Screening, cloning and expression of a novel alginate lyase gene from P. aeruginosa TAG 48 and its antibiofilm effects on P. aeruginosa biofilm.

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 tobramycin and cefixime but not with ciprofloxacin. CONCLUSION: The results indicate that the use of purified novel alginate lyase with antibiotics could be a beneficial alternative for the treatment of P. aeruginosa infections. Elucidation of mechanisms involved in antibiotic resistance and the role of biofilm structure could assist physicians select optimum treatment regimen.

Screening, identification and experimental design to optimization of the selenite bioremediation by new isolated Bacillus sp. Selena 3 in water.

Background and Objectives: Heavy metals pollution is one of the most important concerns in the world. Selenium is one of the most important elements for the life, but if the absorption of this element in cells increases, it acts as a toxic element. Materials and Methods: In this study, bacterial isolates were screened and isolated from selenium-contaminated soil and water. Twenty-five out of 42 isolates were able to reduce Selenite. Also, the response surface method (RSM) was used to evaluate and optimize the biological reduction of selenite by Selena 3. Factors of bacterial inoculation percentage, time, and amount of selenium oxyanion salt concentration were studied at five levels of -α, -1, 0, +1, and +α. Results: Bacillus sp. Selena 3 was able to reduce 80 mM sodium selenite in less than 4 hours compared to other bacterial isolates. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of sodium selenite Bacillus sp. Selena 3 was reported as 160 and 320 mM, respectively. The results showed that with increasing duration, the percentage of selenite reduction by bacteria increases and the percentage of bacterial inoculation does not have much effect on its reduction. Conclusion: Due to the ability of Bacillus sp. Selena 3 for rapid reduction in significant concentration of selenium oxyanion (SeO32-), this bacterium can be used as an efficient candidate in removing selenite from the environment.

Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production.

BACKGROUND: Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. METHODS: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Resuls: The results showed the ability of Bacillus sp. TAG8 to utilize alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. CONCLUSION: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples.