Glutaraldehyde efficacy against biofilm bacteria was investigated using a model system of Pseudomonas aeruginosa entrapped in hydrated gel bead "artificial biofilms." Bacteria in biofilms were clearly less susceptible to glutaraldehyde than the same microorganisms when grown in a conventional suspension culture. For example, using 50 mg/L glutaraldehyde it took only approximately 20 minutes to achieve a 2 log reduction in viable cell numbers in planktonic experiments but almost 600 minutes to achieve this same level of killing in the biofilm. In general, the susceptibility of bacteria in biofilms was reduced by approximately an order of magnitude compared to planktonic bacteria. Biofilm results indicated that glutaraldehyde penetration into the biofilms was retarded and that poor penetration likely contributed to biofilm reduced susceptibility. Treating biofilm bacteria with a brief, concentrated dose of glutaraldehyde was more effective than a proportionally longer treatment at a lower concentration. For example, a 200 mg/L dose of glutaraldehyde for 40 minutes was as effective as a 50 mg/L dose delivered for 600 minutes. The demonstration of unambiguous biofilm resistance to glutaraldehyde highlights the need to continue to employ biofilm testing methodologies in designing and optimizing applications of this agent. The insights obtained in this investigation suggest possible approaches for improving biofilm control with glutaraldehyde.
Microorganisms in biofilms are almost always found to be profoundly less susceptible to antimicrobial agents than are their freely suspended counterparts. Examples of biofilm resistance to glutaraldehyde in particular can be found in the literature1-12. Because biofilms exhibit distinct physiologies and physical properties, the conclusions drawn from antimicrobial experiments performed with planktonic (freely suspended) microorganisms may be inappropriate for biofilm applications. For example, in a comparison of two forms of chlorine, hypochlorous acid and monochloramine, we have shown that monochloramine, which is a weaker disinfectant when tested against planktonic microorganisms, was actually the superior agent when tested against biofilms13. This result underscores the importance of performing biocide efficacy tests against biofilms. In many of the application areas of glutaraldehyde, including oilfield control of souring and corrosion, industrial water treatment, and sterilization of medical instruments, biofilms are known to be present and probably represent the major target for microbiological control. Researchers at the Center for Biofilm Engineering (CBE) have devised a simple, repeatable artificial biofilm test system for characterizing the efficacy of antimicrobial agents against biofilms. Biofilm disinfection experiments at four different glutaraldehyde concentrations were performed using this test system. In addition, several planktonic disinfection experiments were performed. The goal of this project was to obtain antimicrobial efficacy data in a model biofilm test system that would provide information about 1) the nature of biofilm resistance to glutaraldehyde and 2) relative biofilm efficacy of various glutaraldehyde concentration and dose duration treatments.
MATERIALS AND METHODS
Artificial biofilm test system
Artificial biofilms were created by entrapping bacteria, in this case Pseudomonas aeruginosa, in alginate gel beads. The gel beads were then cultured in a nutrient medium overnight to allow for growth of microorganisms in the beads and adoption of the biofilm phenotype. The end result - dense microcolonies dispersed in a highly hydrated gel matrix - simulated the structure of real biofilms. Alginate
