Search

The in vitro effect of nine antibiotic combinations was investigated in Staphylococcus epidermidis biofilms using ATP-bioluminescence for viable bacterial cell quantification. Four slime-producing (SP) strains were used to form biofilms 6, 24 and 48 h old. These biofilms were exposed for 24 h to antibiotics at 4 x, 2 x, 1 x and 0.5 x MIC. Combinations involving tetracycline together with another antibiotic were the most efficient at the biofilm age and concentration range under study. The combination vancomycin-rifampicin produced the highest bactericidal effect on 6 h biofilms at 4 x MIC, but this effect decreased dramatically in older biofilms. To detect possible antibiotic synergy in combinations that had a significant killing effect, antibiotics were studied not only in combination but also individually. Synergic effects were observed in all the combinations tested. Differences between the effect in combination and the sum of individual antibiotic effects (degree of synergy) were significant (mostly P< 0.001) and exceeded 1 log10 cfu/mL in the majority of cases. In 48 h biofilms, antibiotics caused a significant bactericidal effect when applied in combination, but never when used individually. These results indicate that the biofilm test applied allows the detection of synergy between antibiotics and suggests that this assay could be useful in clinical and extensive synergy studies on S. epidermidis biofilms.

Staphylococcus aureusbiofilms formed on medical implants represent a serious problem, being difficult to eradicate with antibiotic therapy and leading to chronic infections. Simplified in vivo and in vitro antibiotic susceptibility assays using biofilm bacteria are needed. In this work, a novel chronic osteomyelitis infection model was developed in rats in the absence of bacterial suspension, requiring the use of only 106bacteria in biofilms at the site of surgery, with a full success in reproducing infection. Stainless‐steel implants pre‐colonized for 12 h with a highly adherent S. aureusisolate were introduced into the rat tibiae. In animals not submitted to antibiotic treatment, infection was found in the implants and spread to bone in all cases, indicating the high efficacy of the model to reproduce osteomyelitis. The effect of a 21‐day treatment with cefuroxime, vancomycin, tobramycin or ciprofloxacin on infection was studied in this model 42 days after surgery. Bone colonization was inhibited by vancomycin and cefuroxime. Cefuroxime (the most efficient antibiotic, able to sterilize 1 out of 8 implants) reduced the number of bacteria in biofilms adhered to implants at a higher extent than vancomycin, trobramycin and ciprofloxacin. Analogous observations were made in this work in vivo and in vitro on the relative antibiotic efficacy against S. aureusbiofilm bacteria, suggesting the usefulness of both tests as a potential tool to study antibiotic suceptibility, and the need for new antimicrobials against these bacteria. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.

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 (> or =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.

A rat experimental osteomyelitis model was used to study the efficiency of antibiotics on biofilm bacteria adhered to implants in relation to the efficiency obtainedin vitro.In the osteomyelitis model, 104bacteria of the strain variant used for thein vitrostudies (a slime-producing variant ofStaphylococcus aureus) were inoculated into the rat tibia at surgery, after implanting a stainless steel canula precolonized for 12 h with this strain. After 5 weeks, a 21-day antibiotic treatment was applied (using cefuroxime, vancomycin, or tobramycin). Subsequently, implant and tibia were studied for presence of bacteria. In this osteomyelitis model, cefuroxime inhibited bone colonization and reduced the number of bacteria in metal and bone at a higher degree (P< 0.05) than vancomycin and trobramycin (the latter antibiotic did not have this reduction effect). Thein vitroassay was applied using three concentrations of each antibiotic (8, 100, and 500 μg/ml) and 6-, 24-, and 48-h biofilms. Bacterial viability was evaluated by ATP-bioluminescence after 24 h of antibiotic treatment. In thisin vitroassay, cefuroxime significantly (P< 0.05) reduced in all cases the number of viable bacteria in biofilms, tobramycin did not affect viability, and vancomycin affected viability except at the lowest concentration used (8 μg/ml, i.e., 8× the minimal bactericidal concentration of this antibiotic) when facing the oldest (48 h) biofilm. These results demonstrate the usefulness of the osteomyelitis model applied in providing evidence for a close correlation between thein vitroandin vivofindings on the effect of three antibiotics under study.