Impact of Bacterial Biofilm Formation on In Vitro and In Vivo Activities of Antibiotics

Bacteria.Two strains of S. epidermidis have been used: a well-described adherence-positive wild-type strain (S. epidermidis RP62A [ATCC 35984] [4, 14, 15]) and its adherence-negative mutant (S. epidermidis M7, described by Schumacher Perdreau et al. [21]).

Drug susceptibility.MICs and minimum bactericidal concentrations (MBCs) were determined with both suspended and adherent bacteria. MICs were determined in both (i) tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) supplemented with 50 μg of Ca²⁺ per ml and 25 μg of Mg²⁺ per ml (TSB-S) and (ii) phosphate-buffered saline (PBS)-GCP (PBS-GCP is 2.38 g of Na₂HPO₄, 0.19 g of KH₂PO₄, 8.0 g of NaCl, 0.9 g of glucose, 1.0 g of Casamino Acids, and distilled water to 980.0 ml, which was separately autoclaved; 20.0 ml of sterile human plasma was then added to the mixture). The suspended bacteria were tested by a macrodilution method with a standard inoculum of 10⁶ CFU/ml (1). MBCs were determined as described by Amsterdam (2). The lowest antibiotic concentration that reduced the inoculum by ≥99.9% was defined as the MBC. The susceptibility of adherent bacteria was tested by a previously described macrodilution method (25, 29) with an inoculum of 5.5 × 10⁵ CFU/bead. The MBCs were the lowest antibiotic concentrations that killed ≥99.9% of the adherent bacteria.

Animal model.The previously described guinea pig tissue cage model was used (30). In brief, four sterile polytetrafluoroethylene (Teflon) tubes (32 by 10 mm) perforated with 130 regularly spaced 1-mm-diameter holes (Ciba-Geigy Ltd.) were each filled with six sinter-glass beads (Sikug 023/300 A; Schott Schleifer AG, Muttenz, Switzerland) and were aseptically implanted in the flanks of albino guinea pigs (weight, 600 to 1,100 g). Experiments started after complete healing of the wound (3 weeks after surgery). Prior to each experiment, the interstitial fluid that accumulated in the tissue cages was checked for sterility. Tissue cages were infected by local inoculation of about 10⁷ CFU. Antibiotic therapy was started 16 h after inoculation. At this time the number of suspended bacteria averaged 5.99 ± 0.3 log₁₀ CFU/ml for strain M7 and 6.02 ± 0.44 CFU/ml for strain RP62A. The kinetics of each drug were measured in 10 to 12 tissue cage fluid samples from three animals. The kinetics were determined at up to seven time points (time zero to 24 h). In this report only the peak and the trough levels are given. The average time course of the measured concentrations was used as a reference to stimulate the kinetics in vitro. Therefore, pharmacokinetic measurements were performed for a total of 12 animals.

In vitro model.A previously described one-compartment pharmacokinetic model was used in the present study (22, 25). This model allows periodic assessment of the bactericidal effect of antibiotic dosing against both adherent and suspended bacteria. A peristaltic pump continuously transported sterile culture medium from the reservoir into the 17-ml culture compartment resulting in an exponential decrease in the drug concentration. The bacteria were exposed in the model to oscillating drug levels simulating the kinetics determined in the cages implanted in the animals.

The system was filled with a special culture medium referred to here as PBS-GCP. In contrast to standard culture medium this medium supports growth at only a limited rate and results in a reduced final bacterial density. The generation time in this medium was 64 min for strain M7 and 81 min for strain RP62A, whereas a generation time of 26 min was observed for both strains in TSB.

The intention was to achieve in the in vitro model an inoculum of suspended bacteria with numbers similar to the numbers in vivo at the onset of treatment. To obtain such an in vitro inoculum, glass beads were exposed to the bacteria in a flask for 16 h before the onset of treatment. This simulated the 16-h delay between in vivo inoculation and the start of therapy. Sterile sinter-glass beads (Sikug 023/300 A; Schott Schleifer AG) were placed for 16 h in a bacterial suspension of 25 ml of PBS-GCP inoculated with three bacterial colonies taken from a blood agar plate (working cultures). The beads covered with bacterial biofilm were separated from the PBS-GCP of the overnight culture by filtration and were rinsed with 10 ml of sterile saline solution to wash off the bacteria suspended within the broth carried over from the culture. Subsequently, 10 beads were placed into the culture compartment with a sterile surgical forceps 30 min before the start of antibiotic treatment. The inocula in the suspensions in the model present at the beginning of antibiotic dosing averaged 5.61 ± 0.37 and 5.20 ± 0.26 log₁₀ CFU/ml for strains M7 and RP62A, respectively. The number of adherent bacteria per glass bead averaged 5.91 ± 0.48 and 5.65 ± 0.25 log₁₀CFU/bead for strains M7 and RP62A, respectively. Control experiments demonstrated stationary-phase conditions. The number of suspended bacteria as well as the number of adherent bacteria of both strains did not change by more than 0.66 log over an incubation period of 48 h in the in vitro model. Each drug and both strains have been tested with three animals with four tissue cages containing a total of 24 beads. The numbers of CFU in one animal were determined at the beginning of the treatment, and the numbers of CFU in a second animal not treated with an antibiotic were determined at the same time point. Since only the data for animals with complete eradication of all microorganisms are reported (see Table 2), the data for the control animals are not presented. Overall, drug efficacy was evaluated with 24 animals (3 animals per drug and strain). Since each bead was processed separately 72 individual microbiological determinations were performed per drug and strain. Due to the need to restrict the use of animals, we believe that it would not have been justified to use more animals.

Antibiotic concentrations and dosage regimens.Table1 summarizes the dosing of the antibiotics in vivo and the concentrations achieved in serum and tissue cage fluid. The kinetics of each drug have been measured in 10 to 12 tissue cage fluid samples from three animals. The kinetics were determined at up to seven time points (time zero to 24 h). In this report only the peak and trough levels are given. The average time course of the measured concentrations was used as a reference to simulate the kinetics in vitro. Therefore, pharmacokinetic measurements were obtained for a total of 12 animals. The peak and trough concentrations obtained in vitro are also listed. The concentrations determined in the samples from the animals in the in vivo study were determined in antibiotic medium 1 (Difco Laboratories) by agar diffusion bioassays. Bacillus subtilis 0453-52-9 (Difco) was used as the test strain. Standard curves were generated from pooled heat-inactivated guinea pig serum with a final concentration of 50%. Results were calculated by microcomputer-assisted exponential regression analysis (Hewlett-Packard HP41V). The concentrations achieved in vitro were defined according to the concentrations obtained in vivo in the infected tissue cages. A one-compartment kinetic model was simulated in vitro on the basis of the in vivo observations over the two 12-h dosing intervals (Table 1). Concentration-time profiles for all four drugs were calculated, and the presence of the calculated concentration profiles within the culture compartments was confirmed by spectrophotometric concentration measurements by using fluorescein sodium as the test substance (PBS [pH 7.4], 23°C, 490 nm;r² = 0.9996; Stasar III; Gilford Instrument Laboratories Inc., Oberlin, Ohio). The kinetics of the in vitro system were determined every 0.5 h over the entire dosing interval. The mean intra-assay and interassay coefficients of variation were less than 5%; the measured concentrations of all four drugs averaged 101% ± 5% of the respective target values.

Quantification of bactericidal activity. (i) In vivo.Six beads were removed from each cage at each sampling point. The number of bacteria adhering to the beads was determined as described previously (29). Each bead was placed on a filter with a sterile forceps and was washed twice with saline. The number of bacteria adhering to the beads was determined by placing the washed glass beads in 2 ml of physiological saline containing EDTA (0.15%) and Triton X-100 (0.1%), followed by vigorous vortexing three times for 15 s each time to remove the adhering bacteria. Thereafter, the tubes were placed in an ultrasonic bath and sonicated for 3 min at 120 W (Labsonic 2000; Bender & Hobein, Zurich, Switzerland). The specimens containing resuspended bacteria were subsequently diluted 100- to 10,000-fold and were subcultured at 37°C onto tryptic soy agar plates. The detection limit was 20 CFU per bead. The term “sterile” is defined here as no bacterial growth in subcultures of individual beads.

(ii) In vitro.Each bead was removed from the compartment after 24 h of antibiotic treatment. The bacteria adhering to the sinter-glass bead were detached by vortexing as described previously (25). The detection limit was 40 CFU per bead. The term “sterile” is defined here as no bacterial growth in subcultures of individual beads.

Data analysis.Probabilities (P values) of <0.05 were considered statistically significant. The chi-square test was used for analysis of the frequency of bacterial eradication.