Assistant Professor University of Alabama at Birmingham, United States
Background: Staphylococcus aureus is one of the most common pathogens in people with cystic fibrosis (PwCF), with methicillin resistant S. aureus (MRSA) being of particular concern. As rates of methicillin sensitive S. aureus(MSSA) decrease, rates of MRSA increase with age. Interestingly, this rise in MRSA corresponds with early manifestations of CF related diabetes (CFRD), the most common comorbidity in PwCF affecting up to 50% of adults. CFRD is associated with worsening lung function and disease outcomes. Studies of non-CF populations have found that hyperglycemia is associated with increased glucose in the airways, which correlates with increased infections with Staphylococcus species, especially MRSA. Our goal is to investigate how the hyperglycemic airway environment affects S. aureus growth and pathogenesis in CF.
Methods: We cultured immortalized CF (CFBE41o-) and non-CF (16HBE) human bronchial epithelial cells at air liquid interface (ALI) for 10-14 days. A hospital associated USA100 MRSA strain was used to infect ALI cultures. Glucose concentrations in the basolateral media were maintained at 5mM or 12.5mM, to mimic the hyperglycemic lung. We collected airway surface liquid (ASL) from ALI cultures for analysis. We also co-cultured ALI cells with S. aureus by inoculating bacteria onto the apical surface. To test responsiveness to antibiotic treatment, rifampicin was added to ALI infections 6 hrs post-inoculation of S. aureus. S. aureus growth was measured by counting colony forming units (CFU). Localization and biofilm development of fluorescent S. aureus was observed via confocal microscopy.
Results: After 24 hrs of infection, the burden of S. aureus following co-culture with CF and non-CF cells in normal or hyperglycemic media did not differ significantly. However, confocal microscopy images indicated cell genotype and glycemic conditions altered development of MRSA biofilms. We observed a slight increase in overall S. aureus growth in ASL from CF cells cultured in hyperglycemic media. However, S. aureus grown in ASL from CF cells forms significantly more biofilm than in ASL from non-CF cells, regardless of glucose culture conditions. Measurement of glucose concentrations revealed CF cells have higher ASL glucose levels than non-CF cells in both normal and hyperglycemic growth conditions. Under normal glucose conditions, S. aureus exhibited decreased sensitivity to antibiotic exposure in co-culture with non-CF cells compared to CF cells. However, culture in hyperglycemic conditions promoted more frequent development of antibiotic resistance in co-culture with CF cells.
Conclusions: Our results suggest co-culture of S. aureus with CF and non-CF cells leads to different growth environments, resulting in altered biofilm formation and responses to antibiotic treatment. Culturing airway cells in hyperglycemic conditions further enhances differences in S. aureus behaviors observed. Ongoing studies are investigating why glucose homeostasis is dysregulated in CF cells and how nutrient availability or altered metabolism in the epithelium is impacting S. aureus transcriptional regulation.
Acknowledgements: This work was supported by the Cystic Fibrosis Foundation [KIEDRO18F5 and ROWE21R3].
