Abstract
The spread of antibiotic resistant bacteria is a worldwide problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed, through molecular techniques, to detect point mutations in chromosomal genes responsible for the quinolones class of antibiotics resistance. A total of 52 isolates from burn infections were identified using specific primers for P. aeruginosa 16S rDNA. Ciprofloxacin minimum inhibitory concentrations (MIC) were estimated using the agar dilution assay. DNA sequences of the quinolone resistance-determining regions of gyrA and parC were determined for detecting the mutations found in these genes and the relations among the isolates by constructing phylogenetic trees. The results revealed that only 43 (82.7%) of isolates were P. aeruginosa, of which 31 (72.06%) were resistant to different concentrations of ciprofloxacin, ranging between 4 and >32 µg/ml. Twenty six isolates were selected for sequencing, including sensitive, intermediately resistant, and highly resistant to ciprofloxacin. The ciprofloxacin sensitive isolates did not exert any amino acid alterations in gyrA or parC genes; however, a single intermediately resistant isolate had a single mutation at each gene. Of the total resistant isolates (20), 6 isolates had no mutations at different MIC levels, While 14 isolates had Thr-83-Ile substitution in gyrA and Ser-87-Leu substitution in parC, only five isolates had a second mutation, namely Asp-87-Asn, in gyrA. The phylogenetic analysis of the studied groups showed divergence from the P. aeruginosa PAO1 and PAO1OR reference strains due to increased mutations and polymorphisms in studied isolates. In conclusion, P. aeruginosa occurrence was increased in burn infections and the fluoroquinolones in current use are not as effective as before; the main resistance mechanism in local clinical isolates of P. aeruginosa is mutations, where the main target of fluoroquinolones is gyrA gene.
Highlights
Burn injury is a worldwide public health issue due to increasing nosocomial infections [1,2]. in developing countries, Pseudomonas aeruginosa pathogen can cause severe nosocomial infections, especially in catheterized, immunocompromised, or burned patients [3, 4] resulting from its containment of intrinsic and acquired resistance to many antibiotic groups [5]
Fluoroquinolones work by inhibiting two enzymes involved in bacterial DNA synthesis, both of which are DNA topoisomerases that human cells lack and that are essential for bacterial DNA replication [9], thereby enabling these agents to be both specific and bactericidal [10]
The main mechanisms of fluoroquinolones resistance in P. aeruginosa are resulting from mutations in DNA gyrase and topoisomerase IV [12], decreased permeability of the cell wall, multidrug efflux pump [13], and plasmid mediated resistance [14]
Summary
Burn injury is a worldwide public health issue due to increasing nosocomial infections [1,2]. in developing countries, Pseudomonas aeruginosa pathogen can cause severe nosocomial infections, especially in catheterized, immunocompromised, or burned patients [3, 4] resulting from its containment of intrinsic and acquired resistance to many antibiotic groups [5]. The main mechanisms of fluoroquinolones resistance in P. aeruginosa are resulting from mutations in DNA gyrase and topoisomerase IV [12], decreased permeability of the cell wall, multidrug efflux pump [13], and plasmid mediated resistance [14]. Most bacteria have both enzymes; gyrase is more sensitive to inhibition by quinolones than is topoisomerase IV in Gram -ve bacteria, whereas, in Gram +ve) bacteria, topoisomerase IV is usually the main target and gyrase is interestingly less susceptible [15]. We aimed at constructing a phylogenetic tree for both genes to observe the effects of mutations on divergence between isolates
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