title: Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study.
abstract: The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance of Staphylococcus aureus biofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0-100 microg ml(-1) at 37 degrees C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells - most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance of S. aureus biofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated with S. aureus biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.
authors.affiliation: Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
authors.forename: Rachna
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
authors.forename: Pallab
authors.initials: P
authors.lastname: Ray
authors.affiliation: Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
authors.forename: Anindita
authors.initials: A
authors.lastname: Das
authors.affiliation: Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
authors.forename: Meera
authors.initials: M
authors.lastname: Sharma
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial; D008826:Microbial Sensitivity Tests; D013211:Staphylococcus aureus / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents