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Tropical Biomedicine 28(3): 651–660 (2011)

Characterization and antimicrobial activities of two

Streptomyces isolates from soil in the periphery of

Universiti Putra Malaysia

Nurul Zarith Mohamad Zin1, Nor Asmara Tasrip1, Mohd Nasir Mohd Desa1,2*, Cheah Yoke Kqueen1,Zainul Amiruddin Zakaria1, Rukman Awang Hamat3 and Mariana Nor Shamsudin3,4

1Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,43400 Serdang, Selangor, Malaysia2Halal Products Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia3Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia4Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia*Corresponding author email: nasirdes@medic.upm.edu.myReceived 30 March 2011; received in revised form 1 July 2011; accepted 15 July 2011

Abstract. This study was to assess the identification and antimicrobial activities of twoactinomycete isolates. The two isolates designated as B8 and C2, were isolated from a patchof soil in the peripheral area of Universiti Putra Malaysia by streaking on starch casein agarafter standard serial dilution procedures. Their antimicrobial activities were first evaluatedagainst eight clinical laboratory strains namely Bacillus sp., Enterococcus sp., Escherichia

coli, Klebsiella sp., Pseudomonas sp., Salmonella sp., Staphylococcus aureus, andStaphylococcus epidermidis by perpendicular streak method on Mueller Hinton and TrypticSoy agar. In both media, a broad-spectrum antibacterial activity was observed for bothisolates, with B8 against all the test bacteria and C2 against five of them (Bacillus sp., E. coli,

Pseudomonas sp., S. aureus and S. epidermidis). Re-assessment against E. coli ATCC 25922and S. aureus ATCC 25923 strains by similar method showed antibacterial activities byisolate B8 against both ATTC strains while C2 only against S. aureus ATCC 25923. Streptomyces

griseus ATCC 10137 was included in the later experiment and showed antibacterial activityagainst both ATCC strains. Subsequently, the two isolates were identified by PCR/sequencingtechniques and phylogenetic analysis to be Streptomyces species (>93% homology based on16S rRNA and rpoB genes). Characterization on cultural characteristic and viable count atdifferent temperatures (37ºC and 28ºC), on different microbiological media (AIA, ISP-2, MHA,NA, PDA and TSA), were performed. More morphological features were observed on ISP-2 forboth isolates. A higher growth yield was also observed at 28ºC in all media but in comparingthat between the two isolates, isolate B8 outnumbered C2 at all experimental conditions. Theobserved variation in cultural traits and growth yield indicate unique properties between thetwo antibiotic-producing isolates.

INTRODUCTION

Infectious diseases account for 13.3 billiondeaths worldwide, which constitute for about25% of all deaths (Cassell & Mekalanos,2001). The chemotherapeutic treatments ofinfectious diseases are also getting limiteddue to the emergence of antibiotic resistantpathogens, which have been widely andcontinuously reported. For example,

methicillin-resistant Staphylococcus aureus

(MRSA), vancomycin-resistant S. aureus

(VRSA), extended spectrum β-lactamases(ESBL)-producing Escherichia coli, andmulti-drug resistant Mycobacterium

tuberculosis (MDR-MTB) are of a majorconcern nowadays (Selvameenal et al.,2009). In consequence, search for newantibiotics has been a major attemptthroughout the world. Environmental

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microorganisms have been the most feasiblesource for new antimicrobial compounds dueto their high abundance and remarkablediversity in nature. Numerous antimicrobialagents of these kinds have been isolatedsuch as streptomycin, aminoglycoside,anthracyclines, chloramphenicol, β-lactams,macrolides and tetracyclines by the membersof actinomycetes, in particular theStreptomyces species (Goodfellow et al.,1988). Streptomycetes are fungus-likefilamentous gram-positive bacteria which areresponsible for the musty odor of soil. Theyhave been the major source of suchantimicrobial compounds and more than70% of all known antibiotics in the market(Ozgur et al., 2008). Due to the biodiversity ofStreptomycetes in both aquatic and terrestriallocales, the variability in antibacterialcompounds is also expected. Thus searchingfor new territory that has yet to be exploredfor new Streptomycetes may lead to thediscovery of new bioactive compounds withantibacterial properties (Mincer et al., 2002).

Meanwhile, the cultivation methods thatcan be considered as a standard forcultivating Streptomyces species are stillelusive. This is due to the complexity of themembers of the actinomycete group thatdifferent isolates from diverse habitats andlocations may require different nutritionalneeds and growth conditions (Rho & Lee,1994; Jensen et al., 2005). Thus, the selectionof media and incubation temperatures in astudy may not be able to accommodategrowth of the different isolates in otherstudies. In view of the important roles ofStreptomycetes as the antibiotic producingorganisms, we have been isolating andcharacterizing the isolates from soil samplesat the Universiti Putra Malaysia andnearby areas (Zin et al., 2010). In this presentstudy, basic conventional and molecularapproaches for the identification of twoactinomycete-resembling isolates with abroad spectrum-activity against variousbacterial pathogens were conducted. Inaddition to morphological and antibacterialcharacteristics, the growth yield on severalcommon laboratory media as well as thosecommercially formulated for actinomycetesat different incubation temperatures were

quantitatively evaluated for a definiteunderstanding on the growth pattern andpreferences of these isolates.

MATERIALS AND METHODS

Isolation and phenotypic characterization

The two actinomycete isolates were from apatch of soil taken at a small forested area atthe periphery of Universiti Putra Malaysia.Isolation was done by standard serial dilutionand spread plate methods using Starch CaseinAgar (SCA) as the primary isolation agar withincubation temperature at 28ºC for 7 to 14days (Zin et al., 2010). Actinomycete colonieson the plates were tentatively identified ascolored, dried, and rough with irregular orregular margin (Williams & Cross, 1971).Based on the morphological characteristicsand musty odor, cultures were selected andfurther purified by several subculturing onISP-2 (International Streptomyces Project)agar medium (Difco, USA). Pure isolates werestudied for their basic colony appearance,Gram-stained, and tested for catalase andoxidase activities (Wu, 1995). Furthermorphological characterizations on aerialmass, substrate mycelium, pigment andspore production of matured cultures onActinomycete Isolation Agar (AIA) (Difco),ISP-2, Mueller Hinton Agar (MHA) (Merck,Germany), Nutrient Agar (NA) (Merck),Potato Dextrose Agar (PDA) (Merck) andTyrptic Soy Agar (TSA) (Merck) were doneas described by Shirling & Gottlieb (1966).Stock cultures were preserved inActinomyces broth (BBL, USA) with 15%glycerol (v/v) at -20ºC.

Antimicrobial screening

The antimicrobial activity of the two isolateswas first evaluated against eight clinicallaboratory strains; Bacillus sp., Enterococcus

sp., E. coli, Klebsiella sp., Pseudomonas sp.,Salmonella sp., S. aureus and Staphylococcus

epidermidis by perpendicular streakmethod on MHA and TSA at 30ºC(Duraipandiyan et al., 2010). Re-assessmentof the antibacterial activity of the two isolateswas done by similar method againstpathogenic E. coli ATCC 25922 and S. aureus

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ATCC 25923 strains. Streptomyces griseus

ATCC 10137 was also included in the laterexperiment as a positive control.

Molecular identification by 16S rRNA and

rpoB gene sequence homology

The two isolates were subjected to molecularidentification by polymerase chain reaction(PCR)/sequencing technique targeting 16S

rRNA and rpoB genes. For each isolate, aloopful of culture containing myceliumand spores was scraped from colonies onISP-2 agar medium and suspended in Tris-EDTA (TE) buffer. Genomic DNA of theisolates was extracted using the GF-1bacterial DNA extraction kit (Vivantis Tech.,Malaysia). The DNA was semi-quantified ona 0.8% agarose gel in 1XTris-borate-EDTA(TBE) and visualized under ultraviolet (UV).Concentration and quality of DNA extractwas checked by using the biophotometer(Eppendorf, Germany). The 16S rRNA genewas detected and amplified using a set ofpublished primers targeting almost the fulllength of the gene: 27F (5’-AGT TTG ATC CTGGCT CAG-3’) and 1492R (5’-ACG GCT ACCTTG TTA CGA CTT-3’) (Duraipandiyan et al.,2010). The reaction was carried out in a 50 µlreaction volume consisting the followingreagents: 10 µl of 5Xi-PCR RED Master Mix(i-DNA Biotech., Singapore), 0.2 µl of forwardprimer, 0.2 µl of reverse primer, 38.6 µl ofwater and 1 µl of template DNA, with thermalcycling conditions as follows: 94ºC, 52ºC and72ºC for 1 min each for 30 cycles followed byanother incubation at 72ºC for 10 min(Duraipandiyan et al., 2010). For rpoB gene,the primers were SRPOF1 (5’-TCG ACC ACTTCG GCA ACC GC-3’) and SRPOR1 (5’-TCGATC GGG CAC ATG CGG CC-3’) (Kim et al.,2004). The reaction was carried out similarlyas described earlier with thermal cyclingconditions as follows: 30 cycles at 95ºC for30 s, 60ºC for 30 s and 72ºC for 45 s. Thereaction was then held at 72ºC for 5 min (Munet al., 2007). PCR amplification was detectedby agarose gel electrophoresis and UVvisualization. PCR product was purified usingthe GF-1 PCR clean-up kit (Vivantis Tech.),checked for purity, and sequenced usingthe same primers for PCR (First Base Lab.,Malaysia). The generated sequences were

searched for sequence homology, using theBLAST software available at the NationalCenter of Biotechnology Information (http://www.ncbi-nlm-nih.gov). The determined16S rDNA nucleotide sequence of the twoisolates were subjected to phylogeneticanalysis in comparison to those of otherrelated species as shown by the BLASTsearches. The later sequences, includingsome of the Micromonospora species toserve as the out-group, were retrieved fromthe GenBank sequence databases. A distancematrix tree was then built by UPGMAclustering method with Needle-Wunschalgorithm and 100X bootstrap analysis, usingthe Bionumerics software version 6.1(Applied Maths, Belgium).

Viable cell count

The two isolates were grown on ISP-2 agarmedium at 28ºC. The spores produced werescraped and transferred into normal salinein sterile test tubes. The density of the sporesuspension was checked at OD600 of 0.10 –0.15 (~0.5 McFarland standard). A 10-foldserial dilution of the spore suspension wascarried out and 0.1 ml of each dilution wasspread onto different media; AIA, ISP-2, MHA,NA, PDA and TSA. Plating was done in twosets for 7 days-aerobic incubation at 37ºC and28ºC respectively. All experiments were donein triplicate and a mean value was obtained.Kruskal Wallis and Mann Whitey tests wereused for statistical analysis with P < 0.05 asthe significant level.

RESULTS

The two isolates, labeled as B8 and C2 werecolored, dried, rough, and irregular in marginwith a strong musty smell on the agar plate.Basic microbiological description on thecolony appearance showed generally raisedand opaque colonies, with filamentous formand rugose surface. Microscopic observationshowed rod shaped-branching filaments witha gram positive reaction to further indicatethe characteristics of actinomycetes. In thebiochemical characterizations, catalase andoxidase tests showed that the two isolateswere positive for the enzymes. The summary

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of the studied phenotypic properties of theisolates was listed in Table 1.

Further morphological characterizationsof the isolates on the different media weresummarized in Table 2. Overall, a colorhomogeneity (pale yellow) was observed forboth aerial mass and substrate mycelium ofisolate B8 on all media except ISP-2 thatshowed a different color (dark brown) forsubstrate mycelium. On the other hand, avariety of color was observed for isolate C2on the different media with a color contrastbetween aerial mass and substrate mycelium(orangish and dark brown) also on ISP-2.Soluble pigment was observed for bothisolates on ISP-2, MHA and TSA whereas thaton PDA was observed only for isolate C2.While the presence of spore mass was

obviously visible only on ISP-2 for bothisolates, and each was of different color.

In the antimicrobial screening byperpendicular streak method (Table 1),isolate B8 showed an inhibition zone againstthe entire test bacteria while isolate C2against five of them (Bacillus sp., E. coli,Pseudomonas sp., S. aureus and S.

epidermidis) on both MHA and TSA. Re-assessment against E. coli ATCC 25922and S. aureus ATCC 25923 strains alsoshowed a similar result on MHA and TSAwhereby isolate B8 had antibacterial effectagainst both ATCC strains while isolate C2only against S. aureus ATCC 25923. S.

griseus ATCC 10137 demonstrated inhibitoryeffect against both ATCC strains on MHAand TSA.

Figure 1. Phylogenetic dendogram of 16S rDNA distance matrix-based analysisshowing the position of isolates B8 and C2 and their phylogenetic neighbors(GenBank accession numbers in parentheses). The above scale indicates thepairwise similarity scores (%) calculated from a total of 30 aligned sequencescomprising Micromonospora species. The later serve as the out-group to rootthe tree. Numbers at the branch nodes indicate the bootstrap values (%)

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Table 1. Phenotypic properties of isolates B8 and C2

Test Result

B8 C2

Colony appearanceOptical character Opaque OpaqueElevation Raised RaisedForm Filamentous FilamentousSurface Rugose Rugose

Cell characteristicGram’s reaction + +Shape Filament Filament

Biochemical testCatalase + +Oxidase + +

Antibacterial activityBacillus sp. + +Enterococcus sp. + –E. coli + +Klebsiella sp. + –Pseudomonas sp. + +Salmonella sp. + –S. aureus + +S. epidermidis + +E. coli ATCC 25922 + –S. aureus ATCC 25923 + +

‘+’ = positive reaction, ‘–’ = negative reaction

Table 2. Cultural characteristics of isolates B8 and C2 on different media

Aerial Substrate Soluble Spore

Medium mass mycelium Pigment

B8 C2 B8 C2 B8 C2 B8 C2

AIA Pale Pale Pale Pale – – – –yellow yellow yellow yellow

ISP-2 Pale Orangish Dark Dark + + + +yellow brown brown brown (White) (Orange)

MHA Pale Pale Pale Pale + + – –yellow yellow yellow yellow

NA Pale Pale Pale Pale – – – –yellow yellow yellow yellow

PDA Pale Pinkish Pale Pinkish – + – –yellow orange yellow orange

TSA Pale White Pale White + + – –yellow yellow

‘+’ = present, ‘–’ = not present

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The presumptively identifiedactinomycete-isolates B8 and C2 weresuccessfully identified as Streptomyces

species (>93%) following sequencehomology search of the generated 16S rRNA

and rpoB gene sequences in the GenBank.Based on 16S rDNA nucleotide sequences,BLAST results showed isolate B8 to havethe highest similarity (99%) to Streptomyces

lavendularectus NBRC 13675 (AB184456)and Streptomyces lilacinofulvus NBRC13677 (AB184458), while isolate C2 has thehighest similarity (99%) to Streptomyces

spectabilis NBRC 15441T (AB184677). Thephylogenetic tree further shows the twoisolates to be in a very close contiguity withtheir respective highest matches ofStreptomyces species as in the BLASTanalysis, with each in a distinct evolutionarycluster. For the out-group, all Micro-

monospora species were collectivelyseparated from the Streptomyces cladesas expected. The 16S rDNA nucleotidesequences of isolates B8 and C2 includingthose for rpoB gene have been submittedinto the GenBank database with accessionno. JN116247, JN116248, JN116249 andJN116250 respectively.

Viable count on the different mediashowed that growth of isolate B8 was about10-fold higher than that of isolate C2 (106 vs.105cfu/ml) at all experimental conditions.

Nevertheless, for both isolates, the growthyield was higher at 28ºC of incubationtemperature than at 37ºC (Tables 3 and 4).In comparing the growth yield on variousmedia for the respective isolates, viablecount differed significantly at bothtemperatures. For isolate B8, the order ofmedia that generated from low to highgrowth yield was as follows: PDA<NA<TSA<MHA<AIA<ISP-2 at 37ºC while that at28ºC was NA<TSA<MHA<AIA<ISP-2<PDA(Table 3). Based on the order of growth yield,a consistent pattern of media preferencewas observed (underlined) at bothtemperatures except for PDA that harboredthe highest growth at 28ºC but turned outto be the lowest at 37ºC. A different mediapreference was nevertheless observedfor isolate C2 as follows: at 37ºC:PDA<MHA<ISP-2<NA<TSA<AIA, and at28ºC: PDA<MHA<AIA<NA<TSA<ISP-2(Table 4). Still, media preference of isolateC2 at the two temperatures was mostlyconsistent (underlined) except for AIA andISP-2 whereby the later was preferred at 28ºCbut not at 37ºC, whereas AIA was preferredat 37ºC but not at 28ºC. As a whole, ISP-2 wasfrequently the best media to harbour highergrowth except for isolate C2 at 37ºC. PDAwas the lowest to harbour growth except at28°C for isolate B8, while the effects of othermedia were variable.

Table 3. Viable cell count (cfu/ml) of isolate B8 on various microbiological media at differenttemperatures

Temperature/ 37ºC 28ºCP-value1*

Medium (mean ± standard deviation) (mean ± standard deviation)

AIA 2.47 ± 0.035 x 106 4.77 ± 0.058 x 106 0.046

ISP-2 2.57 ± 0.036 x 106 5.13 ± 0.058 x 106 0.046

MHA 2.04 ± 0.021 x 106 4.53 ± 0.252 x 106 0.050

NA 1.78 ± 0.042 x 106 3.50 ± 0.265 x 106 0.050

PDA 1.20 ± 0.105 x 106 6.17 ± 0.569 x 106 0.050

TSA 1.88 ± 0.067 x 106 4.27 ± 0.155 x 106 0.046

P-value2* 0.005 0.007

1Comparison of growth at different temperatures on respective media2Comparison of growth on different media at respective temperatures*Significant values in bold

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Table 4. Viable cell count (cfu/ml) of isolate C2 on various microbiological media at differenttemperatures

Temperature/ 37ºC 28ºCP-value1*

Medium (mean ± standard deviation) (mean ± standard deviation)

AIA 6.70 ± 0.173 x 105 6.77 ± 0.153 x 105 0.637

ISP-2 6.07 ± 0.252 x 105 7.50 ± 0.458 x 105 0.050

MHA 5.93 ± 0.306 x 105 6.00 ± 0.200 x 105 0.822

NA 6.40 ± 0.300 x 105 6.80 ± 0.173 x 105 0.105

PDA 3.60 ± 0.400 x 105 5.50 ± 0.458 x 105 0.050

TSA 6.60 ± 0.200 x 105 7.33 ± 0.503 x 105 0.077

P-value2* 0.017 0.012

1Comparison of growth at different temperatures on respective media2Comparison of growth on different media at respective temperatures*Significant values in bold

DISCUSSION

Actinomycetes are a diverse group ofmicroorganisms that identification approachby biochemical tests may be tedious andtroublesome. Due to the specificity ofmolecular technique, it may substitute robustconventional identification by giving a fasterand reliable result. In this study, some basicconventional identification tests forStreptomycetes such as Gram-stain, odor andmorphological examination followed bymolecular detection using PCR/sequencingtechnique targeting 16S rRNA and rpoB

genes were performed. The former genehas been long used and shown to be effectivein identification and evolutionary tracingof various microorganisms includingStreptomyces strains but the later has alsobeen extensively studied as an alternativetarget for species identification due to itsgreater polymorphism to render it morediscriminatory than the former gene (Khamiset al., 2004; Adékambi et al., 2008; Ki et al.,2009). Until a consensus is reached to treatrpoB gene as so, it was used in this study asan additional validation at genus level only.The BLAST analysis and generated 16S rDNAbased-phylogram suggested that isolatesB8 and C2 are Streptomyces species withpotential different lineage of evolution.Considering that the isolates were from apatch of soil that there could be a potential

accumulation and dissemination of clones,some of their phenotypic characters whichare dissimilar in term of colour and growthcapacity on the different media may supportthe phylogenetic inference that they aredifferent isolates.

Streptomyces strains have been knownfor their valuable potential as sources forantimicrobial agents. But their diversityobscures the selection of feasible media fora sustainable growth of various unknownisolates. To maximize the isolation andrecovery of various actinomycetes fromenvironmental samples on agar media, manystudies have been employing various sourcesof carbon, nitrogen and mineral in mediapreparation (Narayana & Vijayalakshmi,2008; Valanarasu et al., 2009), while onestudy used the soil itself as part of the mediacomponent (Hamaki et al., 2005). It is notknown whether these specially formulatedmedia could be equally effective to providegrowth for the Streptomyces strains fromhabitat and location of different origins asthese media were designed to suit the localenvironments in the respective studies. Amajor shortcoming is that these media arenot commercially available to be readilyutilized by different laboratories throughoutthe world. International Streptomyces Project(ISP) has come up with media ISP-1, -2, and -4 to select stable properties of Streptomyces

strains for characterization purposes

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(Shirling & Gottlieb, 1966). In this study,ISP-2 agar medium was used as themaintenance media following primaryisolation on SCA. Pure culture was obtainedafter several subculturings and growthcharacteristics were then evaluated onvarious common commercial micro-biological media; MHA, NA, PDA and TSA.The AIA and ISP-2 media are commerciallyformulated for Streptomyces and growthon these media was compared with those onthe other common media for potentialdifferences. Malaysia is a tropical countrywhich is very warm during the day-time andoften humid, while it is fairly cool during thenight. Thus the incubation temperatures usedin this study were 37ºC and 28ºC to representthe local surrounding temperature. The latertemperature was widely used in many otherstudies but fewer reports elucidated thepattern of growth at 37ºC in comparison tothat at 28ºC in a quantitative manner(Suthindhiran et al., 2009; Valanarasu et al.,2009; Kumar & Kannabiran, 2010).

The ISP-2 agar medium contains yeastand malt extract which are rich in nutrientssuch as nitrogen, amino acids and vitamins,and dextrose to serve as the source of carbon.But the exact amounts of such individualnutritional elements in the extract are notknown. On the other hand, AIA containsdefined amounts of nitrogen source (sodiumcaseinate) and amino acid (asparagines) withadded glycerol (0.5%) as source of carbon.None of these types of nutritional sources inISP-2 and AIA media are present in the othermedia used in this study but TSA containspeptone from yeast. With a standardized sporesuspension (~0.5 McFarland standard) as thestarting inocula, viable counts showed thatgrowth for both isolates B8 and C2 werewithin106 and 105cfu/ml respectivelyindicating that all media were capable ofsupporting growth at certain extents. In termof incubation temperatures, growth yieldat 28ºC were always higher particularlyisolates B8 that reached significant levelin all media conditions. Nevertheless the twoisolates showed different media preferencesthat changed in response to the incubationtemperatures. These indicate that differentisolates may have different capability in

utilizing different sources of nutritionalcomponents and the capability may changeat different temperatures. Frequently, ISP-2medium sustained a good growth yield forboth isolates in comparison to the othermedia which varied. In addition, moremorphological characteristics of the isolateswere exhibited on ISP-2 medium such asthe color variety of aerial mass, substratemycelium and spores, while those on othermedia were generally homogenous. Thissupports ISP-2 as a medium suitable fordescribing discrete morphological featuresof Streptomyces. Nevertheless, as far as thegrowth output is concerned regardless ofincubation temperature, pigment and sporeproduction, MHA may be favorable for isolateB8 whereas TSA for isolate C2 for the choiceof media other than those specifically devisedfor Streptomyces (ISP-2 or AIA). It is difficultto comment on the impact of the individualcomponents of the media used as in general,these are undefined media that utilizeextracts from various sources whereby thespecific amounts and types of element in theextract are not known. But, due to the frequentgood growth and expression of varied culturaltraits on ISP-2 agar media in this study, thepotential sources of nutrient would be thosepresent in the media; the yeast and maltextract, to serve as a basis for furtherexploitation in media formulation. Yeast andmalt extract are commercially available andhave been widely used as supplement incombination with various media to supportgrowth of fastidious organisms.

Although growths of the two isolateswere shown to vary on different media andtemperatures, the question on the amount andquality of antibacterial metabolites producedis still unknown. Primary antibacterialscreening on MHA and TSA performed inthis current study showed the inhibitoryactivity but the ability of the antibacterialmetabolites produced by the two isolates toequally diffuse in the different media may notbe similar due to different media composition.Secondary screening using metaboliteproduced in broth culture followed by disc orwell diffusion method on a standardized testmedia is usually required to further validatethe antimicrobial activities. However, it is still

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controversial as production of theantibacterial metabolites may be affected bythe different growth environment on agarsurface and in broth suspension (Pandey et

al., 2004).On the whole, findings from this study

showed variations in phenotypiccharacteristics, particularly the growthyield on different media at differenttemperatures to indicate unique featuresbetween the two local Streptomyces isolateswith antibacterial properties. In view of thediversity of the genus, a lot more isolateswith distinct characteristics are anticipatedto further complicate the selection of feasiblemedia for their cultivation. How those couldrelate to the production of antimicrobialmetabolites is of much interest.

Acknowledgement. This study was funded byRUGS grant no: 04-01-09-0715RU and FRGSgrant no: 07-04-10-850FR.

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