The CBD produced 96 equivalent biofilms, making it the first assay system truly amenable to antibiotic susceptibility testing for adherent bacterial populations. The reproducibilities of the results for biofilms formed on each of the pegs of the CBD demonstrate the equivalence of the biofilms formed at each of the sites for susceptibility testing. In addition, the reproducible growth curves obtained for each isolate demonstrate that biofilms of a predicted size can be formed on each peg of the CBD lid (Fig. ). It is therefore possible to select a target biofilm size for antibiotic susceptibility testing and to expose the biofilm to multiple antibiotics in a single assay. SEM of the biofilms that formed (Fig. ) on the CBD demonstrates the typical biofilm appearance, as seen on the modified Robbin’s device (MRD) or on catheter surfaces (12
). Therefore, the major advantages offered by the CBD are its multiple equivalent biofilms that can be used for testing and its ease of use. The CBD requires no pumps or tubing, making the process much simpler to set up than the MRD, and eliminates a major source of possible contamination. The availability of multiple testing sites greatly reduced the time required to determine the antibiotic susceptibilities of biofilms from weeks with the MRD to 3 days with the CBD. The CBD is also amenable to automation because it is built on the typical platform for 96-well plates. There were no differences in the MBECs for all three organisms obtained either by reading of the turbidity at 650 nm or by quantitative bacteriology. This eliminates the need in most cases to do quantitative microbiology to obtain MBECs, again contributing to the ease of use of the assay and contributing to its automation potential.
The MICs obtained by NCCLS standard protocols and those obtained with the CBD were similar for almost all antibiotics tested. This is an interesting observation in that the planktonic population tested for antibiotic susceptibility by the assay with the CBD is one that was continually shed from the biofilm, whereas a prescribed inoculum was tested by the NCCLS assay. The data could be interpreted to indicate that the MIC is predictive of antibiotic efficacy against bacteria being seeded from a nidus of infection, such as a biofilm on a catheter or a line. This is consistent with what is often seen in recurrent infections. Antibiotics that are seen to be effective in MIC assays are able to produce symptomatic relief by eliminating the planktonic population; however, because the biofilm is not eliminated by antibiotic treatment, reinfection occurs once the antibiotic is removed.
A clear difference in antibiotic susceptibility was seen between planktonic populations of each of the bacteria tested and the biofilm populations of the same organism. These results obtained with the CBD are in keeping with previously reported results obtained with the MRD and by other methods of biofilm production (8
). The data produced by Morck et al. (12
), who used the MRD, have recently been compared to data derived from the CBD, with the only difference being that the current technology allows a more detailed analysis of susceptibility because the number of samples that could be handled was greatly increased (1
). As a biofilm, each of the isolates had an unique susceptibility to the group of antibiotics tested. E. coli
ATCC 25922 was the most sensitive to tobramycin, with the other aminoglycoside tested (gentamicin) and the β-lactam piperacillin showing some activity against E. coli
. The P. aeruginosa
ATCC 27853 biofilm was the most susceptible to the aminoglycosides tobramycin and amikacin but was not nearly as susceptible to gentamicin. Eradication of the Pseudomonas
biofilm could also be obtained with ciprofloxacin at obtainable concentrations. The biofilm of S. aureus
ATCC 29213 proved to be very difficult to eradicate, with only gentamicin proving to be effective at achievable drug concentrations.
It is clear that the antibiotic susceptibilities of planktonic populations, as determined by MIC methodologies, are not necessarily applicable to effective treatment of the same organism once a biofilm has been established. One problem faced in selecting alternative antibiotic treatment has been the lack of an easy, reproducible assay which could provide a measure of antibiotic activity against a biofilm. The CBD provides a new technology that can be applied to recalcitrant, recurrent, or device-related infections caused by organisms for which MICs have not provided clinically relevant information. The CBD should also prove to be important in the development of new antibiotics selected for their efficacies against biofilms.