Role of Gamma Radiation and Some Natural Products in Alteration of Bacterial Outer Membrane Porins Permeability for Uptake of Certain Antibiotics

Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The bacterial outer membrane proteins (OMPs) that constitute porins play role in the definition of intrinsic resistance in Gram negative bacilli that is altered under antibiotic pressure. It has been noted that the response to prolonged exposure to increasing levels of antibiotic cause major changes in the permeability of the bacterium due to down regulation of porins and over expression of efflux pumps. In this study a total of 92 bacterial isolates of different species were isolated from different sites of cancer and non cancer patients; the microorganisms were identified using API system. The susceptibility test was carried out for all the isolates to detect the multidrug resistant isolates; from this test eleven strains were selected for further studies. Antimicrobial susceptibility of the eleven strains against some selected antibiotics acting on the inhibition of cell wall synthesis before and after in vitro gamma irradiation was carried out. The obtained results showed a clear increase in the number of resistant isolates after irradiation as compared to those before irradiation. The efficacy of the citrus fruits (Citrus limon, Citrus paradise, Citrus reticulate and Citrus sinensis) was tested to improve the performance of the tested antibiotics by increasing its permeability through the porin channels. The dried crushed citrus fruits peels were decontaminated by gamma irradiation at 700 Gray; then the aqueous extract of the citrus fruits were prepared to test its antimicrobial activity against the selected bacterial strains. The obtained results revealed that the aqueous extracts of different citrus fruits peels did not show any antibacterial activities against six bacterial isolates (Acinetobacter calcoaceticus 44, Enterbacter cloacae 51, Escherichia coli 52, Pseudomonas fluorescens 64, Klebsiella pneumoniae 78 and Pseudomonas aeruginosa 90). Therefore, these six isolates were selected for further studies. The synergistic interaction between natural extracts and some β-lactam antibiotics against the selected clinical isolates was carried out to evaluate the effect of these extracts on the bacterial permeability. It was noticed that the citrus fruits extracts improved the tested antibiotics activity against K.pneumoniae 78 and Ps.aeruginosa 90 thus increasing the bacterial susceptibility. The minimum inhibitory concentrations (MICs) of γ the least efficient antibiotics [ampicillin (AMP), amoxycillin (AML) and cefoperazone (CFP)] were determined for the six selected strains. Also, the MICs of these antibiotics after treating the bacterial cells with natural extracts were studied. The results indicated that MIC′s of all antibiotics after treating K. pneumoniae and Ps. aeruginosa with lemon extract were decreased. The results revealed that the treatments of both species with lemon extract is a promising method for increasing the permeability of the strains and thus facilitates the passage of antibiotics through porins and consequently increase the microbial sensitivity. Alterations in permeability were evaluated by outer membrane protein (OMP) analysis of K.pneumoniae 78 and Ps.aeruginosa90. It was noticed that the resistance of both microorganisms to the tested antibiotics is due to loss of porins proteins OmpK 35 and OprF, respectively in the control and irradiated isolates. Treating both microorganisms with aqueous extract of lemon peels resulted in the expression of these porins and subsequently increased its sensitivity. Plasmid profile of the selected bacterial strains (control, irradiated and lemon extract treated isolates) showed differences between control and irradiated isolates, in the molecular weight, Rf, and optical density (OD) of the extracted plasmid whereas the plasmid profile of the treated isolates were relatively unchanged comparing with the control. The charges of the citrus fruits particles were determined. It was noticed that the citrus fruits particles bear positive charges, and the lemon extract particles had the highest positive charges. Also the results revealed that the citrus fruits particles had varied sizes and the lemon particles were the smallest size. Finally, the dielectric properties of Ps. aeruginosa and K. pneumoniae membranes were studied to observe the effect of lemon extract on the cell wall charges. It was noticed that the capacitance and conductance of the two strains decreased after lemon extract treatment comparing with the control which reflects the decrease in overall charge of the cell.