Plasposon mutagenesis in Pseudomonas aeruginosa isolates illustrates the role of ABC transporter in intrinsic resistance to antibiotics
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Abstract
Pseudomonas aeruginosa is an opportunist pathogen most commonly related to nosocomial infections. P. aeruginosa infection therapy poses a significant challenge due to its ability to resist various antibiotics currently available. As a result, excessive use of antibiotics during therapy expedites the development of multidrug resistant P. aeruginosa. Hence, this study aimed to identify novel genes involved in multiple antibiotic resistance using plasposon mutagenesis technique. One hundred and ten P. aeruginosa isolates were collected from various clinical sources involving urine, burns and wound’s pus. An antimicrobial susceptibility test was performed to detect their resistance to 18 antibiotics. Results showed that all isolates were resistant to ampicillin and tetracycline, and the highest resistance ras were detected for nitrofurantoin and sulfamethoxazole (99%), followed by amoxiclav, cefotaxime, cefoxitin, ceftriaxone, and kanamycin (98%). While the lowest resistance rate was towards imipenem (49%). Plasposon mutagenesis was used to detect the genes involved in multi-antibiotic resistance. The pTnMod-Gm was introduced to the recipient P. aeruginosa PA4 isolate via triparental mating using E. coli HB101/ pRK2013 as a helper strain. Mutants were screened for resistance defects by plating them on nutrient agar supplemented with different antibiotics. Two mutants were identified; one (M1) exhibited susceptibility to tetracycline, cefotaxime, and ceftazidime, and the other (M9) to ceftazidime and ceftriaxone. The analysis of these mutants revealed the insertion of the plasposon into an open reading frame for the ABC transporter in P. aeruginosa, which plays a distinctive role in extruding antibiotics out of cells.
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Antibiotic resistance, cloning, P. aeruginosa, plasposon mutagenesis
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