erythromycin, clindamycin, gentamicin,

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1 JMBR: A Peer-review Jurnal f Bimedical Sciences December 2015, Vl. 14 N. 2 pp COMBINATION OF AGERATUM CONYZOIDES LEAF EXTRACTS WITH ANTIBIOTICS AGAINST STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA ISOLATED FROM WOUND INFECTION Etinsa O. Igbinsa* and Osagie A. Erib Abstract There has been increasing prevalence f bacterial resistance t cmmnly used antibitics. The present study investigates the synergistic actin f Ageratum cnyzides leaf extracts and antibitics against Pseudmnas aeruginsa and Staphylcccus aureus islated frm wunds. The disc diffusin methd was used. Extracts were diluted t cncentratins ranging frm t 5.0 mg/ml. The results indicated that ethanlic extract f Ageratum cnyzides shwed antibacterial activity with zne f inhibitin f mm and 11 mm fr Staphylcccus aureus and Pseudmnas aeruginsa respectively. Antibitics were applied in cmbinatin with ethanlic extract f Ageratum cnuyzides and prduced znes f inhibitin higher than that f the individual antibitics and plant extract. Ethanlic extract + ciprflaxicin and ethanlic extract + erythrmycin prduced inhibitin f 29 mm and 30 mm fr Staphylcccus aureus and Pseudmnas aeruginsa respectively. The result revealed that cmbinatin f Ageratum cnyzides extract and antibitics tested culd be useful in treating wund infectins. Intrductin The management f infected wunds is a challenge in terms f ratinal antimicrbial use especially with the presence f a wide variety f antimicrbial drugs. Similarly, a lt f cncern has been generated wrldwide ver bacterial drug resistance, leading t cheap drugs being replaced with 1 mre effective and expensive nes. Sule 2 and Olusanya have reprted the increasing prevalence f bacterial resistance t mst f the cmmnly used antibitics. At present, clinical islates f Staphylcccus aureus are multiple drugs resistant t three r mre agents such as ciprflxacin, KEYWORDS: Ageratum cnyzides, Viability, Staphylcccus aureus, Pseudmnas aeruginsa Etinsa O. Igbinsa* and Osagie A. Erib Applied Micrbial Prcesses & Envirnmental Health Research Grup, Department f Micrbilgy, Faculty f Life Sciences, University f Benin, P.M.B 1154 Benin City, Nigeria * Crrespndence etinsa.igbinsa@uniben.edu +234 (0) erythrmycin, clindamycin, gentamicin, 3 linezlide and vancmycin. Glbal resistance rate f Streptcccus pygenes islates are as high as 80% fr erythrmycin 4 and 50% fr penicillin. Infectins with Pseudmnas aeruginsa are particularly challenging since the bacterium exhibits inherent tlerance t several antimicrbial agents and can acquire additinal resistant mechanism turning ineffective all current antimicrbial drugs. In many pr cuntries, the high cst f such replacement drugs is prhibitive, with the result that sme disease can n lnger be treated in areas where resistance t first line 5 drugs is widespread. Faced with such a challenge, there is need t develp alternative appraches in additin t the search f new antimicrbial cmpunds. Plants are valuable surce f medicinal cmpunds that cntain brad spectrum 6 f bilgical activity. Apprximately 25-

2 Cmbinatin f Ageratum Cnyzides Leaf Extracts With Antibitics Against Staphylcccus Aureus And Pseudmnas Aeruginsa Islated Frm Wund Infectin % f current pharmaceuticals is derived frm plants and shw less side effects than the systemic drugs. Nrmally, during their life cycle, plants encunter varius infectius agents such as viruses, bacteria, fungi and ther parasites and synthesize a variety f secndary metablites capable f destrying the infectius agents. Fr example, single and cmbine plant extract f Rhuscnaria and Thymus vulgaris were bserved t be highly effective against multiple drug resistant Pseudmnas 7 aeruginsa. Ageratum cnyzides has been knwn since ancient times fr its curative prperties. It has been utilized fr treatment f varius ailments and largely, fr its antibacterial prperties against bacterial infectins. In Africa, Ageratum cnyzides is used t treat fever, clic, rheumatism, headache, pneumnia, 8 wunds and burns. Pharmaclgical 9 investigatin by Durdla verified that ether and chlrfrm extracts f Ageratum cnyzides has inhibitry activities against in vitr develpment f Staphylcccus aureus. An in-vitr study f methanlic extracts f whle plants has shwn antibacterial activity against Bacillus subtilis, Escherichia cli, P s e u d m n a s a e r u g i n s a a n d 10 Staphylcccus aureus. The use f single antibitics des nt ften prduce the desired effective inhibitry effects and t vercme this, a cmbinatin f drugs, ften exercise their synergistic effect which surpasses their individual perfrmance. The synergistic effect frm the assciatin f antibitics with plant extracts against resistant bacteria ffers a new chice fr the treatment f infectius diseases. Therefre, this study was undertaken t investigate the synergistic activity f ethanl and aqueus extracts f Ageratum cnyzides with sme antibitics against Staphylcccus aureus and Pseudmnas aeruginsa islated frm wunds infectin. Materials and Methds Plant Cllectin and Identificatin Fresh leaves f Ageratum cnyzides (cmmn name: Gat weed) were cllected arund the University f Benin, Benin City. Cntaminant were handpicked and then washed with clean water t remve sand. The plant materials were identified by the Department f Plant Bilgy and Bitechnlgy, University f Benin, Benin City. Specimen Cllectin Clinical islates f Staphylcccus aureus and Pseudmnas aeruginsa islated frm wund infectin were btained frm the stck culture f the Micrbilgy Labratry f University f Benin Teaching Hspital Benin, Nigeria. Viability test f the islates were carried ut by resuscitating the rganism int a nutrient brth and thereafter sub-cultured int nutrient agar plates and incubated at 37 C fr 24 h. Standard culture based and bichemical reactin methds were used fr the cnfirmatin f the test bacteria. Preparatin f Plant Extract The leaves f Ageratum cnyzides were separated, the plant were cleaned with sterile distilled water; ven dried and finely grund using a grinder mill. Twenty gram (20 g) f the fine pwder frm Ageratum cnyzides was saked in 200 ml f ethanl and distilled water placed in different cnical flasks and allwed t stand fr 24 h. The cntent f each cnical flask cntaining plant materials and the slvent were filtered after 24 h using

3 90...Jurnal f Medicine and Bimedical Research Whatman filter paper, supernatant f each plant extract was transferred int cnical flask. The cnical flask cntaining plant extract filtrates were allwed t dry cmpletely t btain a slvent free dried residue using water bath evapratr. The plant extracts residues were resuspended in 0.5 ml dimethhylsulphxide (DMSO) fr subsequent analysis. Minimum Inhibitry Cncentratin (MIC) The minimum inhibitry cncentratin (MIC) was determined using the disc diffusin. Filter paper was made int disc using sterile puncher. The discs were sterilized in a ht air ven at 160 C fr 1 h. Extracts were diluted t cncentratins ranging frm t 5.0 mg/ml (fr a mixture f the plant extract and the antibitic). The sterilized discs were saked in different cncentratin f the plant extract and the antibitic fr 24 h. Slidified nutrient agar plates were inculated with test rganisms and the saked discs were placed aseptically n the inculated plates. The nutrient agar plates were incubated at 37 C fr 24 h. The MIC was read as the least cncentratin that inhibited the grwth f the test rganism. Minimum Bactericidal Cncentratin (MBC) The minimum bactericidal cncentratin (MBC) was determined by first selecting tubes that shwed n grwth during MIC determinatin; a lp full frm each tube was sub-cultured nt extract-free agar plates, incubated fr further 24 h at 37 C. The least cncentratin at which n grwth was bserved was nted as MBC. Antibitic Susceptibility Testing Single disc diffusin methd were 11 emplyed as described by Bauer et al. was used t examine bacterial susceptibility t antimicrbial agents. The antibitic sensitivity discs used were nrflxacin (10 µg), chlramphenicl (10 µg), ciprflxacin (10 µg) erythrmycin (30 µg), levflxacin (20 µg), gentamicin (10 µg), ampiclx (20 µg), rifampicin (20 µg), amxicillin (20 µg), and streptmycin (30 µg) (Bectn Dicksn, USA). Single bacterial clnies frm vernight culture were suspended in 5 ml nrmal saline. The surface f Muller Hintn agar plates was evenly inculated; the antibitics discs were applied n the surface f the inculated agar plates with sterile frceps. Each disc was gently pressed dwn nt the agar t ensure cmplete cntact with the agar surface; plates were incubated at 37 C fr 24 h. After incubatin, the plates were examined and the diameter f the znes f inhibitin was measured. Susceptibility data were interpreted accrding t the Clinical and Labratry Standard 12 Institute. Antibacterial Assay Susceptibility screening test using agar w e l l d i f f u s i n m e t h d, e a c h micrrganism was suspended in 6 nutrient brth and diluted t 10 clny frming units per ml (cfu/ml). They were spread (inculated) nt the surface f agar plates which were then dried. Fur millimeter agar wells were cut frm the agar using sterile crk-brer and 0.1 ml f the plant extract and the antibitics were inculated int the wells. After incubatin at 37 C fr 24 h, the plates were examined fr any znes f inhibitin. Cmbinatin Actin Assay The antibitics disc were impregnated with the Ageratum cnyzides leave extract and then dried. The pure culture f Staphylcccus aureus and Pseudmnas aerginsa were subculture n nutrient agar media in pre-sterilized petri dish. The Ageratum cnyzides extract and

4 Cmbinatin f Ageratum Cnyzides Leaf Extracts With Antibitics Against Staphylcccus Aureus And Pseudmnas Aeruginsa Islated Frm Wund Infectin...91 antibitics disc were freshly inculated int agar plates carefully. The plates were incubated at 37 C fr 24 h t test efficacy f bth leaf extract and antibitics against the wund islates. Result The plant extracts shwed varying antimicrbial activity. The aqueus leaf extract shwed n activity against P s e u d m n a s a e r u g i n s a a n d Staphylcccus aureus. The ethanl leaf extract revealed an average mean zne diameter f inhibitin f 11 mm ± 1.0 fr Pseudmnas aeruginsa and mm ± 1.53 fr Staphylcccus aureus (table 1). The minimum inhibitry cncentratin (MIC) and minimum bactericidal cncentratin (MBC) f the ethanl extract is presented in table 2. The MIC and MBC was and 6.25mg/mL fr bth Staphylcccus aureus and Pseudmnas aeruginsa respectively. The antibitics sensitivity patterns f Staphylcccus aureus and Pseudmnas aeruginsa frm wund infectin is presented in table 3. Result shwed that Staphylcccus aureus was highly resistant t all antibitic except fr ciprflxacin and erythrmycin while Pseudmnas aeruginsa was nly susceptible t ciprflxacin and gentamicin. The synergistic actin f Ageratum cnyzides extracts with antibitics n the wund islates is given in table 4. Results revealed that the cmbinatin f Ageratum cnyzides leaf extract plus the individual test antibitics prduced znes f inhibitin higher than that f the individual antibitics and extract. Discussin Herbal medicine has been shwn t have genuine utility and abut 80% f rural dwellers depend n its efficacy fr their 5 primary health care. Medicinal plants cntribute an effective surce f bth traditinal and mdern medicines. Staphylcccus aureus is a nrmal flra f the skin and the leading cause f bth 13 surgical and accidental wund infectin. Pseudmnas aeruginsa is frequently present in small numbers as nrmal flra f the intestine and skin f humans. These bacteria are widely distributed in nature and are cmmnly present in mist envirnment in the hspital and are pathgenic nly when intrduced int area devid f nrmal defenses such as when mucus membrane and skin are 14 disrupted by direct tissue damage. The results btained in the study revealed the antimicrbial efficacy f ethanlic extract f Ageratum cnyzides leaves with mean zne f inhibitin f 11 mm ± 1.0 and mm ± 1.53 fr Pseudmnas aeruginsa and Staphylcccus aureus respectively (table 1). There was significant difference in the antibacterial activity between the aqueus and ethanlic extracts f Ageratum cnyzides (p<0.05). This culd suggest that the active ingredients f the plant extract are nly 15 sluble in rganic slvents. Junaid et al. reprted rganic slvents such as hexane as the best slvents fr extracting plant metablites, due t high nn-plar cmpunds. This study shwed that ciprflxacin alne culd cause a zne diameter f inhibitin f 10 mm and 20 mm against P. aeruginsa and S. aureus respectively (table 3) but prduced 24 mm and 29 mm znes f inhibitin when cmbined with ethanl extract f Ageratum cnyzides (table 4). Als, gentamicin was 13 mm and 0 mm zne f inhibitin n P. aeruginsa and S. aureus respectively, but prduced 30 mm and 7 mm against Pseudmnas aeruginsa and Staphylcccus aureus

5 92...Jurnal f Medicine and Bimedical Research respectively when cmbined with ethanl extract f Ageratum cnyzides. 16 Stevanvic et al. reprted an increase in zne f inhibitin frm 23 mm t 30 mm when tetracycline was cmbined with leaf extract f Aegpdium pdagranial. Park et 17 al. als reprted that cmbinatin f antibitic peptides with chlramphenicl inhibited the grwth f Staphylcccus aureus and Pseudmnas aeruginsa. Interactin between antibitics and plant extracts depends upn species f micrrganism, type and cncentratin f 16 extract as well as the MIC value. Studies have shwn that the efficacy f antimicrbial agents can be imprved by cmbining them with crude plant extract against different pathgens including Staphylcccus aureus, Pseudmnas aeruginsa, Escherichia cli, extended spectrum â-lactamase prducing multiple E. cli and vancmycin resistant 18 enterccci (Entercccus faecalis). This synergistic effect may be due t certain cmplex frmatin which becmes mre effective in the inhibitin f particular specie f micrrganisms either by inhibiting the cell wall synthesis r by causing its lysis r death. The cmbinatin f medicinal plant extracts and knwn antibitics ffer significant ptential fr the develpment f antimicrbial therapies and the treatment f several disease caused by micrrganisms. The synergistic activity f plant extract is a psitive interactin that is btained when tw agents in cmbinatin shw inhibitry effect n targeted rganism that is greater than the sum f their individual 19 effect. Synergy with chemtherapeutic agent is highly specified that is synergism with a particular extract may be effective with a specific drug and ineffective with 20 ther drug. Several in-vitr studies have als reprted synergistic effect with significant reductin in the MICs f the a n t i b i t i c s r e s u l t i n g f r m t h e cmbinatin f different crude plant 21 extracts against S. aureus strains. The ability f plant extracts as ptential antibitics has been well knwn, s it is predicted that inhibitin f drug efflux and alternative mechanisms f actin culd be respnsible fr the synergistic interactin between plant extracts and 22 antibitics. Hence, research shuld be fcused twards this directin t identify mre medicinal plants which exhibit synergistic behaviur. Cnclusin The emergence f resistance t antimicrbial agents is a glbal public health prblem particularly in pathgens causing nscmial infectins when antibitics are n lnger effective by itself during therapeutic treatment. This culd be vercme by the assciatin f antibitics with plant extracts t prduce new chices fr the treatment f infectius diseases.

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8 Cmbinatin f Ageratum Cnyzides Leaf Extracts With Antibitics Against Staphylcccus Aureus And Pseudmnas Aeruginsa Islated Frm Wund Infectin Junaid SA, Olabde AO, Onwuliri FC, Okwri AE, Agina SE.. The antimicrbial prperties f Ocimum gratissimum extract n sme selected gastrintestinal bacterial islates. Afri J Bitechnl : Stefanvic O, Cmic L, Stenjevic D, Slujic SS.. Antibacterial ability f Aegpdium pdagranial leaf extracts and interactins between extracts and antibitics. Turk J Bil : Park Y, Kim HJ, Hahm KS.. Antibacterial synergism f nvel antibitic peptides with chlramphenicl. Bichem Biphy Res Cmm : Adwan GM, Abu-Shanab BA, Adwan K. Invitr activity f certain drugs in cmbinatin with plant extracts against Staphylcccus aureus infectin. Pakistan J Sci : Aiyegr OA, Aflayan AJ, Okh AI. In-vitr antibacterial activity f crude extracts f the leaves f Helichrysum lngiflium in cmbinatin with selected antibitics. Afri J Pharm Pharmacl : Ali NH, Kazim SU, Faizi S. Activity f synergestic cmbinatin Amxy-cassia against Salmnella. Pakistan J Pharmacl : Dicksn RA, Hughtn PJ, Hyland PJ, Gibbns S. Antimicrbial resistance mdifying effects, antixidants and free radical scavenging activities. Phytther Rev : Zha WH, Hu ZQ, Okub S, Hara Y, Shimamura T. Mechanism f synergy between epigallchate chingallate and â - lactams against methicillin resistant Staphylcccus aureus. Antimicrbial Agents Chemther :