full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 998-1003 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)999Materials <strong>and</strong> MethodsBb 36Streptomyces isolate <strong>and</strong> testorganismsIn a previous study, Saadoun <strong>and</strong> Gharaibeh(7) characterized two Streptomyces isolates (Bb 36<strong>and</strong> Z 11) which inhibited the growth <strong>of</strong>multiresistant bacterial pathogens (Escherichiacoli, Staphylococcus aureus <strong>and</strong> Klebsiellapneumonia). These pathogens were sensitive toat least one <strong>of</strong> these antibiotics (CTX: Cefotaxime, STR: Streptomycin , CXM: Cefuroxime, GN10:Gentamycin). However they were resistant to atleast five <strong>of</strong> these antibiotics (GN10: Gentamycin;AM10: Amoxicillin; STR: Streptomycin; OFF:Offloxacin (Novecin); COT: Cotimoxazole; ERY:Erythromycin; OXC: Oxaxillin; PEN: Penicillin;VAN: Vancomycin; CLT: Cephaloyhin; AUG:Augmantine; CTX: Cefotaxime; CXM:Cefuroxime sodium; TCP: Teicoplanin; TET:Tetracyclin; CMD: Cefam<strong>and</strong>ole; CHL:Chloramphenicol; TOB: Tobramycin; CPR:Cipr<strong>of</strong>loxacin; SXT: Sulphamethoxazole; IMP:Impecillin; CB100: Carbenicillin).Optimal conditions for antibiotic productionCulture media : Bb 36Streptomyces isolate wasgrown in submerged cultures in 250 ml flaskscontaining 50 ml <strong>of</strong> the following broth culturemedia: Tryptone Yeast Extract (TYE), NutrientBroth (NB), Yeast Extract Malt Extract (YEME)<strong>and</strong> Inorganic Salt Starch (ISS) (8). The flaskswere inoculated with 1 ml <strong>of</strong> Bb 36activeStreptomyces spore suspension <strong>and</strong> incubated at28°C ± 1 for 21 days with shaking at 100 rpm.Agar well diffusion method : The inhibitoryactivity was tested on Muller Hinton agar plates(8). Three cores <strong>of</strong> 8 mm diameter were removedfrom the agar plates that have pre-seeded withthe test organisms using a sterile swab. Wells werefilled up with a 100 µl supernatant broth <strong>of</strong> 3, 5, 7,14 <strong>and</strong> 21 days culture. The plates were incubatedat 37°C ± 1 for 48 h, <strong>and</strong> then inhibition zoneswere visualized <strong>and</strong> recorded.Fermentation : Bb 36Streptomyces isolatethat showed the highest activity against thepathogens in the above media was selected forseed culturing by growing the producer strain(Bb 36) on oatmeal agar at 28ºC ± 1 for 14 days.After growth, the whole aerial mycelium wasscrapped by a loop <strong>and</strong> suspended in 10 ml <strong>of</strong>sterile distilled water. Aliquots <strong>of</strong> 0.5 ml <strong>of</strong> thespore suspension was inoculated in 250 mlErlenmeyer flasks containing 50 ml <strong>of</strong> theproduction medium, pH=7.2. Twenty flasks <strong>of</strong> thebroth medium were used for large-scalefermentation. All seed cultures were grown at28ºC ± 1 with shaking in a rotary shaker incubator(TEQ, Portugal) at 100 rpm for 21 days. Afterfermentation, 5 ml <strong>of</strong> culture broth weretransferred to a sterile tube <strong>and</strong> centrifuged at5000 rpm for 15 min at 4 °C.Extraction : The remaining broth was extractedwith 4 volumes <strong>of</strong> absolute ethanol for overnightat 4ºC. After extraction, the pellet was discarded<strong>and</strong> the supernatant was evaporated using rotaryevaporator (Heidolph, Germany) then the dryweight <strong>of</strong> the ethanol evaporate extract wasdetermined.Determination <strong>of</strong> MIC for the crude ethanolevaporates: Dried ethanol evaporate <strong>of</strong> Bb 36extract was suspended in 10 ml <strong>of</strong> distilled waterto have a concentration <strong>of</strong> 0.1 g <strong>of</strong> the ethanolevaporate/ml. Three cores <strong>of</strong> 8 mm diameter wereremoved from the Mueller Hinton agar plates thathave pre-seeded with the test organisms using asterile swab. Holes were filled up with thedissolved ethanol evaporate extract (100 µl) <strong>and</strong>plates were incubated at 37°C ± 1 for 48 h theninhibition zones were visualized <strong>and</strong> recorded (6).MIC <strong>of</strong> the ethanol evaporate wasdetermined <strong>and</strong> the activity <strong>of</strong> the ethanolevaporate against the pathogens was comparedto the activity <strong>of</strong> the broth production medium.Ismail Saadoun <strong>and</strong> Mohammad alawawdeh