[Investigation of Resistance Nodulation Division (RND) Efflux Pump and OPRD Expression Levels in Antibiotic Resistance of Clinical Pseudomonas aeruginosa Isolates].

The increasing antibiotic resistance in Pseudomonas aeruginosa, responsible for both community-acquired and hospital-acquired infections, is of global significance. The primary mechanisms contributing to resistance development in P.aeruginosa include the increased activity of efflux pumps, decreased permeability of outer membrane porins and the production of carbapenemases. This study aimed to determine the effects of resistance nodulation division (RND) efflux pumps, outer membrane porin D (OprD) outer membrane protein and carbapenemase production on the development of resistance to different antibiotics in P.aeruginosa isolates. Eighty P.aeruginosa isolates obtained from clinical samples in our hospital between 2019 and 2021 were included in the study. Species-level identification of the isolates was performed using MALDI-TOF MS (Bruker Daltonics, Germany). Antibiotic susceptibilities were determined using the VITEK® 2 (bioMérieux, France) system according to the criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The expression levels of the outer membrane porin protein OprD and the regulatory genes of efflux pumps (mexB, mexC, mexE, and mexX) were investigated using real-time quantitative reverse transcriptase polymerase chain reaction (Rt-qPCR). The rpsL gene was used as the reference gene and P.aeruginosa PAO1 strain was used as the control strain in Rt-qPCR. Comparative expression analysis was calculated using the delta-delta cycle threshold (ΔΔCt) method. The presence of blaOXA-48, blaNDM, blaVIM, blaIMP, blaKPC and blaOXA-10 was investigated by PCR. Clonal relationships among the isolates were determined by AP-PCR using the M13 primer. Band profiles were analyzed using GelCompar II (Applied Maths) software. Decreased OprD expression was detected in 63.7% of the isolates and 74% of carbapenem-resistant isolates. The decrease in OprD expression was found to be significant only between the carbapenem-susceptible (n= 30) and carbapenem-resistant (n= 50) groups (p= 0.014). The overexpression rate of mexB was 82% in carbapenem-resistant isolates and 33% in carbapenem-susceptible isolates; 76.6% in multidrug-resistant (MDR) isolates and 45.5% in non-MDR isolates (p= 0.001 and p= 0.004, respectively). The overexpression rate of mexX was 68% and 40% in the amikacin-resistant and susceptible groups, respectively; and 64.3% and 40.4% in the gentamicin-resistant and susceptible groups, respectively (p= 0.02 and p= 0.041, respectively). Overexpression of mexX was found in 61.7% of MDR isolates and 30.3% of non-MDR isolates (p= 0.006). No significant difference was found in the expression of mexC and mexE between antibiotic-susceptible and resistant groups. While blaOXA-10 was detected in 32% (16/50) of carbapenem-resistant isolates, blaOXA-48, blaNDM, blaVIM, blaIMP, blaKPC were not detected in any of the isolates. AP-PCR analysis identified 64 different genotypes and no dominant genotype was observed. The isolates were grouped into 14 different clusters with a clustering rate of 36%. RND efflux systems play a crucial role in the development of antibiotic resistance in P.aeruginosa. This study showed that overexpression of mexB and decreased expression of OprD contributed to the development of carbapenem resistance, while overexpression of mexX contributed to the development of amikacin and gentamicin resistance. Overexpression of mexB and mexX has significant correlation with MDR isolates. The detection of blaOXA-10 in 16 isolates suggests that the presence of this resistance gene may contribute to the development of carbapenem resistance.