A Novel Cell-Associated Protection Assay Demonstrates the Ability of Certain Antibiotics To Protect Ocular Surface Cell Lines from Subsequent Clinical Staphylococcus aureus Challenge

UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15208, USA.
Antimicrobial Agents and Chemotherapy (Impact Factor: 4.48). 05/2011; 55(8):3788-94. DOI: 10.1128/AAC.01828-10
Source: PubMed


In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 μg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 μg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 μg/ml, whereas AZM displayed toxicity to one cell line at 512 μg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Download full-text


Available from: Eric G Romanowski
  • Source
    • "Cytotoxicity assays were conducted as described [24] with some modifications, Human corneal-limbal epithelial (HCLE) [25] cells were cultured in 24-well plates until they were confluent. HCLE cells were grown in Keratinocyte serum-free medium (KSFM) with L-Glutamine, supplemented with 25 µg/ml BPE, 0.2 ng/ml EGF, and 1 mM CaCl2. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ocular infections are a leading cause of vision loss. It has been previously suggested that predatory prokaryotes might be used as live antibiotics to control infections. In this study, Pseudomonas aeruginosa and Serratia marcescens ocular isolates were exposed to the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. All tested S. marcescens isolates were susceptible to predation by B. bacteriovorus strains 109J and HD100. Seven of the 10 P. aeruginosa isolates were susceptible to predation by B. bacteriovorus 109J with 80% being attacked by M. aeruginosavorus. All of the 19 tested isolates were found to be sensitive to at least one predator. To further investigate the effect of the predators on eukaryotic cells, human corneal-limbal epithelial (HCLE) cells were exposed to high concentrations of the predators. Cytotoxicity assays demonstrated that predatory bacteria do not damage ocular surface cells in vitro whereas the P. aeruginosa used as a positive control was highly toxic. Furthermore, no increase in the production of the proinflammatory cytokines IL-8 and TNF-alpha was measured in HCLE cells after exposure to the predators. Finally, injection of high concentration of predatory bacteria into the hemocoel of Galleria mellonella, an established model system used to study microbial pathogenesis, did not result in any measurable negative effect to the host. Our results suggest that predatory bacteria could be considered in the near future as a safe topical bio-control agent to treat ocular infections.
    Full-text · Article · Jun 2013 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK). Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours. Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test. Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process.
    Full-text · Article · May 2013 · Clinical ophthalmology (Auckland, N.Z.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Antibiotic resistance is a world-wide problem of systemic medicine, but it appears to be less problematic in the treatment of ophthalmic infections. The use of topical administration and intraocular injection of antibiotics provides very high antibiotic concentrations in the ocular tissues. These high concentrations are effective in treating bacteria that are deemed resistant using standard interpretations of susceptibility. Although in vitro bacterial resistance, based on serum standard interpretation, is reported for ocular bacteria, these isolates are generally not spread from patient-to-patient and there are antibiotics available to effectively treat and overcome the false resistance.
    No preview · Article · Jan 2014 · Expert Review of Ophthalmology
Show more