Screening selected medicinal plants for antibacterial activity against Methicillin- Resistant Staphylococcus aureus (MRSA)

Transcription

1 Advances in Natural and Applied Sciences, 3(3): , 2009 ISSN , American Eurasian Netwrk fr Scientific Infrmatin This is a refereed jurnal and all articles are prfessinally screened and reviewed 330 ORIGINAL ARTICLE Screening selected medicinal plants fr antibacterial activity against Methicillin- Resistant Staphylcccus aureus (MRSA) 1,2 1,3 1,4 1 1,4 Sahgal, G., Sreeramanan, S., Sasidharan, S., Xavier, R., Ong, M.T. 1 Department f Bitechnlgy, Faculty f Applied Sciences, AIMST University, Kedah, Malaysia, 2 Centre fr Drug Research, University Sains Malaysia, Pulau Pinang, Malaysia, 3 Schl f Bilgical Sciences, University Sains Malaysia, Pulau Pinang, Malaysia, 4 INFORMM, University Sains Malaysia, Pulau Pinang, Malaysia. Sahgal, G., Sreeramanan, S., Sasidharan, S., Xavier, R., Ong, M.T.; Screening se lected medicinal plants fr antibacterial activity against Methicillin-Resistant Staphylcccus aureus (MRSA); Adv. in Nat. Appl. Sci., 3(3): , ABSTRACT Methanlic extracts f Vitex negund (Nchi), Cleus ambinicus (Karpuravalli), Hylcereus plyrhyzus (Dragn fruit) and Andrgraphis paniculata (Hempedu Bumi) were evaluated in an effrt t identify antibacterial cmpunds against clinical islates f Methicillin-Resistant Staphylcccus aureus (MRSA). Cleus ambinicus crude methanlic (CACM) leaves extract inhibited MRSA grwth cmpletely at lwer cncentratin cmpared t ther extracts. This is the first time that the CACM leaves extract was tested and reprted t be effective against MRSA. Hence, CACM leaves extract was chsen fr determinatin f minimum inhibitry cncentratin (MIC), grwth prfile f MRSA, biautgraphic and txicity test against brine shrimp. The MIC -1 value f CACM leaves extract against MRSA was 200µg.ml. CACM leaves extract resulted cmplete inhibitin grwth f the MRSA in grwth prfile and biautgraphy assay. The CACM leaves extract tested shwed cyttxicity activity against the Artemia salina. Therefre, CACM leaves extract culd be used as a ptential antibacterial agent frm the natural plant surces fr the treatment f MRSA infectin. Key wrds: Methicillin-Resistant Staphylcccus aureus; Clues ambinicus; antibacterial activity; Vitex negund; Hylcereus plyrhyzus; Andrgraphis paniculata Intrductin Nature has been a surce f medicinal agents fr thusands f years. Varius medicinal plants have been used fr years in daily life t that disease all ver the wrld. Many f mdern drugs have been islated frm natural surces are based n their use in traditinal medicine. Plants prduce a diverse range f biactive mlecules, making them a rich surce f different types f medicines. Over 50% f all mdern clinical drugs are f natural prduct rigin (Stuffiness and Durs, 1982) and natural prducts play an imprtant rle in drug develpment prgrams in pharmaceutical industry. Staphylcccus aureus is cmmnly assciated with hspital and cmmunity-acquired infectins. It causes superficial skin lesins such as bils, styes and furunculsis with mre serius infectins such as pneumnia, mastitis, phlebitis, meningitis, and urinary tract infectins; and deep-seated infectins, such as stemyelitis and endcarditis. In 1980s, methicillin resistant cases ascend and vancmycin has been used as a last resrt. The prevalence f MRSA islates in several hspitals in Malaysia in 1996 was even higher at 40% (Rhani et al., 2000). In general the frequency f S. aureus nasal carriage frm such settings in Malaysia varies frm 45% t 76%. Antibitics resistance has emerge as a serius prblem in Malaysia hspitals and Methicillinresistant Staphylcccus aureus (MRSA) are reprted t be the majr pathgen since 1995 (Malaysia Medical Crrespnding Authr: Dr. Xavier Rathinam, Department f Bitechnlgy, Faculty f Applied Sciences, AIMST University, Kedah, Malaysia. rxavier77@yah.cm. Tel:

2 331 Assciatin, 2002).Therefre there is an urgent need t develp alternative antimicrbial drugs fr the treatment f infectius diseases. In rder t search antibacterial activity against MRSA strains, fur different plants such as Andrgraphis paniculata (Hempedu bumi), Cleus ambinicus (Oregan), Vitex negund (Chasteberry) and Hylcereus plyrhizus (Dragn Fruit) were selected. Cleus ambinicus belngs t the family Lamiaceae. It is als knwn as regan, r as Karpuravalli intamil. Synnym fr this plant is Cleus armaticus. Cleus ambinicuss Benth, cmmnly knwn as an Indian Brage, is a medicinal plant and several medicinal prperties are attributed t this plant in the Indian system f medicine. It is cnsidered t be an antispasmdic, stimulant and stmachic and is used fr the treatment f headache, fever, epilepsy and dyspepsia (Mrtn, 1992). Andr graphis paniculata r knwn as Hempedu Bumi is frm the family Acanthaceae. Andrgraphis paniculata is als cmmnly knwn as Kalmegh is an imprtant medicinal plant, ccurring wild in India, and is used bth in Ayurevda and Unani system f medicine (Chadha, 1985). The dried herb is a remedy fr a number f ailments related t digestin, hepatprtectin, vermicidal, antiacne, analgesic, anti-inflammatry, antibacterial, antityphid, antibitic activities, hypglycemic, besides immune enhancement (Saxena et al., 2000). Vitex negund is frm family Verbanecea and knwn as Chastetree. The highly dissected leaves f Cut-leaf Chastetree are shaped like Cut-leaf Japanese Maple and were nce believed t have sedative effects. The preparatin f leaves used in catarrhal fever and applied t sinuses and scrfulus sres. Aqueus extract and il f seeds pssessed anti-xidant and anti-inflammatry prperty. Hylcereus plyrhizus a trpical fruit ppular in Sutheast Asia belngs t the climbing cacti (Cactaceae) family. Dragn fruits reputedly imprve eyesight and prevent hypertensin. The seeds f the fruit suppsed help in cntrlling bld glucse levels in peple with nn-insulin-dependent hyperglycaemic cnditins and it is als used t treat stmach and endcrine prblems. Materials and Methds Identificatin f bacterial strains: Twenty three clinical islates f Staphylcccus aureus were btained frm Hspital Besar, Alr Star, Kedah, Malaysia and psitive cntrl Methicillin Resistant Staphylcccus aureus (MRSA) strain was btained frm Universiti Kebangsaan Malaysia (UKM). Viability tests fr each islate were carried ut by resuscitating the rganism in nutrient agar. These clinical islates were recnfirmed by Gram staining plating n Mannitl Salt Agar which is the selective media fr Staphylcccus aureus. Then fllwed by the specific bichemical test fr Staphylcccus aureus such as catalase and cagulase tests were carried ut. Cllectin f plant materials: Cleus ambinicus (Karpuravalli), Andrgraphis paniculata (Hempedu bumi), Vitex nigund (Nchi), and Hylcereus plyrhizus (Dragn Fruit) were cllected frm Kedah. These plant materials were identified and classified by a btanist in the Bitechnlgy Department f AIMST University. Preparatin f plant extract: Cleus ambinicus, Andrgraphis paniculata, and Vitex nigund leaves were cllected. The leaves were washed with tap water and allwed t air dry. This was dne t reduce the micrbial lad f plant material due t handling and transprtatin. The plant materials were dried in shade fr three t fur weeks. Direct sun light shuld nt be given t the plant materials because the sun light radiatin can destry the active cmpunds f plant leaves. After drying, the plant leaves were grund finely. This is t increase the surface area f the plant materials when expsed t slvent. The grund plant materials were weighed and stred in separated cntainer. The Hylcereus plyrhizus was cut int fur pieces and the skin was remved. The fruits were grund by using blender. Methanl was used fr crude extractin purpse using maceratin methd. The mixture is kept fr ninety six hurs with peridic stirring. After fur days, the mixtures were filtered using Whatman N.1 filter papers. The precipitates were discarded and the supernatant was cllected. Each extract was cncentrated using rtary evapratr (EYELA, AS). These extractins are primarily used fr preliminary screening antibacterial activity. Antibitic Susceptibility Test: Antibitic susceptibility test was perfrmed t identify the Methicillin Resistant Staphylcccus aureus

3 332 (MRSA) frm these 23 clinical islates. Mueller Hintn Agar was used fr this test. Each islates was spread n the medium and Methicillin antibitic disc (5µg) was placed n the middle f the culture. It was then incubated at 37 C fr 24 hurs. Similar prcedures were fllwed fr xacillin and vancmycin antibitics. Disc Diffusin Assay: The disk diffusin (Kirby-Baurer) technique, which is f the recmmended standards f the Natinal Cmmittee fr Clinical Labratry Standards (NCCLS), was used fr antimicrbial test. An vernight 8-1 suspensin culture f MRSA was standardized accrding t n. 0.5 McFarland Standard (1 x 10 cell ml ) and spread n Mueller-Hintn agar (MHA) media. All the sterilized discs were placed n agar media. The disc was -1 impregnated with the CACM leaves extract at 100 mg. ml. Methanl was used as negative cntrl and standard antibitic vancmycin (30 µg/ml) as psitive reference t determine the sensitivity f the strain. The inculated plates were incubated at 37 C fr 24 h (Karaman et al. 2003). The antimicrbial activity was evaluated by measuring diameter f the inhibitin zne (mm) arund the disc. Determininatin Minimum Inhibitry Cncentratin (MIC): The inculum f MRSA was prepared frm 18 h Nutrient brth culture and adjusted t n. 0.5 McFarland standard turbidity. The CACM leaves extract was disslved in methanl. Then serial twfld dilutins f test samples were prepared in a cncentratin range frm 6.25 t 400 µg/ml in 10 ml sterile test tubes cntaining Mueller Hintn (MH) brth. The psitive cntrl was MH brth with standard reference antibitic and inculums and negative cntrl was the MH brth and inculums. The test tubes were vrtex gently t mix the cntent and incubated at 37 C fr 24 h (Rland et al. 2007). The MIC value was determined as the lwest cncentratin f CACM leaves extract in the brth medium that inhibit the grwth f MRSA. Determinatin Minimum Bactericidal Cncentratin (MBC): Fr the determinatin f minimum bactericidal cncentratin (MBC), ne hundred micrliter f the culture frm the diluted sample f MIC assay was transferred int nutrient agar. The liquid was spread plate and incubated at 37 C fr 24 h. The MBC was recrded as the lwest cncentratin f the extract that gave cmplete inhibitin f clny frmatin fr the test bacteria at latter cultivatin (Rnald et al., 2007). Time-Killing Prfile f MRSA: The time-killing f MRSA with half, ne and tw fld MIC cncentratin ver time was pltted t asses the bactericidal effect. The CACM leave extract was added t an aliqut f 25 ml nutrient brth in an amunt which wuld achieved the cncentratin f 0.5, 1 and 2 fld MIC. After 18 hurs, MRSA culture randmly chsen was harvested and adjusted by using n. 0.5 McFarland standard. One milliter f adjusted culture transferred int CACM leave extract and nutrient brth and nutrient brth alne (cntrl). The samples were incubated n the water bath at 37 C. After the additin f the adjusted culture, 1 ml f the test sample was transferred nt nutrient agar and spread plates. The plates were incubated at 37 C fr 24 h and determined -1 the CFU ml. The grwth f MRSA was measured every 4 h fr 48 h (Yga Latha et al., 2007). Brine Shrimp Txicity Assay: Brine shrimp eggs, Artemia salina, hatch in artificial seawater. Thus artificial sea water was prepared by disslving 38g f salt in 1 L f distilled water. A ttal f 0.5g Artemia salina were transferred int artificial sea water and incubated fr 24 hurs under rm temperature with xygen supplement and light intensity. MRSA extract was prepared in varius cncentratins fr txicity test. One milliliter frm MRSA wrking slutin was aliqut and disslved in 10 ml f 2% dimethylsulphxide (DMSO) t btained 10 mg/ml cncentratin f extract. Then 1 ml f diluted extract was aliqut and transferred int 10 ml f artificial sea water t btain 1 mg/ml. This methd was fllwed by tw fld serial dilutin until t mg/ml. 15 Artemia salina was transferred int each dilute extracts and incubated fr 12 hurs in light intensity cnditin. The mrtality rate f Artemia salina and lethal cncentratin at 50% (LC 50) were calculated (Carball et al., 2002). Biautgraphy assay:

4 333 Thin Layer Chrmatgraphy (TLC): TLC was perfrmed n a silica gel aluminum plate (10 x 20 cm, Silica gel 60 F 254, Merck) t fractinate active cmpund f 100 mg/ml CACM leave extract. The TLC plates were cut int 2 cm x 8 cm and dried int ven at 90 C fr 10 minutes. This is t activate the TLC plates by absrbing the misture cntent frm the plates. Then the extract was sptted n the bttm f the plates. Then the TLC plates were transferred int beaker which cntains separating slvent. The plates were placed in ascending directin in a beaker with chlrfrm: methanl: distilled water (25:3.5:0.5) slvent as mbile phase. The plates were bserved under UV light. The separated spts were marked and the R value was calculated. f Biautgraphy technique: Biautgraphic methd is anther technique that is used in screening antibacterial activity. Biautgraphic methd is basically t lcalize the antibacterial cmpund frm crude extract int chrmatgram. This is a supprtive and quick search methd fr antibacterial activity cmpund. The TLC plates that were used t separate active cmpunds in CACM leave extract were directly munted n t seed culture medium. The culture plate was kept fr five hur in rm temperature t make sure separated cmpund diffuse int the medium. Then the TLC plates were taken ut and incubated at 37 C fr 24 hurs (Paul Cs et al., 2006.) Statistical analysis: Each experiment was carried ut three times. The data in this study was cmputed and expressed as mean ± standard deviatin and als in SEM. Statistical analysis (ANOVA) was perfrmed using GraphPad Prism Sftware versin 4.0. P 0.05 was cnsidered statistically significant fr all cmparisns. Results and discussin The methanl yield extracts f the Andrgrphis paniculata, Cleus ambinicus, Vitex nigund and Hylcereus plyrhizus were presented in Table 1. The highest quantity f material was extracted frm Cleus ambinicus (5.28%), while the lwest quantity was btained frm Hylcereus plyrhizus (1.22 %). In Mask s antifungal analysis, it was revealed that rganic slvent methanl is the best extractant in quantitatively and qualitatively. The methanl extract is cmpetent t yield greater amunt f extract and als cntain higher plar cmpunds and tannins (Mask et al., 2007) which are generally act as antimicrbial agents. Table 1: The extractive values and % yield f the extracts f the selected medicinal plants Extracts Extractive values (g) % Yield (w/w) Cleus ambinicus (leave) Andrgraphis paniculata(leave) Vitex nigund (leave) Hylcereus plyrhizus (fruit) Identificatin f Bacterial Strains: Hspital islates bacterial strains were identified as MRSA using standardized micrbilgy technique. The clnial appearance f 23 nasal cavity clinical islates was identical and typical. All clnies were glden yellw, paque, circular, smth and creamy clur. Gram-staining revealed that Staphylcccus aureus islates are Gram psitive ccci in grape like clusters (Fig. 1). Qualitative bichemical cagulase test cnfirmed that all the islates are psitive fr S. aurues. Amng these 23 islates, nly 4 islates shwed a psitive catalase reactin and the remaining 19 were catalase negative (Table 2). Recently few catalase-negative Methicilin Resistant Staphylcccus aureus strain have been reprted t cause utbreaks in Brazilian hspitals (Del Alam et al., 2007). Therefre 19 clinical islates f catalase-negative Staphylcccus aureus were used fr antibitics susceptibility test t identify methicillin resistant strains. Antibitic Susceptibility Assay: Antibitic susceptibility test was perfrmed t identify the MRSA strains. In general nscmial MRSA is multi-drug resistant. During 1944, the first reprts f penicillin-resistant S. aureus had appeared and tday all strains f S. aureus are resistant t natural penicillins, aminpenicillins, and antipseudmnal penicillins (Chambers et al., 2001; Carball et al., 2002). Therefre the antibitic test was dne fr the ten catalase

5 334 negative S. aureus with methicillin (5 µg) and xacillin (1 µg). Only ten nasal cavity clinical islates shws psitive results t methicillin and xacillin antibitics. Vancmycin antibitic is used t treat MRSA infectin (Stevens et al., 2002). Therefre vancmycin 30 µg antibitic discs were used t test against all the ten islates which are shwing MRSA psitive. As a result, all the ten islates were susceptible t vancmycin antibitics (Table 3). Table 2: Qualitative bichemical and antibitic tests against Staphylcccus aureus fr the identificatin f Methicillin Resistant Staphylcccus aureus (MRSA). Islated strains Bichemical tests Antibitics Cagulase Catalase Methicillin Oxacillin Vancmycin *PC1 Psitive Negative R R S *PC2 Psitive Negative R R S *PC3 Psitive Negative R R S *PC4 Psitive Negative R R S 1 Psitive Negative S S S 2 Psitive Negative S S S 3 Psitive Psitive S S S 4 Psitive Negative S S S *5 Psitive Negative R R S 6 Psitive Psitive S S S 7 Psitive Negative S S S *8 Psitive Negative R R S 9 Psitive Psitive S S S *10 Psitive Negative R R S *11 Psitive Negative R R S 12 Psitive Negative S S S *13 Psitive Negative R R S *14 Psitive Negative R R S *15 Psitive Negative R R S 16 Psitive Negative S S S 17 Psitive Negative S S S 18 Psitive Negative S S S Nte: R: Resistant twards the antibitics; S: Susceptible twards the antibitics. PC1, 2, 3, and 4 strains resemble as the Psitive Cntrl f Staphylcccus aureus frm University Kebangsaan Malaysia (UKM). The numerical numbers emphasis the islates strains frm hspitalized respiratry infected patients. * Marked strains were used fr further antibacterial screenings. Table 3: Fur different plants including Cleus ambnicus (Karpuravalli), Vitex nigund (Nchi), Andrgraphis paniculata, Hylcereus plyrhizus (Dragn fruit) were used fr the antibacterial screening against MRSA clinical islates. Islates Remarks Inhibitin zne (diameter, mm) E 1 (mm) E 2 (mm) E 3 (mm) E 4 (mm) Cntrl 3 MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain Ntes: E 1: Andrgraphis paniculata (Hempedu bumi); E 2: Cleus ambnicus (Karpuravalli); E 3:Vitex nigund (Nchi); E 4: Hylcereus plyrhizus (Dragn fruit); Cntrl: Methanl. The inhibitin zne mre than 6 mm is sensitive twards the extract. Fig. 1: Staphylcccus aureus Gram psitive ccci in grape like clusters

6 335 Disc Diffusin Assay: The antibacterial activity f fur different plants including Andrgraphis paniculata, Cleus ambinicus, Vitex nigund and Hylcereus plyrhizus methanlic crude extracts were evaluated thrugh standard disc diffusin methd. In the disc diffusin technique, a reservir cntaining the test cmpund at a knwn cncentratin is brught int cntact with the inculated medium. The diameter f the clear zne arund the reservir (inhibitin diameter) is measured at the end f the incubatin perid. In rder t enhance the detectin limit, the inculated system is kept at lwer temperature fr several hurs befre incubatin t favur cmpund diffusin ver micrbial grwth. Thereby it increases the inhibitin diameter efficiently (Paul Cs et al., 2006). After 12 hurs incubatin perid, CACM leave extract cnferred prminent results cmpared t Andrgraphis paniculata Vitex nigund and Hylcereus plyrhizus crude extract. Clear and larger inhibitin znes were frmed surrunding the CACM leave extract (Table 4). This is the first time whereby CACM leave extract has been used fr the evaluatin f antibacterial activity against MRSA strains. Other tw plants methanlic extracts such as Vitex nigund and Hylcereus plyrhizus des nt shw any better inhibitin znes. As CACM leave extract gave prminent results cmpared t ther plants extracts, therefre the same plant extract was chsen fr further tests against MRSA strains. Table 4: Qualitative Minimum Inhibitin Cncentratin (MIC) and Minimum Bactericidal Cncentratin (MBC) f Cleus ambnicus crude methanlic (CACM) leave extract against MRSA clinical islates. Clinical Islates Remarks MIC (µg/ml) MBC (µg/ml) Extract Extract Vancmycin 3 MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain MRSA strain Brth Dilutin and effect f CACM leave extract against MRSA strains: Table 4 shws MIC f CACM leave extract against 11 MRSA clinical islates. MIC is defined as the lwest cncentratin that is able t inhibit any visible micrbial grwth and belw that cncentratin which, there is n further inhibitin. The minimal bactericidal cncentratin (MBC) is determined by plating-ut samples f cmpletely inhibited dilutin cultures and assessing grwth (static) r n-grwth (cidal) after incubatin (Paul Cs et al., 2006). MIC f this extract is 200µg/ml. Hwever, when this cncentratin was lwer, the brth cntaining the extracts was turbid. Grwth f a MRSA strain in brth culture with CACM leave extract was assessed by determining the ptical density at 540 nm in each fur hurs interval fr 48 hurs. The time-killing prfile f MRSA in the presence f extract was shwn in Figure 2. The grwth prfile f MRSA was inhibited at 4 h fr MIC and tw fld MIC (400 µg/ml). The lg phase f MRSA was reduced frm 28h t 20 h in half fld MIC value (100 µg/ml). This exhibits that the extract decelerated the reprductin f MRSA. The time killing prfile f MRSA with CACM leave extract prve that the effectiveness f the extract within 48 h. Brine Shrimp Txicity Assay: The brine shrimp lethality assay was prpsed by Michael et al. 1956; Carball et al., The results revealed that LC 50 f CACM leave extract was 182 µg/ml. In primary txicity evaluatin f plant extracts by brine shrimp lethality biassay, LC 50 values lwer than 1000 ìg/ml are cnsidered biactive (Meyer et al., 1982). Therefre the extract cnsists relatively high amunt f biactive cnstituents. Besides this, the extract als might cnsist f biactive cmpund which is cyttxic t Artemia salina. Therefre fr mre accurate result fr txicity assay, animal mdels and islate pure cmpund shuld be further evaluated. Biutgraphy Assay: Biautgraphy lcalizes antimicrbial activity n a chrmatgram using three appraches such as direct biautgraphy, cntact biautgraphy and agar ver lay biautgraphy (Hamburger and Crdell, 1987,

7 336 Rahalisn et al., 1991, Paul Cs et al., 2006). Separated fragments were visualized under Ultravilet (UV) light and the R f values were calculated. The TLC plates had eleven separated spts. Cntact biautgraphy methd was used t lcalize antibacterial activity cmpunds f CACM leave extract frm TLC plates t an inculated agar plate thrugh direct cntact. Tw separated spts which crrespnds t R f values and revealed inhibitin zne n the culture medium (Fig. 3). Therefre it can be presumed that these tw spts may have antibacterial activity cmpunds in CACM leave extract. Fig. 2: Grwth prfile f MRSA in the presence f Cleus ambinicus methanlic crude extract fr 48 hurs. Fig. 3: Biautgraphy result f MRSA strain in CACM leave extract. There is clear inhibitin zne n the MRSA at and R values. f Discussin MRSA is imprtant nscmial pathgens in Malaysia and several recent reprts have shwn that its prevalence as an endemic nscmial pathgen is increasing. Methicillin-resistant Staphylcccus aureus (MRSA) as a hspital pathgen has presented many clinical prblems in the University Hspital, Kuala Lumpur, Malaysia since It was nted that the incidence f MRSA amng S. aureus islated frm hspital inpatients had increased frm 11.5% in 1979 t 18.8% in 1985 (Rhani et al., 1999). The identificatin f hspital islates bacteria strains is very imprtant t cnfirm the presence f MRSA infectin in patients. During the identificatin cagulase test was carried ut. This is an imprtant test t differentiate S. aureus frm ther species especially frm Staphylcccus epidermidis. S. aureus prduces cagulase, an enzyme like prtein that clts xalated r citrated plasma which is generated in bth esterase and cltting activities. Generally catalase test is used t differentiate Streptcccus frm Staphylcccus.

8 337 Hwever nw it is difficult t verify this hypthesis because there are few cases n catalase-negative MRSA infectins. First case f catalase-negative MRSA was reprted by Xavier and his clleagues in This catalase-negative MRSA specimen was islated frm a 70-year-ld wman, suffering frm an Alzheimer s disease (Xavier et al., 2001). Therefre catalase-negative S. aureus strains were chsen fr identificatin f MRSA strains and warrant fr the further antibacterial screening. The hspitals islates catalase negatives S. aurues exhibit methicillin and xacillin resistant. Resistance t these drugs ccurs because f the acquisitin f genes that encde drug-inactivating enzymes knwn as â-lactamases. The expressin f the meca gene encding lw-affinity penicillin-binding prtein PBP2a cnfers resistance t ther â-lactams in additin t methicillin. Besides that, transfrmatin f a SCCmec type I element int S. aureus strains yielded highly xacillin-resistant (Utsui et al., 1985; Luis, 2006). The MIC and time killing prfile reveal the effectiveness f the CACM leave extract MRSA clinical strains. The MRSA grwth inhibitin is may due t the active cmpund frm the extract. This statement is pretty true due t the cmparisn f the cntrl MRSA grwth fr 48 h with the extract. The reductin in the grwth rate may due t the restrictin f cell divisin in the culture. Therefre again it is prven the effectiveness f the extract against MRSA strain. The brine shrimp assay is based n the ability t kill labratry cultured Artemia salina naupii. The Artemia salina lethality assay is cnsidered a useful tl fr preliminary assessment f txicity. The brine shrimp assay is very useful tl fr the islatin f biactive cmpunds frm plant extracts. The methd is attractive because it is very simple, inexpensive and lw txin amunts are sufficient t perfrm the test in the micrwell scale. In additin, this methd is rapid, simple, reprducible and ecnmical. A wide variety f bilgically active chemical cmpunds, in particular cyttxic agents are txic t brine shrimps. Biactive cmpunds are nearly always txic in high cncentratins and as txiclgy can be described as pharmaclgy at higher dses. This premise has been applied t the screening f medicinal plant extracts in the brine shrimp txicity test. The TLC tw blue clur spts exhibits inhibitin area in the biuautgraphy assay. Under UV light these tw relative spts expressed blue clur. Generally flavnid derivatives express blue clur under UV light. Besides that, Alcaraz and clleagues (2000) reprted that specific grwth rate f MRSA strains linearly decrease with the increase in the cncentratin f flavnids in the culture medium. Therefre, it can be assumed that the antibacterial activity spts in TLC plates may be flavnids r its derivatives. Further evaluatin fr the islatin and identificatin f biactive cmpunds frm CACM leave extract shuld be undertaken. Cnclusin Cleus ambinicus methanlic crude leave extract prnunce prmising results against nscmial MRSA pathgens. In further studies islatin f these tw pure cmpunds which cmpletely inhibit this pathgen shuld be the next line f research. This can give mre prmising results n antibacterial activity f Cleus ambinicus methanlic extract against nasal carriage MRSA pathgens. References Alcarad, Z.L.E., S.E. Blanc, O.N. Puig, F.S. Tmad and F.H. Ferretti, Antibacterial Activity f Flavnids Against Methicillin-resistant Staphylcccus aureus strains. Jurnal f Theretical Bilgy, 205: Carball, J.L., Z.L. Hernández-Inda, Pilar Pérez and M.D. García-Grávals, A cmparisn between tw brine shrimp assays t detect in vitr cyttxicity in marine natural prducts. BMC Bitechnlgy, 2:17. Chadha, Y.R., The Wealth f India: Raw Materials, Vl. 1A. Cuncil f Scientific and Industrial Research, New Delhi, India, pp: 264. Chambers, H.F., The changing epidemilgy f Staphylcccus aureus Emerging Infectius Diseases, 7: Del Alam, L., P.A. d Azeved, A.J. Strb, D.V. Rdriguez-Lpez, J. Mnteir, S.S. Andrade, A.C.C. Pignatari and A.C., Gales, An utbreak f catalase-negative methicillin-resistant Staphylcccus aureus. Jurnal f Hspital Infectin, 65: Hamburger, M.O., G.A. Crdell, A direct biautgraphic TLC assay fr cmpunds pssessing antibacterial activity. Jurnal f Natural Prducts, 50: Karaman, I., F. Sahin, M. Gulluce, H. Ogutcu, M. Sengul and Adiguzel, Antimicrbial activity f aqueus and methanl extract f Juniperus xycedrus L. Jurnal f Ethnpharmaclgy, 85: Luis, B.R, Antimicrbial Resistance in Gram-Psitive Bacteria. The American Jurnal f Medicine, 119(6A): S11 S19.

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