full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 998-1003 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)999Materials and MethodsBb 36Streptomyces isolate and testorganismsIn a previous study, Saadoun and Gharaibeh(7) characterized two Streptomyces isolates (Bb 36and Z 11) which inhibited the growth ofmultiresistant bacterial pathogens (Escherichiacoli, Staphylococcus aureus and Klebsiellapneumonia). These pathogens were sensitive toat least one of these antibiotics (CTX: Cefotaxime, STR: Streptomycin , CXM: Cefuroxime, GN10:Gentamycin). However they were resistant to atleast five of 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: Cefamandole; CHL:Chloramphenicol; TOB: Tobramycin; CPR:Ciprofloxacin; 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 of the following broth culturemedia: Tryptone Yeast Extract (TYE), NutrientBroth (NB), Yeast Extract Malt Extract (YEME)and Inorganic Salt Starch (ISS) (8). The flaskswere inoculated with 1 ml of Bb 36activeStreptomyces spore suspension and 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 of 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 of 3, 5, 7,14 and 21 days culture. The plates were incubatedat 37°C ± 1 for 48 h, and then inhibition zoneswere visualized and 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 and suspended in 10 ml ofsterile distilled water. Aliquots of 0.5 ml of thespore suspension was inoculated in 250 mlErlenmeyer flasks containing 50 ml of theproduction medium, pH=7.2. Twenty flasks of 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 of culture broth weretransferred to a sterile tube and centrifuged at5000 rpm for 15 min at 4 °C.Extraction : The remaining broth was extractedwith 4 volumes of absolute ethanol for overnightat 4ºC. After extraction, the pellet was discardedand the supernatant was evaporated using rotaryevaporator (Heidolph, Germany) then the dryweight of the ethanol evaporate extract wasdetermined.Determination of MIC for the crude ethanolevaporates: Dried ethanol evaporate of Bb 36extract was suspended in 10 ml of distilled waterto have a concentration of 0.1 g of the ethanolevaporate/ml. Three cores of 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) andplates were incubated at 37°C ± 1 for 48 h theninhibition zones were visualized and recorded (6).MIC of the ethanol evaporate wasdetermined and the activity of the ethanolevaporate against the pathogens was comparedto the activity of the broth production medium.Ismail Saadoun and Mohammad alawawdeh