Evaluation of efflux pumps overexpression and β-lactamase genes among colistin resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is one of the major factors in severe opportunistic and hospital-acquired infections. Structural mechanisms such as resistance-nodulation-division (RND) efflux pumps and drug resistance genes such as extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) are factors causing drug resistance. Colistin is the prominent therapeutic strategy with an excellent inhibitory role for combating multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates. Wide usage of colistin and genetics interactions leads to the emergence of colistin-resistant (CoR) PA isolates which is a dangerous occurrence in XDR-PA isolates. The current study investigates the presence and expression of PA-related RND genes and harboring the antibiotic resistance genes such as ESBL and MBL genes in CoR-PA strains. 70 clinical isolates were obtained from different clinical specimens during 2019-2020. The antimicrobial susceptibility testing was performed by the both Kirby-Bauer disk agar diffusion method and the minimum inhibitory concentration (MIC). Presence of bla(TEM-1), bla(SHV, )bla(CTX-M), bla(IMP), bla(VIM )and bla(NDM) was measured in all CoR-PA isolates. Besides, screening of mcr-1, aadA, aadB and efflux pump genes (mexA, mexD, mexE and mexY) was conducted for CoR-PA isolates. To determine the expression level of efflux pump genes, Quantitative real-time PCR (qRT-PCR) reactions were performed. According to the standard antimicrobial susceptibility methods 39 isolates, (55.7%) and 31 isolates (44.3%) were considered as MDR-PA and XDR-PA strains, respectively. 12 isolates (17.1%) displayed resistance to colistin using MIC assessment. All CoR-PA isolates were negative for mcr-1 gene. The bla(SHV) (n = 11, 91.6%) and bla(NDM) (n = 7, 58.3%) were the most prevalent ESBL and MBL genes among CoR-PA, respectively. PCR results showed all CoR-PA isolates were positive for all 4 efflux pumps genes. On the other hand, overexpression of MexA, MexD, MexE and MexY genes was observed in 5 (41.6%), 5 (41.6%), 5 (41.6%) and 6 (50%) CoR-PA isolates by qRTPCR, respectively. 9 (75%) CoR-PA isolates overexpressed at least one of the four efflux pumps and overexpression of altogether 4 efflux pumps was shown in 2 (16.6%) CoR-PA isolates. Overexpression of MexA, MexD, MexE and MexY genes was associated to the resistance toward imipenem, cipmfloxacin, ceftazidime and aminoglycosides, respectively. The current research demonstrated the co-existence of CoR-PA with ESBL and MBL genes besides overexpression of efflux pumps. The distribution of such isolates can lead to a burden on the healthcare system by the incidence of the most crucial situation named pan-drug resistant PA strains.