2218. Low pH and an Intracellular Environ Induce Staphylococcus epidermidis Small Colony Variant Formation
Session: Poster Abstract Session: Microbial Pathogenesis
Saturday, October 29, 2016
Room: Poster Hall
Posters
  • PerezIDWeek.pdf (634.8 kB)
  • Background: Small colony variants (SCVs) are naturally occurring, slow-growing subpopulations of bacteria that emerge in response to diverse environmental pressures. The slow growth of SCVs may compromise antimicrobial activity. In addition, their intracellular survival may shield them from the host immune system and from extracellularly active antimicrobials. Moreover, SCVs may be undetected by conventional diagnostic tests. This may result in discontinuation of antimicrobial treatment before SCVs are effectively cleared, resulting in their persistence. It is unclear how SCVs are induced and maintained in patients. We assessed the effect of low pH and intracellular growth on the selection of S. epidermidis SCVs in vitro.

    Methods: S. aureus strain 6850 (known to form SCVs under low pH conditions), S. epidermidis strain RP62A and two S. epidermidis prosthetic joint infection isolates, IDRL-8933 and IDRL-8864, were studied. Bacteria were grown in EMEM supplemented with 10% FBS at 37°C in 5% CO2 for 5 days with quantitative cultures performed on days 0, 3, and 5. In addition, human lung fibroblast MRC5 cells were infected at a MOI of 1 for intracellular assay. MRC5 cells were washed daily with PBS and lysostaphin (to kill extracellular bacteria). At 0, 3, 5 and 7 days post infection, host cells were lysed and serial 10-fold dilutions of the cell lysates plated onto blood agar plates to detect and quantitate intracellular bacteria. CFU were enumerated after overnight incubation at 37°C and the colony phenotype determined after an additional incubation overnight at room temperature. SCVs were identified on the basis of their size, reduced pigmentation and reduced hemolysis.

    Results: The frequency of SCVs was increased over time as the pH in the medium was decreased for all four strains tested (Figure). Additionally, the frequency of SCVs increased in the intracellular environment of human lung fibroblast cells. After 3 hours of infection, the frequency of SCVs was below 7% for each strain. By day 5, more than 50% of the viable intracellular bacteria were SCVs for all four strains tested.

    Conclusion: Low pH and an intracellular environ promote the formation of SCVs. Acidic environments, such as within lysosomes or phagosomes, may induce S. epidermidis SCV-formation.

    Figure. Induction of small-colony variants (SCVs) during exposure to low pH

    Kimberly Perez, BS, Department of Immunology, Mayo Clinic, Rochester, MN and Robin Patel, MD, FIDSA, D(ABMM), Divisions of Clinical Microbiology and Infectious Diseases, Mayo Clinic, Rochester, MN

    Disclosures:

    K. Perez, None

    R. Patel, None

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