Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro

Four slime-producing isolates of Staphylococcus aureus were used in an anti biotic susceptibility assay for biofilms developed on 96-well polystyrene t issue culture plates. The study involved 11 antibiotics, two biofilm ages ( 6 and 48 h), two biofilm growth media (tryptone soy broth (TSB) and delipid ated milk) and three antibiotic concentrations (4 x MBC, 100 mg/L and 500 m g/L). ATP-bioluminescence was used for automated bacterial viability determ ination after a 24 h exposure to antibiotics, to avoid biofilm handling. Un der the conditions applied, viability in untreated biofilms (controls) was lower when biofilm growth was attempted in milk rather than in TSB. Various antibiotics had a greater effect on viability when used at higher (,100 mg /L) antibiotic concentrations and on younger (6 h) biofilms. Increased anti biotic effect was observed in milk-grown rather than TSB-grown biofilms. Ph osphomycin and cefuroxime, followed by rifampicin, cefazolin, novobiocin, v ancomycin, penicillin, ciprofloxacin and tobramycin significantly affected biofilm cell viability at least under some of the conditions tested. Gentam icin and erythromycin had a non-significant effect on cell viability. Trans mission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofil ms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grown biofilms. A reduced matrix distribution and enh anced cell density were observed as the biofilm aged. The S. aureus biofilm test discriminated antibiotics requiring shorter (3 h or 6 h) from those r equiring longer (24 h) exposure and yielded results which may be complement ary to those obtained by conventional tests.