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Journal of Antimicrobial Chemotherapy (1999) 44, 43-55
© 1999 The British Society for Antimicrobial Chemotherapy

Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro

Beatriz Amorenaa,*, Elena Graciaa,{dagger}, Marta Monzóna, José Leivab, Concepción Oteizab, Marta Péreza, José-Luis Alabarta and José Hernández-Yagoc

a CSIC Department of Animal Health and Production, Agricultural Research Service (SIA-DGA), PO Box 727, 50080 Zaragoza b Department of Microbiology, University Clinics, 31080 Pamplona c Cytology Research Institute, Amadeo de Saboya,4, 46010 Valencia, Spain

Four slime-producing isolates of Staphylococcus aureus were used in an antibiotic susceptibility assay for biofilms developed on 96-well polystyrene tissue culture plates. The study involved 11 antibiotics, two biofilm ages (6 and 48 h), two biofilm growth media (tryptone soy broth (TSB) and delipidated milk) and three antibiotic concentrations (4 x MBC, 100 mg/L and 500 mg/L). ATP-bioluminescence was used for automated bacterial viability determination after a 24 h exposure to antibiotics, to avoid biofilm handling. Under 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 antibiotic effect was observed in milk-grown rather than TSB-grown biofilms. Phosphomycin and cefuroxime, followed by rifampicin, cefazolin, novobiocin, vancomycin, penicillin, ciprofloxacin and tobramycin significantly affected biofilm cell viability at least under some of the conditions tested. Gentamicin and erythromycin had a non-significant effect on cell viability. Transmission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofilms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grown biofilms. A reduced matrix distribution and enhanced cell density were observed as the biofilm aged. The S. aureus biofilm test discriminated antibiotics requiring shorter (3 h or 6 h) from those requiring longer (24 h) exposure and yielded results which may be complementary to those obtained by conventional tests.

* Corresponding author. Department of Animal Health, Agricultural Research Service (SIA-DGA), Montañana Road, 176, 50016, Zaragoza, Spain. Tel: +32-976-57-63-36; Fax: +32-976-57-55-01; E-mail: beatriz{at}mizar.csic.es

{dagger} Present address: Exopol S.L., Polígono Río Gállego, Calle D, Parc. 8; S. Mateo de Gállego, 50840 Zaragoza, Spain.


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