Using the Bacteriophage Cocktail StaphLyse™.

Staphylococcus aureus causes intramammary infections (IMIs), which are refractory to antibiotic treatment and frequently result in chronic mastitis. IMIs are the leading cause of conventional antibiotic use in dairy farms. Phage therapy represents an alternative to antibiotics to help better manage mastitis in cows, reducing the global spread of resistance. A mouse mastitis model of S. aureus IMI was used to study the efficacy of a new cocktail of five lytic S. aureus-specific phages (StaphLyse™), administered either via the intramammary (IMAM) route or intravenously (IV). The StaphLyse™ phage cocktail was stable in milk for up to one day at 37 °C and up to one week at 4 °C. The phage cocktail was bactericidal in vitro against S. aureus in a dose-dependent manner. A single IMAM injection of this cocktail given 8 h after infection reduced the bacterial load in the mammary glands of lactating mice infected with S. aureus, and as expected, a two-dose regimen was more effective. Prophylactic use (4 h pre-challenge) of the phage cocktail was also effective, reducing S. aureus levels by 4 log10 CFU per gram of mammary gland. These results suggest that phage therapy may be a viable alternative to traditional antibiotics for the control of S. aureus IMIs.

Bactericidal Activity of the Bacterial ATP Synthase Inhibitor Tomatidine and the Combination of Tomatidine and Aminoglycoside Against Persistent and Virulent Forms of Staphylococcus aureus.

Tomatidine (TO), a steroid alkaloid, exerts a strong bactericidal activity on the infection-persistent 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 is attributable to both a critical drop in the membrane potential accompanied by a substantial ROS production. In the WT, TO helps GEN uptake and ROS is also important for the synergy. Acquiring resistance to TO significantly impairs virulence. The residual ATP synthase activity of SCVs might represent the Achilles' heel of persistent S. aureus.

Despite Antagonism in vitro, Pseudomonas aeruginosa Enhances Staphylococcus aureus Colonization in a Murine Lung Infection Model.

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 co-isolates 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 virulence-associated 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 α5ß1) 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.