The effect of alginate lyase on the antibiotic sensitivity of mucoid Pseudomonas aeruginosa biofilms

Abstract

Bacteria are single celled organisms that can live together in communities called biofilms. These biofilms consist of a structural matrix consisting of polysaccharide gel secreted by the bacteria, with the bacteria living within the confines of the gel. Biofilms can arise on all sorts of surfaces, including those of medical catheters and equipment as well as the tissues of the body itself. Biofilms act as reservoirs of infection allowing bacteria to set up chronic infections that resist both the host immune system and antibiotics. People with compromised defences, such as cystic fibrosis (CF) sufferers, are particularly vulnerable to infection, especially by biofilm forming bacteria. The lungs of CF patients have thickened, dehydrated lung mucus that prevents the lungs from clearing inhaled foreign particles, including bacteria.

P. aeruginosa is an opportunistic pathogen of humans and one of the most common organisms that infect the lungs of individuals with CF. During chronic infection P. aeruginosa can overproduce alginate, a capsular, highly viscous polysaccharide. This polysaccharide forms a thick capsule around the bacteria which retains water and also prevents complement-mediated killing and phagocytosis by neutrophils. Alginate has been linked to antibiotic resistance by some researchers while others have shown alginate plays almost no role in antibiotic resistance. Alginate can be broken down by the enzyme alginate lyase which eliminates the glycosidic bond between two sugar residues in the polysaccharide chain. The aim of this study was to compare and characterise the effects of two different alginate lyase enzymes on the structure of P. aeruginosa biofilms and the sensitivity of biofilm bacteria to antibiotics. The alginate lyases used in this study were a commercially available lyase from Sphingobacterium multivorans (SBAL) and the ALYI-III alginate lyase from Sphingomonas sp. A1 (ALY) produced via recombinant gene expression in E. coli. The recombinant protein included a 6x Histidine tag on the N-terminal sequence and was purified using immobilised metal affinity chromatography with nickel ion coated beads. Both enzyme preparations were characterised to determine basic kinetic parameters, the ability to degrade cation-alginate gels and tolerance to thermal degradation. Enzyme activity was measured using UV spectroscopy and rheometry based assays. Following enzyme characterisation biofilms were grown both in multi-well plates and on coupons, and were treated with two different alginate lyases along with carbenicillin and gentamicin antibiotics. Biofilm imaging techniques using a LIVE/DEAD cell viability stain to determine if lyases affected biofilm cell viability were trialled unsuccessfully. Alginate lyase, at the concentrations used in the study did not increase antibiotic sensitivity. Treatment with alginate lyase either had no effect on biofilm populations or caused a small but significant increase in viable cell number and overall biomass. The results suggested that the biofilm populations contained a group of antibiotic resistant bacteria that were metabolically active. It was hypothesized that the lyase enzymes aid in the survival and proliferation of these resistant bacteria by enzymatic modification of the alginate biofilm to permit better oxygen diffusion and also by acting as carbon and nitrogen sources for the resistant bacteria. These factors do have a direct effect on bacterial resistance to at least one class of antibiotics, aminoglycosides. Alginate lyase may prove more useful as a means to increase phagocytosis of bacteria by host immune cells.