Abstract
Purpose :
To investigate the hypothesis that Staphylococcus aureus keratitis isolates have antibiotic tolerance mechanisms including persister cell formation and biofilm formation. Bacterial keratitis is the most common cause of infectious keratitis seen in patients and can result in permanent vision loss. S. aureus is one of the most agents, causing up to 40% of all bacterial keratitis cases. Bacteria have antibiotic resistance-independent mechanisms that increase survival when exposed to extracellular stresses like antibiotics and antimicrobial peptides. These include biofilm formation and persister cell formation. While the in vitro biofilm formation of ocular isolates has been evaluated, persister cell formation of ocular isolates remains not well understood. We tested the hypothesis that clinical S. aureus keratitis isolates will display differential ability to form biofilms in vitro, and high biofilm growers will display increased tolerance to clinically used antibiotics.
Methods :
Biofilms were formed on tissue culture treated plates and stained using crystal violet dye. Levels were determined by spectrophotometric reading at 590 nM. Antibiotic susceptibility was determined using MIC test strips on Meuller-Hinton agar. Persister cells were quantified by a modified Kirby-Bauer disc diffusion assay.
Results :
S. aureus keratitis clinical isolates (n=96) biofilm growth was ranked for biofilm formation level. Susceptibility of S. aureus (n=10) was cefazolin- 60%, tobramycin- 90%, vancomycin- 100%, and moxifloxacin- 70%). S. aureus isolates (n=6) which were the highest biofilm growers displayed increased persister cells formation frequency across all antibiotics tested compared to low biofilm formers (bottom 25% of biofilm formation). This was statistically significant for vancomycin (P=0.019) and moxifloxacin (P=0.038), but not cefazolin (P=0.053) or tobramycin (P=0.125) (Figure 1). Biofilm growth and tolerance were correlated for cefazolin r=0.94, vancomycin- r=0.88, and moxifloxacin r=0.83 but not tobramycin r=0.66 using Pearson correlation coefficients.
Conclusions :
S. aureus keratitis isolates displayed multiple antibiotic tolerance mechanisms. Persister cells were able to survive treatment with keratitis-relevant antibiotics. Interestingly, strains which were better at forming biofilms had an increased ability to form persister cells. Persister cells may contribute to chronic or recurring ocular infections.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.