Assessing Phagolysosomal Escape of Staphylococcus epidermidis RP62A in Intracellularly Infected Human Fibroblasts. (4th October 2017)

• Record Type: Journal Article
• Authors: Perez, Kimberly; Patel, Robin
• Is Part Of: Open forum infectious diseases. Volume 4(2017)Supplement 1
• Page Start: S225
• Page End: S226
• Publication Date: 2017-10-04
• Subjects: Communicable diseases -- Periodicals; Medical microbiology -- Periodicals; Infection -- Periodicals; 616.9
• DOI: 10.1093/ofid/ofx163.468
• Languages: English
• ISSNs: 2328-8957

Abstract

Abstract: Background: S. epidermidis causes chronic prosthetic joint infection; identifying how it can evade immune killing and persist to cause chronic infection will inform strategies to prevent and treat S. epidermidis PJI. Previously, we showed that S. epidermidis clinical PJI isolates can persist intracellularly in fibroblasts and osteoblasts, and that they reside primarily within phagolysosomes. Others have shown that some S . Aureus strains are able to escape the phagolysosome and enter the cytoplasm. Here, we used intracellular pH monitoring to assess whether S. epidermidis RP62A can similarly escape the phagolysosome. Methods: S . A ureus strain 6850—known to escape the phagolysosome—and S. epidermidis RP62A were grown overnight in TSB at 37°C and labeled with FITC. Human lung fibroblast MRC5 cells were infected at a multiplicity of infection of 15. Cells were infected for either 2 or 6 hours. Cells were then incubated in the presence or absence of chloroquine and analyzed by flow cytometry. The invasion rate with chloroquine (total internalized bacteria), expressed as arbitrary fluorescence units (AFU), was determined and normalized to the mean fluorescence intensity of each corresponding bacterial preparation. Experiments were done in triplicate and the average AFU were used. Because the quenching of FITC fluorescence is proportional to acidic pH, the difference in AFU (ΔAFU) in the presence or absence of chloroquine, normalized to the invasion rate, was used as readout for pH. Results: Results are shown in the figure. At 2 hours post infection, both S . A ureus 6850 and S. epidermidis RP62A were in acidic compartments. At 6 hours post infection, S . A ureus 6850 appeared to be in the cytoplasm, as indicated by a pH close to neutral. In contrast, the environment of S. epidermidis RP62A had become more acidic suggesting lack of phagolysosomal escape. Conclusion: Differences of ΔAFU suggest escape of S . A ureus 6850 but not S. epidermidis RP62A from a phagolysosomal to a cytoplasmic environment 6 hours following infection of MRC5 cells. As not all S . A ureus strains can escape the phagolysosome, with some requiring longer times before escaping, more isolates and longer time points are needed to definitely conclude that S. epidermidis cannot escape the phagolysosome. Disclosures: R. Patel, ASM: Board Member, None. CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Grant recipient. Curetis: Consultant, Monies paid to my employer. A patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Patents, Patents, any money is paid to my employer. Actelion: DSMB, Money paid to my employer ASM and IDSA: Editor's stipends, Editor's stipends. NBME, Up-to-Date and the Infectious Diseases Board Review Course: NBME, Up-to-Date and the Infectious Diseases Board Review Course, Honoraria. Roche, ASM, and IDSA: Travel reimbursement, Travel reimbursement