full issue - Association of Biotechnology and Pharmacy

More documents

Recommendations

Info

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
Current Trends in Biotechnology and PharmacyVol. 5 (1) 998-1003 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1000Cultural and physiological characterizationsof Bb 36Streptomyces isolate : Streptomycesisolate (Bb 36strain) was characterizedmorphologically and physiologically according tothe International Streptomyces project (ISP) (11)and as described by Saadoun et al. (9).ResultsDetermination of optimum culture media forinhibitory compound(s) production : When theactivity of Bb 36Streptomyces isolate wascompared to 23 standard antibiotics, data revealedthat this isolate may not produce any of the testedantibiotics except cefotaxime and cefuroximesodium. Table 1 shows several broth media thathave been examined to test the ability of the activeStreptomyces strain Bb 36to produce inhibitorybioactive compounds against the tested pathogens.Data showed that strain Bb 36optimally produceinhibitory bioactive compound(s) against E. coliand S. aureus after 5 days when cultured inYEME broth with an inhibition zone diameter of8 and 13 mm, respectively. Also, growth asmeasured by dry weight biomass production inthe YEME broth medium was followed for 14days (Table 1). Results from Table 1 demonstratedthat Bb 36Streptomyces isolate prefer to producethese inhibitory substance(s) in YEME brothmedium at the expense of growth.Partial isolation and purification of the activecompound(s) : It is necessary to extract andpurify the substance responsible for the antibioticactivity, so further tests for evaluation werecarried out with a simple ethanol extraction ofthe antibiotic from cells grown in liquid cultures.Table 2 shows the MIC of the evaporated watersolubleethanol extract against S. aureus and E.coli. Ethanol extraction and evaporation of theaTable 1. Activity of Bb 36isolate at different incubation intervals and culture media.Isolate Broth Final pH Dry Weight (mg/ml) Activity (mm) bMediumDays E. coli S. aureus5 7 14 5 7 14 5 7 14 5 7 14Bb36 TYE 8.53 8.75 8.65 1.2 0.0 0.0 13 - - 10 - -NB 8.36 8.66 8.76 0.2 0.6 1.0 - - - - - -YEME 6.36 6.72 8.15 2.4 2.6 3.5 8 8 8 13 11 11ISS 6.42 6.70 8.08 2.4 2.9 3.3 - - - - - -aTYE: Tryptone Yeast Extract; NB: Nutrient Broth; YEME: Yeast Extract Malt Extract;ISS: Inorganic Salt StarchbNo activity was recorded at 3 and 21 daysTable 2. Activity of ethanol evaporate extract of Streptomyces B36 isolate.Extract Concentration (mg/ml)Bacteria 10 9 8 7 6 5 4 3 2 1S. aureus 13 a 13 13 13 13 11 - - - -E. coli 8 - - - - - - - - -aDiameter of inhibition zone (mm, mean value, N= 3).Inhibitory compounds producing against multiresistant bacterial pathogens
Page 3 and 4: ISSN 0973-8916Current Trends inBiot
Page 5 and 6: Current Trends in Biotechnology and
Page 33: Current Trends in Biotechnology and
Page 85 and 86:
Current Trends in Biotechnology and
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123:
show all

full issue - Association of Biotechnology and Pharmacy

Create successful ePaper yourself

Delete template?

Save as template?