Background: Pseudomonas aeruginosa is an opportunistic pathogen and utilizes several virulence
factors for pathogenesis. One of the most important factors is alginate, found in the biofilm which enables
P. aeruginosa to establish chronic lung infections.
Materials and Methods: In this study, 25 clinical alginate-degrading isolates were selected.
Biochemical and molecular approach were carried out to identify the isolates by 16S rDNA gene
amplification. Growth conditions and enzyme production were the criteria for selection. Since the main
objective of the project was the production and characterization of alginate lyase and its effect on biofilm
elimination, the P. aeruginosa sp.TAG48 alginate lyase-encoding gene was isolated, cloned, sequenced
and expressed in E.coli DH5α. The resultant enzyme was purified by affinity chromatography.
Ciprofloxacin, tobramycin and cefixime were also used to test the effectiveness of these antibiotics on P.
aeruginosa biofilm by minimum biofilm inhibitory concentration (MBIC) and minimum biofilm
eradication concentration (MBEC). The synergistic effects of these antibiotics and the recombinant
alginate lyase on biofilm were evaluated.
Results: Results indicate that the addition of alginate (0.2%-0.8%) and NaCl (0.2-0.5 M) to the medium
significantly increases cell growth followed by higher enzyme production (p≤ 0.05). Moreover, substrate
specificity of alginate lyase produced by P. aeruginosa sp.TAG48 shows the enzyme is capable of
degrading both polyM and polyG alginate and acts bifunctionally. Results from the antimicrobial
characteristics of the antibiotics and the enzyme have shown MBIC for ciprofloxacin, tobramycin,
cefixime and enzyme in the following concentrations 4, 32, 256 and 18.75 μg/ml, and MBEC: 32, 128, ≥
512 and 37.5 μg/ml, respectively. The study of synergism between the antibiotics and the enzyme to
prevent growth and eradication of P. aeruginosa sp.TAG48 biofilm shows that alginate lyase exhibits
synergy with tobram