Investigation of the effects of active efflux pumps and porine proteins in antibiotic resistance mechanism of Pseudomonas aeruginosa isolates

The aim of this study is to evaluate the antibiotic sensitivity patterns of multiple drug, isolated carbapenem and isolated quinolone resistant Pseudomonas aeruginosa isolates and to investigate the effects of active efflux pumps and porine proteins in antibiotic resistance. The isolates isolated from all the other clinical samples that are routinely sent to our laboratory between November 2010 and November 2011 were identified according to their species. The antibiotic sensitivity tests of 50 clinical P .aeruginosa isolates included in the study were carried out by using the Kirby-Bauer disc diffusion method and evaluated in terms of recommendations of CLSI. With Vitek 2 automated system (bioMerieux, France); minimal inhibitor concentrations (MIC) of ceftazidime, gentamicine, piperacilline, ciprofloxacin, imipenem and meropenem were determined. The transcription product levels of mexB, mexD, mexF, mexY, ampC, oprD were identified with real-time polymerase chain reaction (qPRC) method. To be able to evaluate the similarity among the strains, arbitrarirly primed PCR (AP-PCR) were used.Among the 50 isolates that we divided into four different groups in our study, the frequency of genes that belonged to oprD down regulation, efflux and ampC overexpression are determined as; mexD (88%), mexB (76%), mexF (46%), ampC (46%), mexY (40%), oprD (38%) respectively. With the AP-PCR method that we use to show the similarity among isolates, 18 different patterns were determined.Of imipenem resistant 38 isolates, in 26 (68%) mexB, in 32 (84%) mexD, in 15 (39%) mexF, in 19 (50%) mexY overexpression and in 23 (61%) ampC overexpression were determined. oprD mRNA levels were found to be low in 20 of 38 isolates. No oprD were found in nine isolates. In isolated carbapenem resistant group, while in one of the 4 isolates whose oprD mRNA levels showed significant decrease were found to have only oprD down regulation, the other 3 isolates had simultaneous mexB overexpression, one of them had mexD overexpression and in two of them, ampC overexpression was determined.Among the 35 isolates (70%) that are morepenem resistant in our study, the overexpression of MexAB-OprM, MexCD-OprJ, MexEF-OprN ve MexXY-OprM efflux systems were found 25 (71%), 28 (80%), 16 (46%) and 19 (54%) respectively. That determination of at least one efflux system in 33 (94%) of these isolates that are meropenem resistant shows the contribution of these mechanisms in meropenem resistance. Among these 35 (70%) isolates that are meropenem resistant, in 5 of 6 isolates that did not have mexB overexpression, oprD expression was significantly found to be low.Because of the fact that in meropenem resistance emergence, OprD loss and up regulation of MexAB-OprM pump take part, emergence of meropeneme resistance is more unlikely than emergence of imipeneme resistance. 30 of 38 isolates in our study were found to be meropenem resistant. Although the decrease in the amount of oprD in 7 isolates of 9 that were isolated carbapenem resistant together with simultaneous increase seen in imipenem and meropenem MICs, strongly support the effect of oprD in carbapenem resistance, that simultaneous presence of overexpressions of resistance genes were considered the synergistic effect in resistance emergence.In 12 isolates in isolated quinolone resistant group, oprD amount were low in 6 isolates. That mexF expression to be found high in all isolates but one in this group show the possible relation between mexF overexpression and oprD down regulation in these imipenem sensitive isolates and the necessity of further additional studies to support its effect in emergence of imipenem resistance.21 of strains (91%) in which ampC overexpression were seen, were determined in the 1st group that contain isolates resistant to antipseudomonal ß-lactams and aminoglycosides. That overexpression not to be seen in the quinolone resistant isolates in 3rd and 4th group considered that AmpC may not have effect in quinolone resistance.The resistance genes that we analyse in our study have importance in P. aeruginosa mechanisms. These mechanisms that were identified lead to the treatment protocol which is followed in the treatment of infections caused by this microorganism. The very first thing to be done on the way to develop new strategies in struggle with antimicrobial resistance should be to explain which mechanisms regulate the expression of these resistance mechanisms.