Pengaruh Paparan Ekstrak Kasar Alkaloid Daun Pepaya Muda Kering Beku terhadap Empat Isolat Staphylococcus aureus dan Ekspresi Gen sea
Kusumaningrum, Harsi Dewantari
Faridah, Didah Nur
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Staphylococcus aureus produces staphylococcal enterotoxin A (SEA) that causes food poisoning and resistant to the heating process. The presence of S. aureus and SEA on food could be detected by moleculer technique, its conducted quickly and accurately. Molecular detection is based on the genetic information of cell. Alkaloids are bioactive compound in young papaya leaves that can act as antimicrobials. The drying process and extraction method are greatly affecting the total alkaloid content in papaya leaves. Alkaloids as antimicrobial can acts by intercalating into cell wall and double-stranded DNA, and affecting topoisomerase enzymes and DNA-repairing enzymes. The aims of this research were to study the effect of drying methods of young papaya leaves to the yields of crude alkaloid extract, to identify toxin-producing of four S. aureus by molecular technique, and to identify the effect of crude alkaloid extract exposure towards S. aureus cells and relative expression of sea gene of four S. aureus isolated from food origin, i.e. raw milk (S10), balado eggs (TBI), stir-fried chicken intestine (UA13) and chicken offal satay (SJI). Drying of Calina young papaya leaves was conducted with the oven temperature of 55°C and freeze drying. Crude alkaloid extract were extracted by ultrasonication method and detected using Mayer’s reagent. S. aureus isolates that have been stored in ampoules were examined morphologically by growing them on baird parker agar (BPA) and triptyc soy agar (TSA) media. Antibiotic resistance was conducted towards S. aureus isolates using gentamicin, streptomycin, kanamycin, chloramphenicol, tetracycline and oxytetracycline. S. aureus isolates were molecularly detected with conventional PCR. The PCR results were sequenced to see the sequence of bases on each DNA, then the sequence was analyzed with Basic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov. Exposure of crude alkaloid extract with concentrations of 0.25 mg/ml and 0.5 mg/ml towards S. aureus was conducted for 2 hours. The cell number of S. aureus before and after 2-hour exposure was calculated using agar spread-plate method, then the relative expression of sea gene was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Papaya leaves dryed with freeze drying method contained crude alkaloid extract content of 1.34±0.36%. These results were relative higher than the oven drying method that only produced 0.99±0.03% crude alkaloid extract, although statistically they were not significantly different. S10, TBI, UA13 and SJI isolates on BPA media has round-shaped colonies, smooth, convex and gray to black in color. The colonies’ surrounded edge was clear (with bright area). All isolates formed a yellow pigment on TSA media. All isolates were resistant to streptomycin. In addition, there was a different sensitivity of each bacteria toward antibiotic gentamicin, kanamycin, chloramphenicol, tetracycline and oxytetracycline. Analysis of isolates by conventional PCR showed that all isolates were positive for sea gene with the amplicon size 120 bp. S10, UA13 and TBI isolates have similarities with Staphylococcus aureus strain RKI4 and SJI isolate has similarities with S. aureus 08BA02176 genome in GenBank. Exposure of crude alkaloid extract obtained from freeze-dried young papaya leaves at 0.25 mg/ml and 0.5 mg/ml for 2 hours slightly reduced the cell number of S. aureus for approximately 0.6 log CFU/ml and 0.8 log CFU/ml respectively compared with the control group that was not exposed to the crude alkaloid extract. The relative expression of sea gene decreased varied among isolates, in a range of 1.75 to 27.00 times after exposure with 0.25 mg/ml and in a range of 13.59 to 33.24 after exposure with 0.5 mg/ml.
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