الفهرس 
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Abstract
Because of the antimicrobial threat caused by MRSA, this study aims at looking for phages active against it, their characterization, and production optimization. Seventy Staphylococcus sp. isolates were collected from two hospitals in Cairo, Egypt. The isolates were already recovered from various clinical specimens, like pus, nasal swabs, blood, urine, sputum, CSF, and semen. Twenty-three were proved to be methicillin-resistant Staphylococcus aureus (MRSA) and were used as bacterial hosts along the study. All the 23 MRSA isolates were susceptible to only two antibiotics; linezolid and vancomycin; however, most of them were resistant to nearly all 10 remaining antibiotics tested.
Since bacteriophage could be found wherever its host is abundant, 88 miscellaneous environmental samples were collected for bacteriophage isolation purpose. The sources of these samples varied between sewage, wastewater from hospital drains, raw milk, rinses of raw chicken and fish, pond and lake waters. Phage isolation was accomplished on MRSA isolate 15 by the typical isolation procedure with little modification. Five lysates showing lytic activity against MRSA were isolated, and were given the names D3, F2, H10, L10, and L12. All the five phage lysates were qualitatively assayed via the spot test and quantitatively by the plaque assay, and they showed a clear lytic effect with relatively high initial titers.
Next, they were examined for their host ranges against the rest of MRSA isolates (n=22). The two phages isolated from raw chicken and fish rinses, L10 and F2 respectively, showed the broadest activity (100%); therefore, they were selected for further testing. characterization of bacteriophages extends to involve several test parameters. Among them, prime ones were chosen. Visualized via electron microscopy, the two phages, were assigned to two different morphotypes based on the apparent morphology. Phage L10 was suggested to be belonging to Podoviridae, whereas phage F2 might be a member of Siphoviridae. The effect of temperature, pH, some biocidal organic solvents on phage stability was investigated. In general, both proved to be stable towards most of the factors tested. Thermal inactivation point was 60℃; they survived all over the pH scale except for the extreme acidity and alkalinity. Upon testing their sensitivity to UV, they were found to be still viable after 30 minutes of exposure to UV light; however, the clarity of the lytic spots of both phages started to weaken thenceforth till it completely disappeared after 1 hr of exposure. Chemically, both viruses withstood most of the concentrations tested of ethanol, isopropanol, and chloroform; L10 phage was negatively affected only by the 100% concentration of isopropanol, while the lytic activity of F2 started to weaken at 50% ethanol concentration.
In a trial to maximize their production and hence their lytic activity, optimization of the two phages was approached using two modalities; one-factor-at-a-time (OFAT), and response surface methodology (RSM).
OFAT optimization results indicated that both phages agreed on peptone as a preferable nitrogen source, 28℃ as the optimum incubation temperature, and a host initial concentration of 107 CFU/ml as the suitable inoculum size. However, they differed in their favorable carbon source, for L10 it was glycerol, but for F2, sucrose gave higher yields.
For RSM optimization, Design Expert Software was used; and D-optimal design was selected for experimental design. The factors incorporated were pH, carbon source, and nitrogen source concentrations, each at three different levels. Seventeen production experiments were generated by the software for each phage and were performed accordingly. The conditions of maximum production obtained by RSM varied for the two phages, and after experimental verification, it was concluded that RSM optimization succeeded in improving phage production yields by two log-folds.
Overall, the above findings point to the lytic ability inflicted by the isolated phages on MRSA. Apparently, their stability under some of the extreme conditions tested implies their potential to be candidates for pharmaceutical formulation as an anti-MRSA therapeutic tool. Definitely, further delving in the world of these tiny giants is essential to fully uncover their potential. I hope that bacteriophages could tip the balance in favor of the human front in their battle against multidrug resistant pathogens.