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Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa.

ABSTRACT: Background and Objectives:The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from Pseudomonas aeruginosa against different bacterial species. Materials and Methods:The azurin gene was cloned in the pET21a vector. The pET21a-azurin construct was transformed into Escherichia coli BL21. The recombinant Azurin was expressed and purified using affinity chromatography and confirmed by Western blotting. The cytotoxicity of rAzurin was assessed on peripheral blood mononuclear cells. Antibacterial and antibiofilm activity of rAzurin with different concentrations were determined by micro-broth dilution and crystal violet methods, respectively. The effect of rAzurin on bacterial species was statistically analyzed by t-test and spearman correlation. Results:The identity of purified protein was confirmed by blotting and distinguished as a 14 kDa band on 15% SDS-PAGE. The IC50 of rAzurin on Peripheral Blood Mononuclear Cell (PBMC) was determined as 377.91±0.5 ?g/mL in 24 h. Vibrio cholerae and Campilobacter jejuni displayed the most sensitivity to rAzurin (27.5 and 55 ?g/mL, respectively) and the highest resistance (220 ?g/mL) was displayed by P. aeruginosa and E. coli. The MIC for other species was 110 ?g/mL. The Minimum Biofilm Inhibition Concentration (MBIC) was determined as 220 ?g/mL for Salmonella enterica and V. cholerae, 300 ?g/mL for Shigella sonnei, Shigella flexneri and P. aeruginosa and 440 ?g/mL for the other species. The antimicrobial effect of rAzurin on bacterial species were significant (p value<0.05) and correlation coefficient was negative. Conclusion:The rAzurin appears to be an appropriate choice and a new strategy for prevention of bacterial infection. It inhibits bacterial growth and biofilm formation and candidates as antimicrobial peptides.

SUBMITTER: Mohammadi-Barzelighi H

PROVIDER: S-EPMC6635317 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from <i>Pseudomonas aeruginosa</i>.

Mohammadi-Barzelighi Hajar H Nasr-Esfahani Bahram B Bakhshi Bita B Daraei Bahram B Moghim Sharareh S Fazeli Hossein H

Iranian journal of microbiology 20190401 2

<h4>Background and objectives</h4>The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from <i>Pseudomonas aeruginosa</i> against different bacterial species.<h4>Materials and methods</h4>The <i>azurin</i> gene was cloned in the pET21a vector. The pET21a-<i>azurin</i> construct was transformed into <i>Escherichia coli</i> BL21. The recombinant Azurin was expressed and purified using affinity chromatography and confirmed by Western blotting. The c ...[more]

PMID: 31341572

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Project description:Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens.

Dataset Information

Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa.

Publications

Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from <i>Pseudomonas aeruginosa</i>.

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