The Antibacterial Potential of Pineapple Core Extract (Ananas comosus (L.) Merr) Against Methicillin-resistant Staphylococcus aureus (MRSA)

Size: px

Start display at page:

Download "The Antibacterial Potential of Pineapple Core Extract (Ananas comosus (L.) Merr) Against Methicillin-resistant Staphylococcus aureus (MRSA)"

Job Leonard
6 years ago
Views:

1 MICROBIOLOGY INDONESIA ISSN , eissn Vl.11, N.3, September 2017, p The Antibacterial Ptential f Pineapple Cre Extract (Ananas cmsus (L.) Merr) Against Methicillin-resistant Staphylcccus aureus (MRSA) 1* 2 3 BOBY PRATAMA PUTRA, DANTI NUR INDIASTUTI, AND DEBY KUSUMANINGRUM 1 Schl f Medicine, Faculty f Medicine, Universitas Airlangga Jalan Prf. Dr. Mestp N. 47, Surabaya 60132, East Java, Indnesia; 2 Department f Pharmaclgy and Therapeutics, Faculty f Medicine, Universitas Airlangga Jalan Prf. Dr. Mestp N. 47, Surabaya 60132, East Java, Indnesia; 3 Department f Medical Micrbilgy, Faculty f Medicine, Universitas Airlangga Jalan Prf. Dr. Mestp N. 47, Surabaya 60132, East Java, Indnesia. Staphylcccus aureus is ne f majr pathgens causes serius infectin. Penicillin antibitic is ne f therapies against Staphylcccus infectin. Hwever, inadequate and irratinal use f antibitic causes resistance and emerges incidence f methicillin-resistant Staphylcccus aureus (MRSA). Herbal medicine frm pineapple, especially frm its cre extract, is hpefully can reduce the incidence f antibitics resistance because it cntains brmelain, flavnid, sapnin, and tanin, which have antibacterial effect. This research was cnducted t investigate the antibacterial ptentiality f pineapple cre extract against MRSA. This research is true experimental with pst-test cntrlled grup design. Pineapple cre was extracted by maceratin methd. Pineapple cre extract's cncentratins used were 750, 500, 250, 187.5, 125, and 62.5 mg ml. The minimum inhibitry cncentratin (MIC) and minimum bactericidal cncentratin (MBC) were determined by brth dilutin test with five replicatins. Vancmycin was used as cntrl grup. MIC was determined visually by cmparing turbidity f slutins after incubatin at 37 C fr 24 h. Then these slutins were cultured n nutrient agar plates at 37 C fr 24 h. MBC was determined visually by inspecting the presence f bacterial clnies grwth. The Minimum Inhibitry Cncentratin (MIC) culd nt be determined due t n turbidity changes. Vancmycin cntrl cannt be used fr determining MIC. Cultures n nutrient agar plates had n clnies grwth in cncentratins f 750 and 500 mg ml. In summary, pineapple cre extract has antibacterial ptentiality against methicillin-resistant Staphylcccus aureus (MRSA) with MBC f 500 mg ml. Key wrds: antibacterial, dilutin susceptibility test, methicillin-resistant Staphylcccus aureus (MRSA), pineapple cre, vancmycin Staphylcccus aureus merupakan salah satu patgen yang menyebabkan infeksi serius. Penisilin merupakan salah satu terapi infeksi Staphylcccus. Penggunaan antibitika yang tidak tepat dan irrasinal mengakibatkan resistensi dan munculnya methicillin-resistant Staphylcccus aureus (MRSA). Obat herbal dari nanas, terutama ekstrak dari bngglnya, diharapkan dapat menurunkan insidensi resistensi antibitika karena mengandung brmelain, flavnid, sapnin dan tanin, yang memiliki efek antibakteri. Penelitian ini dilakukan untuk menguji ptensi antibakteri ekstrak bnggl nanas terhadap MRSA. Penelitian ini merupakan eksperimental murni dengan pst-test cntrlled grup design. Bnggl nanas diesktraksi dengan metde maserasi. Knsentrasi ekstrak bnggl nanas yang digunakan antara lain 750, 500, 250, 187,5, 125, dan 62,5 mg ml. Knsentrasi Hambat Minimum (KHM) dan Knsentrasi Bunuh Minimum (KBM) ditentukan dengan uji dilusi sebanyak lima replikasi. Vancmycin digunakan sebagai kelmpk kntrl. KHM diamati secara visual dengan membandingkan kekeruhan suspensi setelah inkubasi pada suhu 37 C selama 24 jam. Tiap suspensi dikultur pada media agar nutrien pada suhu 37 C selama 24 jam untuk melihat media yang tidak ada pertumbuhan klni bakteri agar dapat menentukan KBM. Knsentrasi Hambat Minimum (KHM) tidak dapat diamati karena tidak ada perbedaan kekeruhan. Kntrl vancmycin tidak dapat digunakan untuk menentukan KHM. Kultur pada media agar nutrien menunjukkan tidak ada pertumbuhan klni pada knsentrasi 750 dan 500 mg ml. Ekstrak bnggl nanas memiliki ptensi antibakteri terhadap methicillin-resistant Staphylcccus aureus (MRSA) dengan KBM 500 mg ml. Available nline at DOI: /mi Kata kunci: antibakteri, bnggl nanas, methicillin-resistant Staphylcccus aureus (MRSA), uji dilusi, vancmycin Infectius disease is ne f glbal health prblems especially in trpical areas. Staphylcccus aureus is *Crrespnding authr: Phne: , bby_pratama_putra@yah.c.id ne f the cmmnest and ptentially dangerus human pathgens (Miller and Diep 2008). One f therapies against Staphylcccus aureus infectin is using beta-lactam antibitics. Hwever, inadequate and irratinal use f the antibitics causes

2 90 PUTRA ET AL. Micrbil Indnes Staphylcccus aureus resistances against beta-lactam antibitics and emerges incidence f methicillinresistant Staphylcccus aureus (MRSA). Metaanalytical study by Taccnelli et al. (2008) shws crrelatin between frequency f antibitic use and incidence f antibitic resistance. Based n the research cnducted by Jarvis et al. (2012) invlving 67,412 hspitalized patients in the United States, the prevalence f MRSA clnizatin r infectin is 66.4 per 1000 patients, as many as 61.8% has been clnized by MRSA and 38.2% were infected by MRSA. MRSA infects mstly the integument system and sft tissue, and is largely nscmial infectin. MRSA infectin epidemilgically has higher mrbidity and mrtality that infectin caused by methicillin-sensitive Staphylcccus aureus (MSSA) (Grdn and Lwy, 2008). In additin, decrease in incidence f MRSA infectin wuld reduce the treatment cst (Rubin et al. 1999). Therefre, it is necessary t search fr alternative medicines frm natural materials t reduce the incidence f MRSA, ne f them is pineapple cre extract. Pineapple is ne f the cmmdities in Indnesia that has been cnsumed by many peple. Hwever, pineapple cre is ften discarded as waste, while Gautam et al. (2010) thrugh experiments came t the cnclusin that brmelain activity in pineapple stem tissue is higher than the activity f the enzyme in the pineapple fruit. Pineapple cre cntains prtelysis enzyme called brmelain that lyse bacterial cell wall (Ali et al. 2015). Besides, pineapple cre als cntains flavnid, sapnin, and tannin that have antibacterial effect. MATERIALS AND METHODS Extractin. Pineapple in the study was btained frm pineapple plantatin in Pnggk District, Blitar Regency, East Java, Indnesia. The extractin methd used was maceratin methd. A ttal f 400 grams f pineapple cre pwder was added 500 ml f ethanl 96% then hmgenized at 120 rpm fr ne hur, then left fr 24 h. After 24 h, the slutin was filtered using a Buchner filter and repeated three times. The filtrate was evaprated by using a rtary evapratr at 70 C t btain crude extracts. Vancmycin Stck Preparatin. One vial f the vancmycin 500 milligrams was disslved with aqua pr injectin slutin f 10 ml btained a cncentratin f 50 mg ml. The slutin is disslved with serial dilutin t btain a cncentratin f mg ml r 5 μg ml. The final step was making vancmycin stck with cncentratin f 2 μg ml by disslving 4 ml vancmycin 5 μg ml in 6 ml aqua pr injectin. Methicillin-Resistant Staphylcccus aureus (MRSA) Preparatin. Methicillin-resistant Staphylcccus aureus (MRSA) clny was btained frm Labratry f Micrbilgy, Faculty f Medicine, Universitas Airlangga. MRSA culture with age f 24 h was taken amunt f 0.1 ml 0.5 McFarland 8 (1.5 x 10 CFU ml ) then placed in tube cntaining sterilized brth dilutin. Anti-bacterial Dilutin Susceptibility Test. The methd used in the research was brth dilutin test with five replicatins. The first serial is pineapple cre extract with cncentratin f 1500 mg ml then diluted until btained cncentratin f 375 mg ml, but the tube with cncentratin f 750 mg ml was discarded. The secnd serial is pineapple cre with cncentratin f 1000 mg ml then diluted until achieved cncentratin f 125 mg ml. All the test tube was added 1 ml f inculum thus btained final cncentratin f 750, 500, 250, 187.5, 125, 62.5 mg ml. The sterility cntrl cnsists f 1 ml extract and 1 ml sterile medium, the grwth cntrl cnsists f 1 ml inculum and 1 ml sterile medium, and antibitic cntrl cnsists f 1 ml vancmycin stck and 1 ml inculum. Then, bth test and cntrl tubes were incubated at 37 C fr 24 h fr determining the MIC. After bservatin, each suspensin was streaked n nutrient agar medium then incubated at 37 C fr 24 h fr determining the MBC. RESULTS Extractin. 400 grams f pineapple cre pwder was extracted with maceratin methd and btained 150 grams f pineapple cre crude extract that has characteristics f viscus and dark brwn-clred. The cntaminatin test was negative. Anti-bacterial Dilutin Susceptibility Test. Dilutin susceptibility test was used fr determining MIC was summarized in the Table 1. Frm the result, the MIC culd nt be determined because the extract's dark brwn clr and high turbidity interfere the interpretatin. After each suspensin was cultured n nutrient agar plate, the result was summarized in the Table 2. The result shwed n clnies grwth in cncentratins f 750 and 500 mg ml. Thus, the MBC f pineapple cre extract against the MRSA is 500 mg

3 Vlume 11, 2017 Micrbil Indnes 91 Table 1 Dilutin Susceptibility Test fr Determining Minimum Inhibitry Cncentratin (MIC) Extract Cncentratin (mg ml ) Observatin Bacterial Grwth in sterilized brth dilutin Replicatin 1 Replicatin 2 Replicatin 3 Replicatin 4 Replicatin X X X X X 500 X X X X X 250 X X X X X X X X X X 125 X X X X X 62.5 X X X X X S- X X X X X G+ X X X X X A X X X X X (Nte: S- : sterility cntrl, G+ : grwth cntrl, A : Vancmycin 2 μg ml, X : cannt be assessed). Table 2 Dilutin Susceptibility Test Culture fr Determining Minimum Bactericidal Cncentratin (MBC) Extract Cncentratin (mg ml ) Bacterial Grwth n Nutrient Agar Plates Replicatin 1 Replicatin 2 Replicatin 3 Replicatin 4 Replicatin , , S G A (Nte: S- : sterility cntrl, G+ : grwth cntrl, A : Vancmycin 2 μg ml, + : viable grwth, - : n viable grwth). ml. Test tubes with cncentratin f 250 mg ml culd nt be referred as the MBC because nly killed the bacterial clnies nly n the first replicatin, while the ther fur replicatins did nt. DISCUSSION In this research, the MIC culd nt be determined because there was n significant difference f turbidity between befre and after treatment. The use f vancmycin cntrl was hpefully culd be used t determine the MIC by cmparing the bacterial clnies grwth n nutrient agar plate between the cntrl and test tubes. The dse f vancmycin used was based n literature study that vancmycin 2 μg ml is the MIC f vancmycin against MRSA (Sakulas et al. 2004). Unfrtunately, there was n significant similarity f culture results between antibitic cntrl and test tubes thus the MIC still culd nt be determined. While the MBC in this research was 500 mg ml. Fr further investigatin especially fr determining MIC, we can cnsider using agar dilutin methd r micrdilutin methd. The pineapple cre extract used in this research is crude extract. Based n literature studies, pineapple cre cntains sme antibacterial substances; they are brmelain, tannin, sapnin, and flavnid. Brmelain is prtelysis enzyme that disrupt the peptide bnd n bacterial cell wall thus lyse the bacterial wall (Ali et al. 2015). Pineapple brmelain extract can inhibit Streptcccus mutans with cncentratin f 2 mg ml, Prphyrmnas gingivalis with cncentratin f 4.15 mg ml, and Aggregatibacter actinmycetemcmitans with cncentratin f 16.6 mg ml (Praveen et al. 2014). Besides, pineapple cntains flavnid that inhibits peptidglycan synthesis n bacterial cell wall and induces prtein denaturatin n bacterial membrane cell (Eumkeb et al. 2012). Flavnid als disrupts bacterial cell wall induces bacterial + metablites leakage and H ins frm flavnid will react with plar grups s alters the phsphlipid n membrane cell (Retnwati, 2011). Audies (2015) states that pineapple cntains tannin, which is a phenl cmpund and wrks at the plypeptide wall f

4 92 PUTRA ET AL. Micrbil Indnes bacteria causes shrinkage walls f bacteria. Based n ther literature, pineapple cre als cntains sapnins which can increase the permeability f the bacterial cell membrane s that it can alter the structure and functin f membrane, disrupt the surface tensin f the cell wall which allws the antibacterial substances enter easily int cells then interfere bacterial metablism, and cause denaturatin f prteins n bacterial membrane (Karlina et al. 2013). Retnwati (2011) als stated that the damage t the bacterial membrane cause the release f enzymes and metablites resulting in decreased metablism that decreases ATP prductin resulting in bacterial cell death. Purificatin f each substance frm the crude extract may be cnsidered fr further investigatin thus the mechanism and cncentratin f each substance fr antibacterial against MRSA can be achieved. In summary, pineapple cre extract has antibacterial ptentiality against methicillin-resistant Staphylcccus aureus (MRSA) with MBC f 500 mg ml, while MIC can be determined in further investigatin using agar dilutin methd r micrdilutin methd. ACKNOWLEDGMENT The authrs are s thankful t the Labratry f Phytchemistry and Pharmacgnsy, Faculty f Pharmacy, Universitas Airlangga, wh helped in extractin prcess. Besides, the authrs wuld like t thank the staffs f the Labratry f Micrbilgy, Faculty f Medicine, Universitas Airlangga, wh helped in preparing fr the dilutin susceptibility test. REFERENCES Ali AA, Milala MA, Gulani IA Antimicrbial effects f crude brmelain extracted frm pineapple fruit (Ananas cmsus (Linn.) Merr.). Adv Bichem. 3(1):1 4. di: /j.ab Audies A Uji efektivitas antibakteri ekstrak kulit nanas (Ananas cmsus. L) terhadap pertumbuhan Streptcccus mutans penyebab karies gigi [Antibacterial effectivity testing f pineapple peel extract (Ananas cmsus. L) against Streptcccus mutans causing dental caries] [undergraduate thesis]. Padang (ID): Universitas Andalas. Caesarita DP. (2011). Pengaruh ekstrak buah nanas (Ananas cmsus) 100% terhadap bakteri Staphylcccus aureus dari piderma [The effect f pineapple extract (Ananas cmsus) 100% n Staphylcccus aureus bacteria frm piderma] [undergraduate thesis]. Semarang (ID): Universitas Dipnegr. Eshamah H Antibacterial effects f prteases n different strains f Escherichia cli and Listeria mncytgenes [Dissertatins]. Clemsn (US): Clemsn University. Eumkeb G, Siriwng S, Phitaktim S, Rjtinnakrn N, Sakdarat S Synergistic activity and mde f actin f flavnid islated frm smaller Galangal and Amxicillin cmbinatins against Amxicillinresistant Escherichia cli. J App Micrbil. 112(1): di: /j x. Gautam SS, Mishra SK, Dash V, Gyal AK, Rath G Cmparative study f extractin, purificatin and sstimatin f brmelain frm stem and fruit f pineapple plant. Thai J Pharm Sci. 34(2): Grdn RJ, Lwy FD Pathgenesis f methicillinresistant Staphylcccus aureus infectin. Cli Infect Dis. 460(5): di: / Jarvis WR, Jarvis AA, Chinn RY Natinal prevalence f methicillin-resistant Staphylcccus aureus in inpatients at United States health care Am J Infect Cntrl. 40(3): di: /j.ajic Karlina CYY, Ibrahim M, Guntur T Aktivitas antibakteri ekstrak herbal krkt (Prtulaca leraceae L.) terhadap Staphylcccus aureus dan Escherichia cli [Antibacterial activity f herbal krkt extract (Prtulaca leraceae L.) against Staphylcccus aureus dan Escherichia cli]. J Lentera Bi. 2(1): Miller LG, Diep BA Clnizatin, fmites, and virulence : rethinking the pathgenesis f cmmunityassciated Methicillin-resistant Staphylcccus aureus infectin. Cli Infect Dis. 46(1): di: / Praveen NJ, Rajesh A, Madan M, Chaurasia VR, Hiremath NV In-vitr evaluatin f antibacterial efficacy f pineapple extract (brmelain) n peridntal pathgens. J Int Oral Health. 6(5): Retnwati Y, Balangi N, Psangi NW Pertumbuhan bakteri Staphylcccus aureus pada media yang dieksps dengan infus daun sambilt (Andrgraphis paniculata) [Staphylcccus aureus bacterial grwth n media expsed with infused bitter leaves (Andrgraphis paniculata)]. Saintek. 6(2):1-9. Rubin RJ, Harringtn CA, Pn A, Dietrich K, Greene JA, Miduddin A The ecnmic impact f Staphylcccus aureus infectin in New Yrk City hspitals. Emerg Infect Dis. 5(1):9 17. Sakulas G, Mise-Brder PA, Schentag J, Frrest A, Mellering RC, Elipuls GM Relatinship f MIC and bactericidal activity t efficacy f Vancmycin fr treatment f methicillin-resistant Staphylcccus aureus bacteremia. J Clin Micrbil. 42(6): di: /JCM

5 Vlume 11, 2017 Micrbil Indnes 93 Taccnelli E, Angelis GD, Catald MA, Pzzi E, Cauda R Des antibitic expsure increase the risk f methicillin-resistant Staphylcccus aureus (MRSA) islatin?: A systematic review and meta-analysis. J Antimicrb Chemther. 61(1): di: /jac/dkm416.

5.1. What do we need to know before we start planning a canine rabies control programme?

5.1. What do we need to know before we start planning a canine rabies control programme? 5.1. What d we need t knw befre we start planning a canine rabies cntrl prgramme? Yu need t knw abut: The epidemilgy f rabies in yur area The reservir species in yur area Hw rabies is transmitted. This