Activity of crude alkaloid extract of papaya leaves toward staphylococcus aureus and staphylococcal enterotoxin a gene expression

Abstract

Papaya plant is widely grown in Indonesia. Papaya leaves are used by people as vegetable, meat tenderizer and traditional herbs. Extracts of papaya leaves have been reported to have antimicrobial activity against both Gram positive and Gram negative bacteria. This antimicrobial activity was related to phytochemical content of papaya leaves. Tannins and terpenoids can make cell membrane disruption, whereas alkaloids have been shown to intercalate into DNA and inhibit DNA synthesis. Therefore, besides as meat tenderizer, papaya leaves can be an alternative natural preservatives in food. Staphylococcus aureus is known pathogen causing intoxication by producing staphylococcal enterotoxins (SEs) in food. Staphylococcal enterotoxin A (SEA) is one of SEs that is commonly implicated in staphylococcal food poisoning. SEA is synthesized from sea gene and expressed from the midexponential phase of growth. In addition to the ability to inhibit S. aureus growth, there is a potential action of papaya leaves extract, i.e the ability to inhibit SEA production through the alkaloids activity. This study was conducted to investigate the ability of crude alkaloid extract from papaya leaves to inhibit SEA expression. Crude alkaloid extract was extracted from grounded dried papaya leaves using ultrasonic-assisted extraction. S. aureus was isolated from ready to eat food and raw cow milk using modified Bacteriological Analytical Manual (BAM) method. Isolates with typical colony of S. aureus on Baird-Parker Agar (BPA) and Mannitol Salt Agar (MSA) and produced coagulase were identified using API Staph test and PCR of 16S rRNA gene, followed by sequencing. Those 16S rRNA gene sequence were analyzed using BLAST. Minimum Inhibitory Concentration (MIC) of crude alkaloid extract were determined by broth macro-dilution method. Furthermore, S. aureus isolate was exposed to crude alkaloid extract at 0, 1, and 2 folds of MIC, and subsequently the expression of sea gene was analyzed using a quantitative reverse transcription PCR (qRT-PCR). The results demonstrated that the yield of crude alkaloid extracts was 0.48% to 1.82% per dry weight of papaya leaves. SEA-producing S. aureus were obtained from some foods, i.e raw milk, fried egg in chilli sauce, sauteed chicken intestine, and chicken liver satay. The MIC of crude alkaloid extract to S. aureus was 0.25 mg/ml. After exposure to the crude alkaloid extract at 0.25 mg/ml and 0.5 mg/ml for 2 h, significant increase in cycle threshold (CT) values of sea gene was observed. These results indicated a decrease of initial amount of sea cDNA which meant lesser mRNA was synthesized from the sea gene. The sea gene was expressed 29 and 41 times less when S. aureus was exposed to crude alkaloid extract at 1 and 2 folds MIC, respectively, determined relatively to the calibrator sample and an internal control using comparative CT method. The crude alkaloid extract of papaya leaves possessed antibacterial activity against S. aureus which was not only inhibited the bacteria but also the toxin formation by suppressing the expression of sea gene.