الفهرس 
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Abstract
Efflux pumps play an important role in the development of MDR in bacteria. The four multidrug efflux pumps in P. aeruginosa are involved in the ejection of hazardous compounds and contribute to diminished antibiotic sensitivity. Furthermore, overexpression of these multidrug efflux pumps in P.aeruginosa is directly associated with resistance to most anti-pseudomonal medications and may impair the efficacy of new classes of therapies produced against P. aeruginosa.
This study aimed to detect efflux pumps in P.aeruginosa by both phenotypic and genotypic methods and examining the ability of efflux pumps to inhibit efflux pumps so restoring antibiotic sensitivity.
Between February 2022 and June 2023, 350 samples had been gathered from Minia university hospitals. P.aeruginosa isolated from 29.7% of collected samples. The isolates collected from different samples type and its prevalence was 28.69%, 25% and 65% as isolated from wounds, urine samples and burns respectively. It was identified by cultural characteristics on cetramide agar, Microscopic examination and biochemical reactions. Regarding demographic data of study patients, the study revealed that P.aeruginosa infections common among males more than females and the mean age of patients was 35 year.
Antimicrobial susceptibility testing was done for all isolates by disc diffusion and Microtitre dilution method. It showed varying levels of resistance to the tested antimicrobial agents and showed a rate of MDR in 55.7% of isolates.
The highest level of antibiotic resistance was against Ceftazidime, Pipracillin and Meropenem by which 62.5%, 51%and 47.1% of isolates were resistant respectively, followed by Pipracillin–Tazobactem, Amikacin, Cefipime, Gentamycin and Levofloxacin 46.2%, 45.2%, 43.3%, 35.6% and 34.6%respectively . However Colistin is considered as the most sensitive agent of all tested antibiotics with resistence rate 17.3%.Combination of different antibiotic with Zinc sulfate by Microtitre dilution test gave synergistic and addition results with best synergistic effect occurred in combination with Gentamycin and Colistin, it was 94.6% and 94.4 % respectively.
Efflux pumps detected in 75.8% of MDR isolates as detected by cartwheel test. Application of ¼ MIC of Zinc sulfate in Et.Br cartwheel assay test found to inhibit efflux pump activity in all tested isolates as all isolates show flurescence.
Genotypic detection of efflux pumps done by conventional PCR to detect efflux pump genes and Real time PCR which measured the expression of these genes. Regarding detection of efflux pump genes by conventional PCR, mexR, nfxB, mexE and mexT and were the most detected genes with 84.5%, 84.5%,81% and 77.6%. Other efflux pump genes detected as 75.9%, 74.1%, 72.4%, 65.55%, 63.8%, 58.6%, 53.4% and 34.5% for mexB, mexZ, mexY, mexD, mexA, mexC, mexX and oprM respectively.
Real-Time PCR measured the expression of efflux pump genes before and after application of Zinc sulfate. mexT, mexE, mexC, mexX, mexZ, mexB and mexR expressed in 100% of tested isolates. Statistical analysis revealed a significant downregulation in expression mexA, mexB, mexR, mexX, mexC, mexT and mexE after treating with Zinc sulfate.
However, mexA, oprM, oprD, mexY and nfxB expressed in 90.9%, 44%, 44%, 91% and 63% of tested isolates respectively.
Based on the findings of this study and comparing with other studies, it has been suggested that increased expression of the RND efflux pump is one of the main mechanisms in antibiotic resistance and a risk indicator that additional research in this field is required. This is the first study in Egypt that uses phenotypic and genotypic assays to investigate the antibacterial effect of zinc sulfate on the efflux pump of P. aeruginosa.
Conclusion
Finally we conclude that P.aeruginosa infections are prevalent at Minia university hospitals mainly in patient with burn infections and UTI. Antimicrobial susceptibility tests revealed that Colistin was highly effective as anti pseudomonal agent followed by Gentamycin.
Application of Zinc sulfate combinations with antibiotics give synergistic and additive result which ensures that it had a great bactericidal effect in P. aeruginosa. Cartwheel test is a simple technique used for detection efflux pump phenotypically. Addition of sub inhibitory concentrations of Zinc sulfate to cartwheel test found to inhibit efflux pump activity in all tested isolates.
This study reported the great effect of Zinc sulfate in gene expression of RND efflux pumps as measured by Real-Time PCR, there was down regulation of some genes and upregulation of repressors genes.
Recommendations
We recommend the following:
• We recommend that antibiotic resistance to be monitored continuously to catch new resistant strains, unnecessary antibiotics be avoided, and antibiotic susceptibility be tested before antibiotic prescription.
• All of the isolates used in this study were extremely resistant and expressed efflux pumps. Combining efflux pump inhibitors with antibiotics would be beneficial to get over this challenge. To better treat infections carried by P. aeruginosa, research into new antibiotics and efflux pump inhibitors could be a desirable approach.
• Additional investigations, that include pharmacokinetic and pharmacodynamic studies, as well as in vivo efficacy evaluation in experimental models of infection, including dosage and safety, are required to validate the value of Zinc sulphate in the treatment of MDR P. aeruginosa infection. Therefore, it was suggested that the anti-bacterial activity of Zinc sulfate was due to the inhibition of efflux pump genes expression.