الفهرس 
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Abstract
P.aeruginosa is a major hospital associated pathogen. It can tolerate a wide variety of physical conditions and many antibiotics by different resistance mechanisms. The fluoroquinolone (FQ); levofloxacin and ciprofloxacin remain the preferred anti- pseudomonal agents. One of the FQ resistance mechanisms is the mutations leading to the over-expression of multi-drug efflux pumps that extrude FQ out of the cell. P. aeruginosa exhibits several efflux pump systems that allow it to be resistant to several antimicrobial agents. The RND family is the most common multi-drug efflux pump in P. aeruginosa. It belongs to the secondary transporters that utilize the proton motif force for drug expulsion. The RND pump is a trimeric structure: an inner membrane- embedded antiport protein connected to the OMP in the outer membrane through the membrane fusion protein (MFP) in the periplasm. A single pump can extrude a wide range of antibiotics and a single organism can show overexpression of more than one pump. MexAB-OprM was the earliest reported RND in P. aeruginosa followed by other RND transporters, of them the most commonly found in clinical isolates are MexCD-OprJ, MexEF-OprN and MexXY-OprM. Bacterial efflux pumps are an important target to overcome bacterial MDR which could be achieved by different efflux pump inhibitors (EPI). Phenylalanine arginyl β-naphthylamide (PAβN) is an EPI that acts by competitive inhibition and can inhibit several multidrug efflux pumps in P.aeruginosa.
This study aimed to detect the efflux pump activity in FQ resistant clinical isolates of P. aeruginosa from inpatients in Alexandria Main University Hospital by both phenotypic and genotypic methods, investigate the role of efflux pump mediated fluoroquinolone resistance in MDR clinical isolates of P. aeruginosa and assess the effect of PAβN as a combination treatment option for MDR P.aeruginosa infection. Over the year 2016, one hundred strains of P. aeruginosa, that were isolated from different clinical specimens delivered to the AMUH routine microbiology lab, were screened after culture for detection of P.aeruginosa fluoroquinolone resistant isolates.
The antimicrobial susceptibility of all P.aeruginosa isolates was determined by the Bauer-Kirby disk diffusion technique according to CLSI guidelines (2018). All the isolates were found to be MDR. All the isolates were sensitive to colistin and the least resistance was found against aztreonam and piperacillin/tazobactam (57%) each. Seventy seven and 73 isolates were resistant to ciprofloxacin and levofloxacin respectively.
Forty two (42/ 73,57.5%) of the 73 P.aeruginosa isolates that were found resistant to both ciprofloxacin and levofloxacin shared the same antibiotic resistance profile, where they were resistant to all tested antibiotics except colistin, and (13/ 73,
17.8%) shared a nearly similar profile except that in addition to colistin susceptibility, they were also sensitive to aztreonam. These antibiotic resistance profiles distribution could give a preliminary indication that the ciprofloxacin and levofloxacin resistant isolates may share the same antibiotic resistance mechanism,i.e. FQ resistant isolates could be in coordinate with resistance to other antipseudomonal agents through overexpression of FQ mediated multidrug efflux pumps. On the other hand, the 27
P.aeruginosa isolates that were susceptible to ciprofloxacin and/or levofloxacin showed
18 different antibiotic resistance profiles. A total of 13 profiles were unique but only 5
strains shared the same antibiotic resistance profile (ceftazidime, gentamicin, tobramycin, piperacillin, amikacin, aztreonam, cefepime, imipenem, and meropenem).
The role of PAβN peptidomimetic efflux pump inhibitor (EPI) as a phenotypic test was evaluated. The MICs of ciprofloxcin and levofloxacin acting on the73 isolates that were resistant to both drugs by the Bauer Kirby disc diffusion technique were tested in the presence and absence of (20µg/ml) PAβN, by the microbroth dilution technique as recommended by CLSI (2018).In the absence of PAβN ciprofloxacin MIC
50 was 256µg/ml and levofloxacin MIC 50 was 128µg/ml. After the addition of PAβN, (70/73, 95.9%) and (69/73, 94.5%) of the isolates showed a reduction in MICs of ciprofloxacin and levofloxacin respectively, (6/73, 8.20%) and (20/73, 27.4%) of the isolates restored their susceptibility to those drugs respectively and (6/73, 8.20%) of the isolates were reverted to levofloxacin intermediate susceptibility breakpoint. The number of isolates that reverted to levofloxacin susceptible breakpoint was found to be statistically significant.
Using Reverse transcription RT PCR for detection of mexA, mexC, mexE and mexX genes overexpression as representatives of the mexAB-OprM, mexCD-OprJ, mexEF-OprN and mexXY-OprM efflux pumps respectively, mexE (51/73,69.8%) was found to be the most common efflux pump overexpressed among the ciprofloxacin and levofloxacin resistant P.aeruginosa isolates, followed by mexX (49/73,67.1%) and mexA was represented in (43/73, 58.9%) of the isolates, whereas mexC (39/73,53.4%) was the least overexpressed efflux pump gene among those isolates. Eight isolates (8/73, 10.9%) showed no overexpression of any of the four tested RND efflux pump genes. Twenty isolates (20/73, 27.4%) overexpressed all four tested RND efflux pump genes mexC+ mexE+ mexX.
Comparing both phenotypic and genotypic methods for detection of efflux pump, the current study showed that (62/64, 96.8%) and (61/64, 95.3%) of the isolates showed both overexpression and reduction of ciprofloxacin and levofloxacin MIC respectively. One isolate (1/9, 11.2%) showed neither efflux pump overexpression nor reduction in ciprofloxacin and levofloxacin MIC. Whereas among the 9 P.aeruginosa isolates that showed no overexpression of any of the four tested efflux pumps as detected by RT PCR, (8/9, 88.8%) P.aeruginosa isolates showed reduction in both ciprofloxacin and levofloxacin MICs after the addition of PAβN.
A roc curve to evaluate the PAβN as a phenotypic method for detection of efflux pump overexpression revealed that the addition of PAβN to levofloxacin can significantly discriminate efflux pump overexpressing from non-overexpressing isolates. It has fair diagnostic performance at the cut- off point of ≥4 fold reduction in MIC with sensitivity of 70% and specificity of 67%.While ciprofloxacin with PAβN cannot significantly discriminate between efflux pump overexpressing and non- overexpressing isolates.