In natural environments, bacteria frequently grow in structured communities called biofilms. Biofilms are defined as bacterial populations adherent to each other and/or surfaces encased within a three dimensional matrix of extracellular polymeric substances [EPS] 1. Biofilms can constitute a major problem to human health with many clinicians citing them as the cause of a variety of chronic bacterial infections 2. Bacterial cells are protected by growing in a biofilm and although antibodies produced in response to biofilm antigens may eliminate the planktonic cells shed from the biofilm, they cannot reach the sessile cells within the biofilm and may damage surrounding tissue instead 3. Similarly, antibiotic therapy often fails to eradicate biofilms, suppressing only the symptoms of infection by killing the planktonic cells 4. Consequently, infections in animals and humans may persist for years with recurring symptoms after each period of antibiotic treatment until the colonised surface is surgically removed.

Whether in humans or animals, the antibiotic resistance of biofilms has a significant impact on health including increased morbidity and mortality 5. The prolonged treatment of diseases and infections causes increased health costs and serious implications for both human and animal welfare. Currently, antibiotic selection is based on an antibiotic sensitivity test using the Kirby-Bauer disc diffusion method, developed in 1966 by Bauer and others 6. Other methods have since been developed but the disc diffusion technique was adopted by the National Committee for Clinical Laboratory Standards [NCCLS] in 1975 and is still used today as the basis for disc diffusion standards 7.

Although the disc diffusion method of antimicrobial sensitivity testing has been described as a reliable, easy and inexpensive method of evaluating antimicrobial efficacy 8, recent research has indicated that the results from the disc diffusion test are open to interpretive error and that it is only useful as a preliminary screen for susceptibility testing 9. Costerton et al. 3 stated that culturing bacteria for use in the susceptibility test transforms a biofilm forming pathogen into a planktonic lab-adapted strain. Thus, the problem with the standard antibiotic susceptibility test is that bacterial growth on agar is not representative of how bacteria grow naturally in tissue sites. Consequently, the current method of antibiotic selection assesses bacterial sensitivity in an unrealistic state.

In this present study poloxamer F127, a di-block copolymer of polyoxyethylene and polyoxypropylene, was used as a medium on which bacteria could be grown as a biofilm phenotype and express the characteristics more appropriate to the 'real world'. An initial experiment was undertaken to determine the molecular weight of the outer membrane proteins of P. aeruginosa grown on standard agar, poloxamer gel and in a biofilm on a microtitre plate to confirm whether bacteria express a biofilm phenotype on poloxamer as was found by Gilbert et al. 10. The second experiment then involved antibiotic sensitivity testing on standard agar and poloxamer gel to compare results for a range of bacterial species.

In this present study two approaches were used to study the effectiveness of antimicrobial dressings on microorganisms. Firstly a wide range of aerobic bacteria and yeasts were tested using a standard agar assay [Kirby Bauer disc diffusion method [6] and a second method used a poloxamer technique to encourage the same strains of microorganisms to exhibit a more clinically relevant biofilm phenotype. Gilbert and others determined that P. aeruginosa cells grown on poloxamer hydrogel ('true' biofilm form) express outer membrane proteins between 78 and 87 kDa, which are not evident in cells grown on standard nutrient agar ('planktonic/quasi-sessile state') 10. Consequently poloxamer gel cultures mimic many of the properties of biofilm-grown Pseudomonas aeruginosa 10. This indicates that there is a phenotypic difference between P. aeruginosa cells grown on poloxamer hydrogel and nutrient agar, with only poloxamer grown cells resembling biofilm cells. It was found from Wirtanen's study 11 that bacteria which are grown in poloxamer have biofilm properties and associated enhanced biocide resistance 11. Gilbert and colleagues suggested that bacteria grown in poloxamer hydrogels could be exposed to biocides to provide a reproducible method for testing the antimicrobial efficacy of biocides against biofilm bacteria 10. Evidence of biofilm growth in the poloxamer model was also confirmed using confocal laser microscopy 12. Sincock and other found that using microscopy, bacteria within poloxamer hydrogels grew to high densities, formed microcolonies and exhibited a biofilm phenotype. The poloxamer hydrogels have also been used to study biofilms of Streptococcus mutans in plaque 13, to look at homoserine lactones and biocide efficacy in biofilms 14 and also to study biofilms and coaggregation in the freshwater bacteria Blastomonas natataria and Micrococcus luteus 15.

In the current study we have utilised and adapted the science of Wirtanen's biofilm model 11 to provide a more clinically relevant method to test the effectiveness of antimicrobial dressings on biofilm microorganisms. The aim of this research was to test a more clinically relevant biofilm model for assessing the efficacy of antimicrobial agents against microorganisms of clinical and veterinary importance.

The treatment of infections with topical or systemic antibiotics is becoming increasingly problematic due to the existence of biofilms 1718. Antibiotic sensitivity testing by traditional methods on agar are used to diagnose the best antibiotic to treat an infection. However, the choice and concentration of antibiotic are often unsuccessful at clearing the infection 19. This is due to the fact that bacteria growing in a biofilm state are very recalcitrant to antibiotic treatment.

Gilbert et al. 10 suggested the use of poloxamer as a substitute for antimicrobial susceptibility testing and hypothesized that bacteria would grow in a biofilm state in poloxamer as opposed to a lab adapted 'planktonic' state. In their study OMPs from poloxamer grown and biofilm grown Pseudomonas aeruginosa had a number of identical OMPs which were not found in the bacteria when it was grown on agar and in broth (planktonic state). Within our study were have also discovered two outer membrane proteins in Pseudomonas aeruginosa at 87 kDa and 112 kDa in the biofilm and poloxamer grown state. These were not present in the MH agar grown cells which suggests that the protein profile of Pseudomonoas aeruginosa biofilm cells are different to that of MH agar grown 'planktonic/quasi-sessile' cells. The data generated in this paper supports the findings of Gilbert et al. 10, who found that poloxamer and biofilm grown Pseudomonas aeruginosa cells expressed outer membrane proteins between 78 and 87 kDa, which were not evident in MH agar grown cells. An additional protein between 71 and 72 kDa was found in the biofilm and poloxamer grown cells that was not found in the agar grown cells. This protein may represent the protein OprC [70 kDa] that was found in biofilm cells by Gilbert et al., 10. This protein was not evident in the planktonic cells and imply that there is a phenotypic difference between P. aeruginosa cells grown on poloxamer gel and MH agar, with poloxamer gel grown cells resembling biofilm cells.

Overall for all the bacteria studied in this paper unique OMPs were identified when the bacteria were grown on poloxamer and in the biofilm state, that were not evident when the bacteria were grown on MH agar or in MH broth. OMPs were also identified from bacteria grown in MH agar and broth that were not found on poloxamer and bofilm grown bacteria. This suggests that bacterial cells display a biofilm phenotype in the presence of poloxamer. Consequently, this suggests that the sessile bacteria when grown on poloxamer express OMPs which are biofilm specific.

Having identified phenotypic similarities between poloxamer and biofilm grown cells an antimicrobial susceptibility test was conducted on a range of bacterial organisms grown in parallel on MH agar and poloxamer gel, in order to determine if a difference existed between the two different growth media. It was found that there was a significant difference [P < 0.05] between the growth diameters of the zones of inhibition on MH agar and poloxamer gel. The zones were generally smaller when the bacteria were grown on poloxamer gel and the antibiotic efficacy often differed between the two different media. For example, imipenem was the most efficient antibiotic against MH agar grown Actinobacillus equuli [35.4 mm mean diameter inhibition zone], whereas meropenem was the most effective antibiotic against the poloxamer gel grown form of the bacterium producing a 26.87 mm mean zone of inhibition. Not only was there a difference in the extent of antibiotic efficacy on both MH agar and poloxamer gel between antibiotics but the degree of efficacy also differed for the same antibiotic. Notably this was demonstrated in the case of penicillin G where organisms tested showed susceptibility when grown on MH agar but complete resistance when grown on poloxamer gel. It is also important to note that in this study the zones sizes are not comparable between the different antibiotics particularly as the methods employed are not quantitative, although gross differences can be concluded.

The differences in results relating to the two types of media calls into question the applicability of the traditional Kirby Bauer antibiotic susceptibility test which has been used widely in microbiology laboratories over the last forty years or so. Incorrect antibiotic concentrations can increase antibiotic resistance mutation rates in bacteria 20. Generally, the use of ineffective antibiotics, whether due to class or dosage, to treat bacterial infections, will apply selection pressure to a population which will favour resistant strains. With the increasing threat of epidemic resistant organisms such as Methicillin Resistant Staphylococcus aureus (MRSA) the need for appropriate antibiotic selection is currently of prime importance to both clinical and veterinary science 2122.

Overall, this study has shown that the efficacy of antibiotics is reduced when bacteria are grown in the presence of poloxamer gel, as a biofilm phenotype. It has already been established that biofilm bacteria are resistant to antibiotics 23, however current susceptibility tests only use agar media that encourage bacteria to grow more within a 'planktonic/quasi-sessile' state than as a 'true' biofilm phenotype. The findings of this study suggest that poloxamer gel could be considered as an alternative medium on which to conduct antibiotic susceptibility tests as it enables bacteria to be grown in a biofilm state more representative of a biological surface infection (e.g. chronic infected wound). However, further studies are necessary to substantiate this claim particularly a quantitative version of this technology to aid clinicians and microbiologists to make informed decisions regarding prevention and treatment of serious biofilm infections.

SLP and JD are employees of ConvaTec Wound Therapeutics™.

ALC and JD performed experimental work. ALC, CC and SLP designed the study, collected and analysed the data and drafted the manuscript. All authors read and approved the final manuscript