الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
Abstract
Aminoglycosides are natural or semisynthetic antibiotics that were introduced for routine clinical use. They have bactericidal effect on bacteria as they bind to the ribosomal decoding site hence cause misreading of mRNA. The central structure 2DOS is a key feature of the aminoglycoside antibiotics. Accordingly, the goal of our study was optimization of paromomycin production through physiological optimization, genetic mutation and molecular cloning, as an attempt for molecular optimization. The study was also concerned with the partial purification of the produced antibiotic and determination of the in vitro antibacterial effects of paromomycin either alone or in combination with other antimicrobial agents.
Production of paromomycin and ribostamycin from their producing strains S. rimosus subsp. paromomycinus NRRL 2455 and S. ribosidificus NRRL B-11466, respectively, was evaluated using agar plug and well diffusion methods. The antibacterial activities of the produced antibiotics were tested against standard S. aureus ATCC 25923. Inhibition zones appear after overnight incubation at 37˚C indicating their antibacterial activities.
Optimization of nutritional and environmental conditions was achieved leading to higher paromomycin production by S. rimosus subsp. paromomycinus. For the production culture, different culture media were tested. Agitation rate and incubation temperatures for fermentation process were also investigated. Results showed that aminoglycoside production medium (A6) was the optimum medium for paromomycin production (equivalent to 0.36 mg/ml paromomycin). The highest cell growth and paromomycin production were found at 28°C incubation temperature and 200 rpm agitation rate.
Using Response Surface Methodology (RSM), other factors including, pH, inoculum size and incubation period were optimized using the statistical Design Expert software package. At the end of each run, the antibacterial activity of paromomycin was bioassayed against S. aureus ATCC 25923. A Quadratic model and response surface method showed that the optimum conditions for enhancing the paromomycin production were an initial pH of 6, an incubation time of 8.5 days and an inoculum size of 5.5% v/v using the optimized medium. This optimization resulted in about 14 fold improvement of paromomycin production as compared to the initial level fermentation in the basal medium. The resulting model predicted data points that corresponded well to the experimental values.
For genetic optimization, UV irradiation of strains, S. rimosus and S. ribosidificus, was carried out. The results showed that the variants of both strains lost their activity in the original conditions however, they show low antibacterial activities in the optimized condition compared to the wild type. Moreover, subsequent culturing of all UV-induced variants showed unstability in their antibacterial activities that are eventually lost during successive culturing.
S. rimosus subsp. paromomycinus was subjected to gamma rays resulted in mutant coded 5M that showed 200 fold increase in paromomycin production as compared to that of the wild type.
For an attempt of molecular optimization, the two genes namely parC and parS, coding for the key enzymes in the biosynthetic pathway of paromomycin, were successfully cloned into pUCPU21 (cloning plasmid) and pUWL201PW (shuttle expression plasmid). The resulted recombinant plasmids, pUWL201PW-parC and pUWL201PW-parS were transformed into S. rimosus protoplasts to test their effect on paromomycin production. Unfortunately, no transformants could be detected on the selection plates.
Partial purification of the produced paromomycin from S. rimosus culture broth was done by extraction using ethyl acetate which was then evaporated till dryness to give yellowish brown residue that was redissolved in methanol and bioassayed for its antibacterial activity. Ethyl acetate was optimum for the extraction process as showed by the high antibacterial activity of the culture extract, however, the supernatant left after the extraction process showed no antibacterial activity.
The effect of combining paromomycin with different antimicrobial agents was tested by the checkerboard assay against six MDR clinical strains: P. aeruginosa isolates (coded PS1 and PS2); K. Pneumoniae (KP); E. coli (EC); MRSA (SA1) and MSSA (SA2). Combination of paromomycin with ampicillin/sulbactam, ceftriaxone, ciprofloxacin, azithromycin, clindamycin and doxycycline showed 45.83% synergistic effect, 41.67% additive effect and 12.5% indifferent effect against the tested clinical isolates. All the combinations tested against E. coli isolate showed synergistic effect. Combination of paromomycin with ciprofloxacin showed synergistic effect against all the tested isolates except P. aeruginosa and MSSA. Fortunately, no antagonistic effects were observed with any of the combinations tested.