Detection of possible mutations in gyrA gene of ciprofloxacin-resistant Pseudomonas aeruginosa isolates from patients with lung infection

Abstract

Chronic obstructive pulmonary disease (COPD) and nosocomial pneumonia are the most frequent and persistent lung infections. Chronic obstructive pulmonary disease (COPD) and pneumonia have become the leading cause of child and adult death for many decades in developing countries. Although antibiotics are largely prescribed during COPD exacerbation and pneumonia to combat against bacteria, antibiotic resistance has become a major problem to combat these lung infections and has become the burning question among the public health issues and modern science. One of the prime pathogens of lung infection is Pseudomonas aeruginosa. Though it seems uncomplicated to treat this pathogen, antibiotic resistance has made it too much difficult. The purpose of this study was to detect possible mutations in the gyrA gene of ciprofloxacin-resistant Pseudomonas aeruginosa and to observe changes in its products. A number of 45 lung infecting Pseudomonas aeruginosa samples were collected and studied against 14 antibiotics of different classes. After the primary screening of antibiotic susceptibility, they were categorized into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pan drug-resistant (PDR) pathogens where 15 isolates were MDR, 5 were XDR and 3 isolates were PDR and 20 were ciprofloxacin-resistant. Further, among the 15 MDR isolates, 4 isolates were selected and among the 20 ciprofloxacin-resistant isolates, 4 isolates were selected for DNA extraction. Furthermore, PCR with gyrA specific primer and gel electrophoresis were done after DNA extraction of those 8 isolates of Pseudomonas aeruginosa. Next, of these 8 isolates, 4 isolates were selected for DNA sequencing where 2 isolates were ciprofloxacin-sensitive strains and 2 isolates were ciprofloxacin-resistant strains. After DNA sequencing the sequences were analysed by some bioinformatics tools and software which confirmed the isolated DNA were gyrA gene. Finally, by using bioinformatics tools the possible mutation sites in gyrA gene of resistant samples were detected by comparing it with the gyrA gene of susceptible samples. The changes in the amino acids in resistant samples were also identified and restriction mapping patterns were analysed to observe the changes between the sensitive and the resistant samples. Moreover, a phylogenetic tree was constructed to analyse the relatedness among the Pseudomonas aeruginosa isolates. Although a further extension of in-vitro and in-vivo study needs to be done in order to confirm the mutation sites and changing pattern of the amino acids, this study is certainly a novel one to combat upcoming ciprofloxacin-resistant Pseudomonas aeruginosa.