Luu, J. M. | Search bioRxiv

bioRxiv2025

Growing Staphylococcus aureus in Synthetic Cystic Fibrosis Medium Promotes Colonization in a Murine Pneumonia Model

Staphylococcus aureus is a leading cause of bacterial infections worldwide and can lead to diseases such as osteomyelitis, skin, and lung infections in humans. Murine mouse models have been essential to the study of S. aureus virulence but are not without their limitations. In murine pneumonia models, colonization of the lungs by S. aureus are generally not well maintained. To increase the level and duration of S. aureus respiratory colonization, various methods have been employed including embedding the bacteria in agar beads and suppressing the mouses immune system. These modifications have improved colonization, but they do not accurately represent clinical infections of diseases such as cystic fibrosis (CF). We hypothesize that culturing S. aureus in a different media such as Synthetic CF Medium 2 (SCFM2) would increase colonization compared to growing the bacteria on standard rich media agar plates. We observed that culturing 6 different S. aureus strains in SCFM2 led to either a neutral or increased level of lung colonization compared to agar plates. For one strain, WU1, culturing in SCFM2 improved colonization in the oropharynx compared to agar plates and led to a sustained long-term infection in the lungs. Finally, when cultured in SCFM2 compared to agar plates, infection with WU1 led to increased inflammation in both the left and right lung lobe. Overall, we have shown that culturing S. aureus in different conditions prior to infection impacts colonization and host response. IMPORTANCEStaphylococcus aureus is a bacterial pathogen that can infect multiple anatomical sites in humans. To study S. aureus virulence, murine mouse models have been an essential tool. However, it has been difficult to establish and maintain these infections. To improve S. aureus pneumonia murine models, changes to the bacterial dose and mice have been utilized but limits its accuracy to represent clinical infections. In this study, we compared how culturing S. aureus in two different conditions prior to infection impacts colonization. We found that the effects of growing S. aureus in these different media on acute infections is strain specific. Following up with one of these strains, we showed that Synthetic Cystic Fibrosis Medium 2 (SCFM2) increases S. aureus colonization in the oropharynx and leads to a sustain long-term infection in the lungs. Additionally, we showed how culturing S. aureus in these conditions impacts the host response.

microbiology↗

bioRxiv2023

Experimentally Evolved Staphylococcus aureus Survives in the Presence of Pseudomonas aeruginosa by Acquiring Mutations in the Amino Acid Transporter, GltT

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacterial pathogens isolated from cystic fibrosis (CF) related lung infections. When both of these opportunistic pathogens are found in a coinfection, CF patients tend to have higher rates of pulmonary exacerbations and experience a more rapid decrease in lung function. When cultured together under standard laboratory conditions, it is often observed that P. aeruginosa effectively inhibits S. aureus growth. Previous work from our group revealed that S. aureus from CF infections have isolate-specific survival capabilities when cocultured with P. aeruginosa. In this study, we designed a serial transfer evolution experiment to identify mutations that allow S. aureus to adapt to the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa strain, PAO1. After 8 coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompete wild-type S. aureus when glutamate is absent from chemically-defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two bacteria are cultured together. ImportanceStaphylococcus aureus and Pseudomonas aeruginosa are the two most common bacterial pathogens that infect people with the genetic disease, cystic fibrosis (CF). They are often found together in CF-associated polymicrobial infections that are associated with worse patient prognosis. Understanding how these very different opportunistic pathogens influence each other in a shared environment is pertinent to improving the treatment of polymicrobial infections. While much attention has been brought to the interspecific interactions between S. aureus and P. aeruginosa, few studies have used experimental evolution methods to identify determinants of their competition and coexistence. Here, we use a serial transfer experimental evolution approach and identified a single genetic change associated with improved survival of S. aureus in the presence of P. aeruginosa. Our findings implicate metabolism of shared resources as an important factor in S. aureuss ability to survive in the presence of P. aeruginosa.

microbiology↗