Evaluating antibiotics for use in medicine using a poloxamer biofilm model

Abstract

BackgroundWound infections, due to biofilms, are a constant problem because of their recalcitrant nature towards antibiotics. Appropriate antibiotic selection for the treatment of these biofilm infections is important. The traditional in vitro disc diffusion method for antibiotic selection uses bacterial cultures grown on agar plates. However, the form of bacterial growth on agar is not representative of how bacteria grow in wounds and other tissue sites as here bacteria grow naturally in a biofilm. The aim of this research was to test a more appropriate method for testing antimicrobial efficacy on biofilms and compare with the standard methods used for antibiotic sensitivity testing.MethodsOuter Membrane Protein analysis was performed on E.coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis and Acinetobacter juni when grown on Mueller Hinton agar ('quasi-biofilm state') and 30% Poloxamer hydrogel ('true- biofilm state). Susceptibility to antibiotics on 28 clinical isolates was determined using the modified Kirby Bauer disc diffusion method, on agar and 30% Poloxamer.ResultsSimilar outer membrane proteins [OMPs] were identified in bacteria grown in a biofilm state and on a 30% poloxamer hydrogel, which were very different to the OMPs identified in bacteria grown on Mueller-Hinton agar and broth. There was a significant difference between the means of the clearance zones around the antibiotic discs on standard agar and poloxamer gels [P < 0.05]. The zones of clearance were generally smaller for poloxamer-grown bacteria than those grown on standard agar. Diffusion distances of various antibiotics through agar and 30% poloxamer showed no significant difference [P > 0.05].ConclusionThe findings of this experiment suggest that poloxamer gel could be used as an appropriate medium on which to conduct biofilm antibiotic susceptibility tests as it enables bacteria to be grown in a state representative of the infected surface from which the culture was taken.

Signal diffusion and the mitigation of social exploitation in pneumococcal competence signalling

“…Of particular relevance to this study, Pluronic has been used to simulate bacterial growth within biofilms by generating an artificial biofilm matrix and to simulate conditions that might prevail within the nasopharyngeal mucosa. Outer membrane protein expression patterns of Pluronic grown biofilm cells of many species are consistent with the induction of biofilm physiology (Gilbert et al 1998;Clutterbuck et al 2007) and are distinct from either planktonically grown cells or those grown upon an agar surface (Gilbert et al 1998;Clutterbuck et al 2007). Pneumococcal growth rates and those of other species (Gilbert et al 1998;Wirtanen et al 1998) are not changed when growing within a medium containing Pluronic when compared with Pluronic-free cells (mean Vm + s.e.…”

“…Outer membrane protein expression patterns of Pluronic grown biofilm cells of many species are consistent with the induction of biofilm physiology Clutterbuck et al 2007) and are distinct from either planktonically grown cells or those grown upon an agar surface Clutterbuck et al 2007). Pneumococcal growth rates and those of other species Wirtanen et al 1998) are not changed when growing within a medium containing Pluronic when compared with Pluronic-free cells (mean Vm + s.e.…”

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

“…All aggregates enlarged with time, but their size increased unevenly. At later times (15,24, and 30 h), the clusters adjacent to the open end of the capillary and therefore closest to the nutrient and oxygen source were larger than the clusters more deeply embedded in the gel.…”

“…There is a substantial history of using gel-entrapped microorganisms as model biofilms (10), particularly as a means of simulating reduced susceptibility to biocides, disinfectants, and antibiotics (11)(12)(13)(14)(15). Gel matrices that have been used to create artificial biofilms include alginate, agarose, agar, poloxamer, gelatin, and collagen.…”

A Novel Qualitative and Quantitative Biofilm Assay Based on 3D Soft Tissue

“…Several in vitro biofilm methods are based on solid substratum such as 1.5% agar [ 6 ] or 30% poloxamer [ 7 ]. However, these substrata are made of polysaccharides and are not close to the clinical setting where the main component is collagen, a major protein in soft tissues existing in, for example, skin.…”