Isolation and antibacterial susceptibility test of pathogenic gram-negative bacilli
Bacterial isolation from different sample sources and sensitivity test using the Kirby-Bauer method (Disk diffusion test) against 23 antibiotics representing several modes of action were carried out as mentioned in [4].
Detection of extended-spectrum beta lactamase (ESBL) activity by different methods
ESBL production was examined by Double disk synergy tests (DDST) [10], Modified double disk synergy tests (MDDT) [11], Phenotypic confirmatory tests (combined disk method) [12], and the Nitrocef disks method [13]. Fifteen identified selected bacterial isolates from previous study [4] were used for further experiments in this study and representing different species; [Escherichia coli (3), Acinetobacter baumannii (3), Pseudomonas fluorescens (1), Pseudomonas aeruginosa (4), Klebsiella pneumoniae (1), Enterobacter sakazakii (1), and Enterobacter cloacae (2)] to distinguish between beta lactamase producer and non-producer isolates.
Examination of antibacterial activity of certain permeabilizers and natural beta lactamase inhibitors (phytochemicals)
Antibacterial activity of the permeabilizers [gallic acid (500, 600, 700 μg/mL), ellagic acid (30, 40, 50 μM), thymol (400, 500, 600 μg/mL), chitosan (50, 100, 250 ppm), Ethylenediaminetetraacetic acid (EDTA) (0.1, 0.5, 1 mM) and sorbic acid (2, 5, 10 mM)], and natural beta lactamase inhibitors [quercetin (25, 50, 100 μg/mL), and epigallocatechin gallate (25, 50,100 μg/mL) were determined (50 μL each) against the studied bacterial isolates, as described by [14], using both disk diffusion (6 mm filter paper discs) and agar well diffusion (10 mm diameter wells) in nutrient agar (NA) plates.
Reduction of antimicrobial resistance
Evaluation of synergistic interactions between non-antibacterial permeabilizers, natural beta lactamase inhibitors and different antibiotics
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a:
Non-beta lactamase producing isolates (antibacterial permeability and antibiotics).
Ten identified multidrug resistant pathogenic isolates were subjected to increasing levels of several non–antibacterial permeabilizers [Chitosan 100 ppm, (EDTA) 0.1 mM, Ellagic acid 40 μM, Gallic acid 600 μg/mL, Sorbic acid 5 mM, Thymol 500 μg/mL] to assess their susceptibility to different selected antibiotics with different modes of action as described by [15]. The selected concentrations had no baseline antibacterial activity.
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b:
Beta lactamase producing isolates.
Five multidrug resistant isolates were subjected to testing of the effects of natural beta lactamase inhibitors (epigallocatechin gallate 50 μg/mL and quercetin 50 μg/mL) alone and in combination with permeabilizers (gallic acid 600 μg/mL and thymol 500 μg/mL) to reduce their bacterial cell wall resistance to beta lactam and standard beta lactamase inhibitor antibiotic discs [piperacillin (PRL) (100 μg), piperacillin/ tazobactam (TZP) (100/10 μg), cefoperazone (CFP) (75 μg), cefoperazone/sulbactam (SCF) (75 /30 μg)] as described by [16].
Irradiation source
A cesium 137 (137Cs) Gamma cell 40, Atomic Energy of Canada Limited, commercial product located at the National Center for Radiation Research and Technology (Nasr City, Cairo, Egypt) was used for irradiation of several identified Gram-negative bacterial isolates.
Effect of in vitro gamma irradiation on the studied multidrug-resistant isolates
By using the linear quadratic (LQ) formula described by [17], an in vitro low radiation dose equivalent to 24.4 Gy, which is biologically equivalent to the 70 Gy in vivo fractionated multiple therapeutic dose used in the cancer treatment protocol of certain cancer patients, was used for irradiation. This source was used at a dose rate of 0.75 rad/sec for the experiments.
Bacterial cultures were divided into two groups, one before irradiation (control group) and the other after irradiation, and each test was performed twice before and after irradiation.
Effect of gamma irradiation on the production of ESBLs
The five studied multidrug resistant producing isolates were assessed for their production of ESBLs by DDST and MDDT after in vitro gamma irradiation.
Determination of minimum inhibitory concentrations (MICs) for selected antibiotics
Four non-beta lactamase producers [Escherichia coli 3, Acinetobacter baumannii 5, Pseudomonas aeruginosa 10, and Enterobacter sakazakii 14] and three producer bacterial isolates [Escherichia coli 2, Pseudomonas aeruginosa 8, and Enterobacter cloacae 13] were investigated in this experiment using antibiotics that were determined in previous work [4] to be highly resistant for these organisms [18]. MICs break points were determined according to [19].
Evaluation of MICs of selected antibiotics in the presence of non-antibacterial permeabilizers and natural beta lactamase inhibitors for certain bacterial isolates
The activity of gallic acid and thymol as a modulator of antibiotic resistance against four selected irradiated and non-irradiated non-beta lactamase producing bacterial isolates, as described by [20], were determined in combination with cefotaxime (CTX, Avants, Cairo, Egypt), cefoperazone (CFP, Pfizer, Cairo, Egypt) and erythromycin (E, National Organization for Drug Control and Research [NODCAR], Cairo, Egypt) by the agar diffusion method. Additionally, the MICs of cefoperazone (CFP) and piperacillin (PRL, National Organization for Drug Control and Research [NODCAR], Cairo, Egypt) in the presence of natural beta lactamase inhibitors (epigallocatechin gallate and quercetin) alone and in combination with selected permeabilizers (gallic acid and thymol) were assessed for three selected beta lactamase producers before and after irradiation.
Permeability assay of the outer membrane in the presence of lytic agents of non-beta lactamase producers treated with gallic acid and thymol
Two multidrug resistant non-beta lactamase producing bacterial isolates were subjected to permeability assays. This method was utilized to determine the permeability properties of the outer membrane in the presence of thymol and gallic acid via their increased susceptibility to the bacteriolytic action of detergents [Triton X-100 & sodium dodecyl sulphate (SDS)] and to the cell wall-degrading action of lysozyme. All bacteriolytic agents were purchased from Sigma-Aldrich, St. Louis, USA. The bacteriolytic effect was assayed on Nunclon microtiter plates (Nunc) by measuring the OD630 of bacterial cultures as previously described [21]. Cell death caused by the sudden influx of these lytic agents was determined by measuring the decrease in optical density (OD) (Relative turbidity %).
Release of lipopolysaccharide
The potential release of OM components (e.g., LPS) was investigated by silver staining of cell-free supernatants after treatment with radiation or permeabilizers [thymol (500 μg/mL), gallic acid (600 μg/mL), EDTA (0.1 mM) and chitosan (100 ppm)]. The samples were analyzed by SDS-PAGE via precast 12% acrylamide gels. Ten μL of lysate was applied to the gel. The gel with proteinase K-treated samples was stained with silver staining [22].
Beta lactamase assays using the peroxidase-chromophore decolorization method of irradiated and non-irradiated beta lactamase producers
This testing was carried out on two multidrug-resistant isolates producing beta lactamase enzyme before and after 24.4 Gy in vitro gamma irradiation in the presence of a permeabilizer (thymol) and a beta lactamase inhibitor (quercetin) for qualitative and quantitative analysis of the production of the open beta lactam ring end product resulting from the hydrolysis of a beta lactam antibiotic, as described by [23].
Statistical analysis
Statistical analysis was performed via computer using the Paired t test, Chi-squared test and One-way analysis of variance (One-way ANOVA). The means and standard deviations (SD) were calculated using SAS software version 9 (SAS Institute, Cary, N.C., USA).