Global Sequence Analysis and Expression of Azurin Gene in Different Clinical Specimens of Burn Patients with Pseudomonas aeruginosa Infection.

Mohammadi Barzelighi, Hajar; Bakhshi, Bita; Daraei, Bahram; Fazeli, Hossein; Nasr Esfahani, Bahram

Aim The purpose of this study was to analyze the sequence of azurin gene in relation to its expression in Pseudomanas aeruginosa strains isolated from different clinical specimens of burn patients. Moreover, in silico sequence analysis of azurin gene using globally reported sequences was intended. Materials and Methods Fifty-nine multidrug-resistant P. aeruginosa isolates were selected from different clinical specimens of patients suffering from burn wound infections in two university hospitals and subjected to antibacterial susceptibility testing. The frequency and genetic diversity of the azurin gene was determined by polymerase chain reaction (PCR) and Sanger sequencing. The azurin gene sequences were compared with the sequence data from other countries. The expression level of azurin gene in P. aeruginosa isolates with different azurin sequences from different clinical specimens was evaluated by real-time PCR. Results and Conclusion About 98%-100% of the isolates were resistant to gentamicin, tobramycin, cefoxitin, ciprofloxacin, amikacin, and imipenem, while 100% and 23.9% of the isolates were susceptible to colistin and ceftazidime, respectively. Only eight point mutations were detected with amino acid substitutions in only two positions (81 and 102). In global analysis, 93% of strains showed missense mutation at positions 81 (alanine to threonine). The majority (81%) of Iranian strains were allocated to two major clusters distinct from the rest of world, which may suggest that strains from Iran have made a distinct genetic stockpile through point mutations which has established them separate from the other counties. However, 19% were distributed in different clusters together with the strains from different countries of North and South America, Europe, South and East Asia. The expression level of the azurin gene was statistically higher in the isolates collected from the blood of burns patients with systemic infection compared to the isolates collected from other specimens (wound, catheter and tissue), which shows a positive correlation between azurin gene expression and increased pathogenicity and capability for dissemination. This study may open new insight about azurin genetic variation and significance in P. aeruginosa pathogenesis.