Subjecting Local Lactobacillus Isolates to Mutagensis for Improving Inhibitory Activity Against Staphylococcus aureus.

number: 
2717
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Ibtesam Salim Mahdi
Supervisor: 
Dr. AbdulWahid B. Al-Shaibani
Dr.Shayma’a Hussain
year: 
2011
Abstract:

Sixteen isolates of Lactobacillus spp. were obtained from different sources including dairy products, vinegar, pickles and two locally vaginal isolates by primary culturing on De Mannes Rogoza Sharp (MRS) agar before subjection to cultural, microscopic and biochemical tests. To examine their inhibitory activity against methicillin resistant Staphylococcus aureus (MRSA), all isolates were cultured once on solid and other in liquid MRS media. Results revealed that high inhibitory effect was detected by the isolates that propagated in liquid medium than on the solid. Upon such result, the MRS broth was used to propagate Lactobacillus isolates in the next experiments. After propagation, filtrates of isolates were obtained. No observable inhibitory effect was detected against the test bacteria when the unconcentrated Lactobacillus filtrates were used. While, after concentration, the one-fold concentrated filtrates gave positive results, especially after 24 hours (hrs.) of incubation period. Among the 16 isolates, Lactobacillus acidophilus (Lb.G2) and Lactobacillus gasseri (Lb.G1) from vagina origin gave the best inhibitory activity, therefore, they were chosen for the further experiments. Inhibitory effect of the two-fold concentrated filtrates was increased to (17) millimeter for L. acidophilus and (18) millimeter for L. gasseri in comparison to (12) millimeter and (11) millimeter, respectively, for the one-fold filtrates after incubation at 37C for 24 hrs. Moreover, the three-fold concentrated filtrates gave the highest inhibitory activity with inhibition zones of (21) millimeter for L. acidophilus and (23) millimeter for L. gasseri. When the effective compounds produced by the two lactic isolates were evaluated, organic acids were found to be the most responsible factor for the inhibitory effect against methicillin resistant Staphylococcus aureus. Susceptibility of the lactic isolates toward (13) commonly-used antibiotics was also determined. Results declared that they were resistant to streptomycin, ciprofloxacin, gentamycin, ampicillin and nalidixic acid, but sensitive to chloramphinicol, cephotaxime and amoxicillin, while they were varied in their sensitivity towards the rest antibiotics. After subjection of the two isolates to mutagenesis for obtaining mutants with improved inhibitory effect against test microorganism, physical mutagenesis by ultra violet led to increase their antagonistic effect against the methicillin resistant S. aureus when their inhibition zones increased to (16.5) millimeter for L. acidophilus and (15) millimeter for L. gasseri by comparison with their wild type (12 and 11 millimeter) respectively. On the other hand, results of chemical mutagenesis revealed that inhibitory activity of the two isolates increased after treatment with ethedium bromide when the inhibition zones against S. aureus raised to (15.5) millimeter for L. acidophilus and to (14) millimeter for L. gasseri compared with the controls (12 and 11 millimeter), respectively. In contrast, treating with the acridin orange resulted in no effect on the isolates inhibitory effect. Upon isolation and visualization of the plasmid contents of wild and mutant types, plasmid profile was found to be identical when each native isolate and its mutant carried one plasmid band.