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  Anti-MRSA activity and Optimal Culture Condition of  Streptomyces  sp.isolate, SUK 25S. . A!mad c , S. Sudi , #. M. Side$  , %. &. 'asri a , and (.M. )in a * a School of Diagnostic and Applied Health Sciences , Faculty of HealthSciences, Universiti Kebangsaan Malaysia, UKM, Jalan Raa Muda A! A i ,#$%$$ Kuala &u'pur, Malaysia  b  School of (ioscience ) (iotechnology Study, Faculty of Science )*echnology, Universiti Kebangsaan Malaysia, +%$$ UKM (angi, Selangor,Malaysia c School of (o'edicine and Diagnostic Sciences, Faculty of Medicines andHealth Sciences, Universiti Sultan -ainal Abidin, Jalan Sultan Mah'ud,.$+$$, Kuala *erengganu, *erengganu, Malaysia +.A stracta'ac$round of study *he potential of secondary 'etabolites e/tracted fro' Strepto'ycessp! for treat'ent of bacterial infections including infections 0ithStaphylococcus aureus has previously been docu'ented! 1n our o0nlaboratory, 0e sho0ed significant anti'icrobial activities associated0ith endophytic Strepto'yces sp! isolated fro' 'edicinal plants in2eninsular Malaysia! O /ectives 3 *his 0or4 ai'ed to deter'ine anti5Methicillin resistant5 Staphylococcus aureus  6MRSA7 activities fro' Streptomyces sp!isolates! cMaterials 0 Met!ods Disc diffusion and Mini'u' 1nhibitory 8oncentration 6M187 assay0ere used to deter'ine the antibacterial activity! 9pti'i ation of  1   8orresponding author: e'ail3 nora;'edic!u4'!'y  . fer'entation para'eters for the 'ost potent anti5MRSA e/tract inter's of 'ediu' type, pH, aeration rate and culture period 0as alsocarried out! &astly, to/icity of the e/tract against 8hang liver cells 0asdeter'ined e'ploying an M** 6%56+,#5di'ethylthia ol5.5yl75.,#5diphenyl tetra oliu' bro'ide7 assay .d1!e results *he screening results sho0ed 'ost potent e/tract as anti5MRSA 0as SUK .#, 0hile SUK .< 0as found as least potent e/tract!Disc diffusion assay revealed that spread plate techni=ue 0as 'oreefficient in screening anti5MRSA activity co'pared to pour plate 6p5value > $!$#7 and thus used in further studies! For SUK .#, *hronton'edia 0as de'onstrated to be the best 'ediu' for enhancing anti? MRSA activity in SUK .# 0ith M18 value, .!++ @ $!$ BgC'l! *helo0est M18 of !#BgC'& 0as obtained 0hen SUK .# 0as culturedfor < days and aerated at at pH < using this 'ediu' 0ith an aerationrate of +$ rp'! *he crude e/tract 0as not to/ic to0ards 8hang liver cells 618 #$  value E +%!%@ !.+ BgC'l7! eConclusion 3 *he SUK .#s culture condition 0as opti'i ed using*hrontons 'edia, at initial pH < and aerated at +$rp'! Further isolation and identification of bioactive co'pounds 0ill allo0develop'ent of the isolate for anti5MRSA therapeutics! fSinificance and impact of t!is study  Go0days, MRSA infectiontreat'ent fro' co''ercial drug could be to/ic at highdosage!*herefore,alternative 'edicine fro' natural source highlyreco''ended! Key0ords3 Streptomyces , MRSA, *hronton 'edia, M18 ++.+ntroduction  ndophyte bacteria is an organis' that lives in plant tissues, act assy'biont 0ith host and secreted benificial products 6.<7! For e/a'ples, 'etabolites 0ith 'edicinal value 0ere ta/ol, cryptocin andcryptocandin 6.I7!*hese secondary 'etabolites 0ere e/tensively  % studies and antibiotic co'pounds obtained 0ere reported byresearchers and phar'aceutical agencies 6.7! *reat'ents for MRSA infection for alternative 0ays need to beinitiated! Metabolites fro' Streptomyces  sp! 0ere reported to act asantibacterial agents against pathogenic bacteria! 9ur previous 0or4 had found, endophytic Streptomyces sp isolated fro' 'edicinal plantsin 2eninsular Malaysia had sho0n significant anti'icrobial activities6 13 7! *herefore, the present study 0as carried out to deter'ine the'ost potent Streptomyces  sp! isolates as anti5MRSA agent and evaluateits opti'al culture conditions as 0ell as the cytoto/ic effect! +++.Materials and Met!ods a7*est 'icroorganis'3*he sa'ple of endophytic actinobacteria  , SUK 6Strain of UKM7 andMRSA 0ere obtained fro' the Govel Antibiotic laboratory at UKM, Kuala&u'pur! *he SUK collection na'ely SUK .#, SUK .<, SUK .I, and SUK %$ and MRSA culture 0ere MRSA A*88 %%#, MRSA A*88 +%%$$ andMRSA ++<! *hese endophytic Streptomyces  sp! 0as previously isolatedfro' 'edicinal plants in 2eninsular Malaysia6 13 7! *he SUK .# and SUK .< 0as isolated fro'  Zingiber spectabile, 0hile SUK .I fro' *heactinobacteria spore fro'  Sarcandra glabra  and SUK %$ fro' Oroxylumindicum. *he + days culture on 1S2 1nternational Streptomycetes  2roect. agar for + days at .I 8 and 'aintained at 5I$ 8 in .$ glycerolsolution 6 25 7, 0hile MRSA strain 0as 'aintained on Mueller Hinton Agar 6MHA7 supple'ent 0ith . Sodiu' 8hloride, Ga8l at I8! actinobacteria0ere cultured on 1S2 . agar for + days prior anti'icrobial testing! b7Anti5bacterial screening 3(ioassay screening of + isolates of Actinobacteria fro' various sources of 'edicinal plants 6SUK .#, SUK ., SUK .<,SUK .I, and SUK %$7 0erescreened against MRSA A*88 strain %%#, +%%$$ and ++< 6 13 7! Acubic 6 c' % 7 of 'atured actinobacteria 0as placed on nutrient agar that  + 0as la0n 0ith MRSA! *he inhibition one 0as 'easured after overnightincubation in 0hich Lanco'ycin 0as used as positive control! 9ut of thesenine actino'yces, + isolate 6SUK .#, SUK .<! SUK .I, SUK %$7 0ere proceeded for fer'entation in nutrient broth follo0ed by e/traction andtested against MRSA through disc assay 'ethod! *he inhibition one 0as'easured for each plate and Lanco'ycin 0as used as a positive control!c78ultural condition of SUK isolates38ulture conditions for the production of anti5 MRSA 0as deter'ined byinoculation of #5 cubic 6 c' % 7 'atured SUK .# fro' 1S2. 'edia intoone5third of  & rlen'eyer flas4s 0hich each flas4 contained a sterili ed+$$'l broth! *he flas4 0as incubated for < days at .I8 0ith aeration rate$rp'! ight fer'entation 'ediu' 0ith for'ula 'odification 0ere usedna'ely A%M Media 6 20 7, (n5. Media 6 20 7, 1S2  Media 6 22 7, 8 ape45Do/ Media 6 24 7, (ennette Media 6 14 7, *hrontons Media 6 30 7, ArneyHeydorn Media 6 15 7 and Gutrient (roth 6Merc47! After that, selected'edia 0as opti'i ed based on anti5MRSA activities, the para'etersinvolved 0ere incubation period, pH level of the 'edia and aeration rate!d7thyl acetate e/tractionthyl acetate e/traction 6 34 7 0as e'ployed to harvest secondary'etabolite fro' fer'ented broth after < days incubation! 8ulture filtrates0ere e/tracted 0ith three half5volu'e of ethyl acetate! After that, solvent phase 0as concentrated 0ith rotary evaporator 6(uchi,   S0it erland7 at +$8 and 0as left to dry! 8rude e/tracts obtained 0ere suspended 0ith'ethanol and 0ere used for M18 testing against MRSA 6 9 7! *he e/tractsof SUK .# e/ploited fro' different 'edia 6na'ely, A%M Media, (n5.Media, 1S2  Media 8 ape45Do/ Media, (ennette Media *hrontonsMedia, Arney Heydorn Media and Gutrient (roth7 0ere proceeded 0ithM18 test! *he concentration used at $!+II BgC'&5$$$BgC'&!e78ytoto/icity *est


Jul 23, 2017
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