Biofilm Studies

Bacterial biofilms are responsible for several chronic diseases that are difficult to treat. Examples are: cystic fibrosis, endocarditis, cystitis, and infections caused by indwelling medical devices. Biofilm bacteria show much greater resistance to antibiotics than their free-living counterparts and our interest is to investigate the mechanistic basis of this phenomenon. One potential reason for this increased resistance is the penetration barrier that biofilms may present to antimicrobials. We invented a real-time method to visualize biofilm cells coming in contact with tetracycline, and showed that all the cells in the biofilm were almost instantaneously exposed to the antibiotic, and yet they were more resistant than their planktonic (free-living) counterparts. See illustration 1.

To identify the genes that may be specifically involved in increased resistance of biofilm cells, we have isolated several mutants of uropathogenic E. coli, and Staphylococcus aureus and which form "normal" biofilms, but which do not possess increased resistance.

One E. coli mutant has been studied in detail, using both the uropathogenic strain as well as the K12 strain; both give similar results. The architecture of the wild type and the mutant (001C3) biofilms is shown in Illustration 2). These mutants are loss of function mutants in the rapA gene. The RapA protein belongs to the SWi/SNF superfamily of helicase-like proteins, which has been implicated in chromatin remodeling in eukaryotic cells. RapA has never before been implicated in bacterial antibiotic resistance. In the RapA-deficient mutant, several genes were down regulated, including the yhcQ and yeeZ genes. The former is putatively concerned with encoding a multidrug resistance pump and the latter with an unknown envelope function. Detailed studies of these mutants lead to the conclusion that the enhanced penicillin resistance of the wild type biofilm is due to a dual strategy: impaired penetration of the biofilm through its matrix, and rapid efflux of the antibiotic that still manages to penetrate. (See Illustration 3 for differential penetration.) As these mechanisms can be generally protected, other antibiotics were tested. It was found that the mutant biofilm had impaired resistance to several other biofilms with different mechanisms of action. Thus, while the matrix may not be important in increased biofilm resistance to some antibiotics, it plays a role in other resistances. We have recently developed new high throughput systems for isolating mutants affected in their biofilm sensitivity to antimicrobials, both in static and flow-through biofilm configurations. The ms has been submitted.

S.V. Lynch, L. Dixon, M.R. Benoit, E.L. Brodie, M. Keyhan, P. Hu, D.F. Ackerley, G.L. Andersen and A. Matin. 2007. Role of the rapA Gene in Controlling Antibiotic Resistance of Escherichia coli Biofilms. Published on line, 30 July 2007. Antimicrobial Agents and Chemotherapy [PDF]

Illustration 3

Penetration of a (green) fluorescent functional homologue of penicillin (Bocillin) through the mutant (top) and wild type (bottom) biofilms. A/D: biofilm top; B/E, biofilm center; C/F biofilm bottom. Much more green fluorescence is seen at the bottom of the mutant biofilm than of the wild type.

We have also examined E. coli biofilms under low-shear/microgravity conditions, using HARV systems that we have modified. Simulated microgravity biofilms are more copious (see Illustration 4) and more resistant both to 'general' stressors, as well as to antibiotics. Resistance to the former is controlled by sigma S, the latter evidently not. Many routes of infection in the human body are low shear environments; thus, these studies are relevant also to controlling diseases on Earth.

Illustration 4. E. coli AMS6 normal gravity (top panels) and low shear simulated microgravity (LSSMG)-grown biofilms (stained with BacLight viability stain [green, viable; red, nonviable) visualized pre- and post NaCl (A) or ethanol (B) stress. LSSMG biofilms were more resistant also to antibiotics.

Stone, G., P. Wood, L. Dixon, M. Keyhan and A. Matin. 2002. Tetracycline rapidly reaches all the constituent cells of uropathogenic Escherichia coli biofilms. Antimicrobial Agents and Chemotherapy 46: 2458-2461.[PDF]

Lynch, S.V., K. Mukundakrishnan, P. Ayyaswami, and A. Matin. 2006. Escherichia coli biofilms formed under low shear simulated microgravity in a ground-based system. Applied & Environmental Microbiology 72 (12): 7701-7710 [PDF file]

Illustration 1

Illustration 2