Antibacterial Activity of Red yeast rice Extract against Propionibacterium acnes ATCC 11827 and Methicilin-Resistant Staphylococcus aureus ATCC BAA-1683

Propionibacterium acnes (P. acnes) ATCC 11827 and Methicillin-Resistant Staphylococcus aureus ATCC BAA-1683 are bacteria that cause skin infections, namely acne vulgaris and skin and soft tissue infection/SSTI. The increase in the number of resistant bacterial strains, such as MRSA, requires the search for alternative antibiotics, including using natural ingredients. Red yeast rice is a product of rice fermentation by Monascus purpureus, which is known to have antibacterial, antifungal, antitumor, antioxidant, anti-inflammatory, anti-cholesterol and immunomodulator. This study aimed to determine the antibacterial activity of several red yeast rice extracts against these bacteria that cause skin infections. The research was carried out through the stages of collecting materials and identifying the yeast isolates, extracting of red yeast rice, phytochemical screening of red yeast rice extract, confirmation of bacterial test, preparation of bacterial test suspension, testing for bacterial resistance, testing for antibacterial activity of red yeast rice extract and determining the value of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the most active extract. The results showed that the ethyl acetate and ethanol extracts had antibacterial activity against P. acnes ATCC 11827 and MRSA ATCC BAA-1683. Both extracts contain compounds from the polyphenols, flavonoids, quinones and saponins group. Ethyl acetate extract was the most active extract with MIC values against P. acnes ATCC 1182 and MRSA ATCC BAA-1683 of 50 mg/mL and 12.5 mg/ mL. The MBC values of ethyl acetate extract against these bacterial tests were 100 mg/ mL and 25 mg/mL. The ethyl acetate extract is more active against MRSA ATCC BAA1683 than against P. acnes ATCC 11827. From the results of this study it is known that red yeast rice has activity against bacteria that cause skin infections, especially against MRSA


Introduction
Acne or acne vulgaris is a skin disease that affects many Indonesians, especially teenagers. One of the bacteria that causes acne is Propionibacterium acnes (P. acnes), which is normal flora in the pilosebaceous glands, which under certain conditions can turn into opportunistic pathogens. 1 In mild cases, acne can be treated using benzoyl peroxide or retinoids, while in moderate cases, a combination topical form of benzoyl peroxide or retinoids can be used with topical antibiotics. In severe cases, acne treatment needs to be added with oral antibiotics in the form of tetracycline, azithromycin and erythromycin. 2 Other skin infections that often occur in the community are skin and soft tissue infections (SSTI), such as impetigo, abscesses and cellulitis. SSTI is caused by the bacteria Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Enterococcus species. MRSA is a strain of S. aureus that is resistant to beta-lactam antibiotics, including methicillin, as well as other antibiotics such as oxacillin, penicillin and amoxicillin.2 If the cause of SSTI is MRSA, then the antibiotic of choice can be clindamycin, deoxycycline, ceftaroline or vancomycin. 3 Currently, around 46% of antibiotics are used inappropriately and with incorrect indications. 4 This condition triggers an increase in the number of bacterial strains resistant to antibiotics, so that it requires the search for alternative antibiotics, including those from natural ingredients. One of the natural ingredients that has potential as an antibiotic is red yeast rice. 5 Red yeast rice is a product of rice fermentation by Monascus, especially Monascus purpureus (M. purpureus). In Asian countries, red yeast rice is used traditionally as a colorant, flavoring and natural food preservative as well as medicine. This fermented product of M. purpureus is known to contain various secondary metabolites, such as flavonoids, lovastatin (monakolin K), citrinin and various pigments from the polyketide group. These compounds are known to have activity as antibacterial, antifungal, antitumor, antioxidant, anti-inflammatory, anticholesterol and immunomodulatory. 6 Various pigments of red yeast rice are known to have activity against Gram-positive bacteria such as Bacillus subtilis, Bacillus meganterium, Bacillus mycoides and Staphylococcus aureus, also against Gramnegative bacteria, such as Escherichia coli. This pigment also has antifungal activity against Aspergillus, Mucor, Penicillium citrinum and Fusarium. Until now, there is no information about the antibacterial activity of red yeast rice against P. acnes and MRSA. 5,7,8 In this study, the antibacterial activity of various extracts of red yeast rice were tested against P. acnes ATCC 11827 and MRSA ATCC BAA-1683. This study aimed to determine the potential of red yeast rice as a natural antibiotic to treat the infection of the two bacteria.

Material Collection and Identification of Mold Isolates
Molds were isolated from red yeast rice and identified macroscopically and microscopically by comparison with M. purpureus ITBCCL61. 9 As much as 1 g red yeast rice finely crushed, then suspended 5 mL of sterile distilled water. Red yeast rice suspension and M. purpureus ITBCCL61 culture each was inoculated onto the SDA surface in a petri dish, then incubated at room temperature for 5-7 days. Macroscopic observations were made on the shape and color of the colonies of the two molds.
Microscopic observations were made of the molds grown using the moist chamber method. The forms of hyphae, conidia and ascospores of both fungi were observed and compared under a microscope.

Extraction of Red Yeast Rice
Red yeast rice was extracted by multilevel maceration method using three solvents with different polarity, namely n-hexane (non polar), ethyl acetate (semi polar) and ethanol 96% (polar). 10 A total of 2 kg of red yeast rice was macerated with 2 L of n-hexane for 24 hours at room temperature. The liquid extract was filtered, then the maceration process was repeated several times until a clear filtrate was obtained. The extract residue was dried, then macerated in the same way using ethyl acetate and 96% ethanol. Each extract was thickened using a rotary evaporator at a speed of 60 rpm at 50 °C until the weight was constant. The three extracts were observed organoleptically, then the yield was determined.

Phytochemical Screening of Red Yeast Rice Extract
Phytochemical screening of red yeast rice extract was carried out using the Fransworth method (1966). 11 Screening was carried out on alkaloid, tannin, polyphenol, flavonoid, steroid, triterpenes, monoterpenes, sesquiterpenes, quinone and saponin class compounds.

Confirmation of Test Bacteria
The test bacteria in the form of P. acnes ATCC 11827 and MRSA ATCC BAA-16 were confirmed through microscopic observation using Gram stain and through biochemical tests. 12 The biochemical tests that were carried out included the carbohydrate fermentation test, the MR-VP test, the citrate test, the motility test and the urea test.

Preparation of Test Bacteria Suspension
P. acnes ATCC 11827 and MRSA ATCC BAA-1683 were inoculated onto the MHA surface, then incubated at 37 °C for [18][19][20][21][22][23][24] hours. Both bacterial cultures were suspended in a 0.9% (w/v) sodium chloride solution. The turbidity of the suspension was adjusted to the turbidity of the McFarland standard solution of 0.5 which was equivalent to 1.5 x 108 CFU/mL.

Bacteria Resistance Testing
The MRSA ATCC BAA-16 resistance test to oxacillin was carried out using the agar diffusion method with the paper disc technique. 14 The bacterial suspension was inoculated on the surface of the MHA in a petri dish, then a 1 µg oxacillin antibiotic disc was placed on the agar surface. The plates were incubated at 37 °C for 18-24 hours, then the diameter of the inhibition was measured using a caliper.

Testing Antibacterial Activity of Red Yeast Rice Extract
Red yeast rice extract was tested for its antibacterial activity against P. acnes ATCC 11827 and MRSA ATCC BAA-1683 using the agar diffusion method with the perforation technique. 13 The three extracts were diluted using 2% DMSO (v/v), resulting in several variations in extract concentration. A total of 20 µL of each tested bacterial suspension was put into a sterile petri dish, then 20 mL of liquid MHA was added. The mixture was shaken slowly until homogeneous, then allowed to solidify.
The medium was perforated using a perforator with a diameter of 8 mm, then filled with 50 µL of various extract concentrations. As a comparison, a petri dish containing MHA (negative control), MHA inoculated with the test bacteria (positive control) was prepared, and MHA inoculated with 2% DMSO (v/v) (solvent control). All petri dishes were incubated at 37 °C for 18-24 hours. The antibacterial activity of the extract in the form of the inhibition area around the hole was measured using a caliper. Measurement results were compared between extracts to determine the most active extract.

Determination of Minimum Growth Inhibitory Concentration (MIC) and Minimum Killing Concentration (KBM) of Red Yeast Rice Extract
The most active red yeast rice extract is determined by the Minimum Growth Inhibitory Concentration (MIC) and the Minimum Killing Concentration (KBM) using the microdilution method. 15 The extract was dissolved in 2% DSMO (v/v) as a stock extract solution. The concentration of the tested bacteria was adjusted, so that each well contained 3.75 x 105 CFU/mL. Column 1 to column 12 of microplates each inserted 100 µL MHB. Column 3 and 4 were added by 100 µL of Red yeast rice extract, then homogenized. The stratified dilution was carried out by pipetteing 100 µL from column 4 to column 5, then the mixture was homogenized. The next dilution was carried out in the same manner until the column 12, then the 100 µL from the last dilution was discarded. To the column 2, 4 until column 12, 10 µL of the tested bacterial suspension were added. Column 1 functions as negative control, column 2 functions as positive control, while column 3 functions as extract control. The microplates were covered using cellophane plastic, then incubated at 37 °C for 18-24 hours. MIC value was determined from the column with the smallest extract concentration that appeared clear (no bacterial growth).
Determination of the KBM value of the extract is carried out by scraping the solution from the last few columns that appeared clear on 5 mL MHA in a petri dish. Petri dishes were incubated at 37 °C for 18-24 hours. The MBC value was determined from the extract concentration which did not show the growth of bacterial colonies.

Material Collection and Identification of Mold Isolates
Macroscopic observations of mold isolates and M. purpureus ITBCCL61 showed colonies with white hyphae, which a change in color to orange and then to red, with increasing incubation time (Figure 1). The same colony shape and color is also shown by M. purpureus isolated by Manan, et al. (2017). The growth medium also turned red, due to the presence of M. purpureus pigments which was diffused into the medium. 9 The microscopic observations of the two mold cultures show the same characteristics of hyphae, conidia, and ascospores ( Figure  2). Both have somatic hyphae, which were composed of branched mycelium, clear to  9 Based on these observations, it is found that the mold isolate was M. purpureus.

Red Yeast Rice Extraction
The results of organoleptic observations of red yeast rice extract showed that the three extracts were in the form of a thick liquid, distinctive smell, with n-hexane extract with a slightly orange color, ethyl acetate extract with a red color, and ethanol extract in red. Of the 2 kg of red yeast rice, 53.36 g of n-hexane extract (2.67% w/w), 84.22 g of ethyl acetate extract (4.21% w/w), and 192.09 g (9.60% w/w) of ethanol extract were obtained. The extract with the highest yield was ethanol extract, followed by ethyl acetate and n-hexane extracts. The same results were obtained by Ismail, et al. (2016) which produced a yield of 9.44% w/w of the ethanol extract of red yeast rice. 16

Phytochemical Screening of Red Yeast Rice Extract
The results of phytochemical screening of red yeast rice extract show that the n-hexane extract contained compounds from the flavonoid and quinone groups, while the ethyl acetate and ethanol extracts contained compounds from the polyphenol, flavonoid, quinone and saponin groups (Table 1). A research by Kongbuntad & Asenphet (2016) showed that the water extract of red yeast rice contained terpenoids, triterpenoids, flavonoids, phenols, plobatin, coumarin and saponin research, while the research of Ismail, et al (2016) showed that the ethanol extract of red yeast rice contained flavonoids, alkaloids and saponins. The difference in results is due to differences in the red yeast rice that are used as research objects. 16,17 Based on the results of phytochemical screening, it is suspected that the antibacterial activity of red yeast rice extract comes from polyphenol compounds, flavonoids, quinones and saponins. Polyphenol and flavonoid compounds are known to cause extracellular and intracellular proteins to form complexes with bacterial cell walls. The formation of this complex causes damage to the microsomes, lysosomes and bacterial cell walls. Flavonoids can also interfere with the intercalation Quinones can form irreversible complex compounds with nucleophilic amino acid residues in transmembrane proteins, also with polypeptides in the cell wall and with various enzymes on the surface of the cell membrane, thereby disrupting the stability of bacterial cells. 19 Saponins also reduce surface tension, thereby increasing cell wall permeability and resulting in the release of intracellular compounds. 18

Confirmation of Bacteria Test
Microscopic observations of the results of Gram stain show that P. acnes ATCC 11827 is a Gram-positive rod-shaped bacterium, while MRSA ATCC BAA-1683 is a round Gram-positive bacterium with a grape-like arrangement. The results of biochemical tests showed that P. acnes ATCC 11827 was a motile bacterium that ferments sucrose and lactose, with a positive VP result. MRSA ATCC BAA-1683 is a bacterium that ferments glucose, lactose, mannose and maltose, with a positive VP result (

Antibacterial Activity of Red Yeast Rice Extract
The extract concentrations used for the    (Table 6). From the results of MIC and MBC determination, it was found that ethyl acetate extract is more active against MRSA ATCC BAA-1683 than against P. acnes ATCC 1827.

Conclusion
Based on the results of this study, it can be concluded that the ethyl acetate extract and ethanol extract of red yeast rice have antibacterial activity against P. acnes ATCC 11827 and MRSA ATCC BAA-1683. Both extracts contain polyphenol compounds, flavonoids, quinones and saponins. Ethyl acetate extract is the most active extract with MIC values against P. acnes ATCC 1182 and MRSA ATCC BAA-1683 of 5 mg/mL and 12.5 mg/mL, respectively. The MBC values of ethyl acetate extract against P. acnes ATCC 11827 and MRSA ATCC BAA-1683 are 100 mg/mL and 25 mg/mL, respectively. The ethyl acetate extract of red yeast rice is more active against MRSA ATCC BAA-1683 than P. acnes ATCC 11827. Based on the results of this study, it is found that red yeast rice has the potential to be used as an alternative antibacterial for bacteria that causes skin infections, especially against MRSA.