Research Article Bioactive Potential of …downloads.hindawi.com/archive/2014/812974.pdfResearch Article Bioactive Potential of Actinomycetes from Less Explored Ecosystems against

Research ArticleBioactive Potential of Actinomycetes from Less ExploredEcosystems against Mycobacterium tuberculosis and OtherNonmycobacterial Pathogens

Radhakrishnan Manikkam1 Gopikrishnan Venugopal2 Balaji Subramaniam3

Balagurunathan Ramasamy2 and Vanaja Kumar1

1 Centre for Drug Discovery and Development Sathyabama University Chennai Tamil Nadu 600 119 India2Department of Microbiology Periyar University Salem Tamil Nadu 636 011 India3 Department of Bacteriology National Institute for Research in Tuberculosis Chennai Tamil Nadu 600 031 India

Correspondence should be addressed to Vanaja Kumar vanaja kumar51yahoocoin

Received 23 May 2014 Accepted 20 October 2014 Published 9 November 2014

Academic Editor Michael J Palladino

Copyright copy 2014 Radhakrishnan Manikkam et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Bioactive potential of actinomycetes isolated from certain less explored Indian ecosystems againstMycobacterium tuberculosis andother nonmycobacterial pathogenswas investigated Actinomycetes were isolated from the soil samples collected fromdesert coffeeplantation rubber forest and hill area and their cultural and micromorphological characteristics were studied Crude extractswere prepared by agar surface fermentation and tested against M tuberculosis isolates by luciferase reporter phage (LRP) assayat 100120583gmL Activity against nonmycobacterial pathogens was studied by agar plug method Totally 54 purified cultures ofactinomycetes including 43 Streptomyces and 11 non-Streptomyceswere isolatedWhile screening for antitubercular activity extractsof 39 actinomycetes showed activity against one or moreM tuberculosis isolates whereas 27 isolates exhibited antagonistic activityagainst nonmycobacterial pathogens In particular crude extracts from sixteen actinomycete isolates inhibited all the three Mtuberculosis isolates tested Findings of the present study concluded that less explored ecosystems investigated in this study arethe potential resource for bioactive actinomycetes Further purification and characterization of active molecule from the potentialextracts will pave the way for determination of MIC toxicity and specificity studies

1 Introduction

Tuberculosis (TB) caused by Mycobacterium tuberculosis isa highly prevalent infectious disease with almost one-thirdof global population believed to be infected [1] Accordingto statistics India is the 17th among the 22 high burdencountries in terms of TB incidence rates [2] Emergence ofdrug resistance amongM tuberculosis isolates and long termtherapy using combination of drugs for its treatment are themajor problem in TB control Hence there is an urgent needfor new antitubercular drugs to fight against drug resistantMtuberculosis strains [3] The new anti-TB drugs are expectedto have less side effects and improved pharmacokinetic prop-erties with extensive and potent activity against drug resistantstrains andor should be able to reduce the total duration of

treatment [4] Secondary metabolites frommicrobial sourceshave a long history in the treatment of TB [5] Actinomycetesare aerobic filamentous Gram-positive bacteria with trueaerial hyphae belonging to the phylumActinobacteria (orderActinomycetales) [6] Actinomycetes are common soil inhab-itants with an unprecedented ability to produce clinically use-ful secondary metabolites including antibiotics Of the totalmicrobial bioactive metabolites around 50 are reportedfrom the members of actinomycetes [7] From the discoveryof streptomycin first antibiotic used for anti-TB therapy fromStreptomyces griseus numerous anti-TB antibiotics such askanamycin and rifampicin have been reported from actino-mycetes of terrestrial origin In recent years exploration ofactinomycetes from routine ecosystems frequently results inreisolation of known actinomycetes and antibiotics Instead

Hindawi Publishing CorporationInternational Scholarly Research NoticesVolume 2014 Article ID 812974 9 pageshttpdxdoiorg1011552014812974

2 International Scholarly Research Notices

bioprospecting of unless explored ecosystems like marinedesert forests caves and hills has been proved as usefulmethod for tapping innumerable number of bioactive com-pounds from novel bioactive actinomycetes [7ndash10] includinganti-TB metabolites [11] Bioprospecting of actinomycetesfrom certain less explored ecosystems in India with specialreference to antitubercular activity was attempted in thepresent work Antagonistic activity of actinomycetes againstnonmycobacterial pathogens was also studied

2 Materials and Methods

21 Description and Characterization of Actinomycetes Acti-nomycetes were isolated from soil samples collected fromthe rare ecosystems namely (i) Thar desert Rajasthan(ii) Rubber forest Kerala (iii) Coffee plantation Kerala(iv) Western Mountain Thirukurungudi Tamil Nadu (v)Thotabeta hills Tamil Nadu (vi) Siruvani hill area TamilNadu (vii) Yercaud hills Tamil Nadu and (viii) Munnar hillarea Tamil Nadu The collected samples were dried at 28∘Cfor 2 d Actinomycetes were isolated by adopting standardspread plate method using starch casein nitrate agar medium[12] supplemented with nalidixic acid and nystatin to retardthe growth of bacteria and fungi respectively Growth ofactinomycetes was maintained on yeast extract malt extract(YEME) agar (ISP medium 2) [13] as well as in 30 glycerolbroth

Cultural characterization was done by inoculating allthe actinomycete cultures into YEME agar medium [13]Micromorphological characteristics were studied by adopt-ing slide culture method [14] Based on growth patternof actinomycetes on YEME agar medium and microscopicappearance similar actinomycete isolates were discarded andthe ones that were exhibiting different characteristics wereselected for further investigations

22 Small Scale Production of Bioactive Metabolites Bioac-tive metabolites from actinomycetes were produced by agarsurface fermentation All the actinomycete cultures wereinoculated into two YEME agar plates each and incubated at28∘C for 10 d After scraping out themycelial growth the agarmedium was cut into pieces and metabolites secreted extra-cellularly into the agar medium were extracted using 50mLof methanol for 24 h The methanol portion was collectedand concentrated using eppendorf concentrator at 30∘C andquantified [15] One mg per mL of working concentrationof crude extract was prepared using 10 dimethyl sulfoxide(DMSO) prepared in sterile distilled water and filtered using045 120583 filters

23 In Vitro Screening for Antitubercular Activity Anti-tubercular activity of actinomycete extracts was studiedagainst standard laboratory strain Mycobacterium tuberculo-sis H37Rv SHRE (streptomycin isoniazid rifampicin andethambutol) sensitive and SHRE resistant clinical isolatesof M tuberculosis by adopting LRP assay [16] All the Mtuberculosis isolates were obtained from the Department ofBacteriology National Institute for Research in Tuberculosis

Viability of all the isolates was maintained on LJ slopes Hightitre of mycobacteriophage phAE129 used in this study wasprepared using M smegmatis mc2155 in Middlebrook 7H9complete medium [17]

About 350 120583L of G7H9 broth supplemented with 10albumin dextrose complex and 05 glycerol was taken incryovials and added with 50 120583L of crude extract in orderto get the final concentration of 100 120583gmL 100 120583L of Mtuberculosis cell suspension was added to all the vials Theabove procedure was followed for all the threeM tuberculosisisolates DMSO (1) was also included in the assay as solventcontrol All the vials were incubated at 37∘C for 72 h Afterincubation 50120583L of high titre phage phAE129 and 40 120583L of01M CaCl

2solution were added to the test and control vials

All the vials were incubated at 37∘C for 4 h After incubation100 120583L fromeach vial was transferred to luminometer cuvetteAbout 100 120583L of D-Luciferin was added and relative light unit(RLU) was measured in luminometer (Monolight 2010)

Percentage RLU reduction

=Control RLU minus Test RLU

Control RLUtimes 100

(1)

Extracts showing RLU reduction by 50 or more whencompared to control were considered as having antituber-cular activity Actinomycetes which showed activity againstall the three M tuberculosis isolates were selected and testedin triplicate against the same M tuberculosis isolates andnonmycobacterial pathogens The mean values of the resultswere calculated

24 In Vitro Screening against Nonmycobacterial PathogensTest pathogens used in this study include standard strainsStaphylococcus aureus MTCC96 Bacillus subtilis MTCC441clinical isolates of Escherichia coli Salmonella typhiKlebsiellapneumoniae Proteus vulgaris Pseudomonas aeruginosa andCandida albicans All the nonmycobacterial pathogens wereobtained from the Department of Microbiology PeriyarUniversity Tamil Nadu Test pathogens were inoculated ontoMuller Hinton Agar (MHA) plates using sterile cotton swabActinomycete cultures were inoculated onto YEME agarplates (20mLplate) and incubated at 28∘C for 10 d Afterscraping the mycelial growth 5mm diameter agar plugswere taken and placed over MHA plates seeded with testpathogens All the plates were incubated at 37∘C for 24 hfor bacteria and 48 h for fungi Agar plug prepared fromuninoculated YEME agar was included as medium controlZone of inhibition was expressed in millimetre in diameter[15]

3 Results

31 Isolation andCharacterization of Actinomycetes Seventy-two actinomycete isolates were isolated from soil samples col-lected from different ecosystems Based on the microscopicand cultural characteristics 54 different actinomycete isolateswere selected after dereplication of 18 isolates Fifty out of 54isolates produced good growth with powdery consistency on

International Scholarly Research Notices 3

Table 1 Growth and morphological pattern of actinomycetesisolated from different rare ecosystems

Characteristics Appearance Number ofisolates ()

Growth Good 50 (92)Moderate 4 (7)

Consistency Powdery 45 (83)Leathery 9 (16)

Aerial mass colour

White 26 (48)Gray 20 (37)Green 2 (3)Blue 1 (2)

Orange 2 (370)Pink 3 (555)

Reverse side pigment 17 (3148)Soluble pigment 18 (3333)

Micromorphology

Aerial and substratemycelium 53 (9814)

Substrate myceliumalone 1 (185)

Spore chainmorphology

Rectus flexible (RF) 34 (6296)Retinaculum apertum

(RA) 6 (1111)

Spirals (S) 2 (370)Others 12 (2222)

YEME agar Majority of the isolates produced either white(48) or gray (37) colour aerial mycelium Reverse sidecolour and colour of soluble pigments produced by theseactinomycetes include brown yellow pink green blue andorange In micromorphological study except the strain S28all the actinomycete isolates showed the presence of substrateand aerial mycelium with different length and arrangementIn 34 (6296) isolates the aerial mycelium was of rectiflex-ibile (RF) type (Table 1) Based on the observed phenotypiccharacteristics 43 actinomycete isolates (7962) were tenta-tively identified as Streptomyces sp and 11 (2037) isolateswere identified as nonstreptomycesrare actinomycetes

32 Production and Antitubercular Activity of ActinomyceteExtracts All the actinomycete isolates showed growth onYEME agar medium Overall about 30ndash40mg of crudeextract was obtained from 50mL of YEME agar mediuminoculated with actinomycetes Extracts from 39 (7222)out of 54 isolates inhibited one or more of M tuberculosisisolates tested Extracts of 16 (2962) actinomycete isolatesinhibited the growth of all the three strains namely standardstrain M tuberculosis H37Rv SHRE sensitive and SHREresistant M tuberculosis isolates Extracts of 24 (4444)actinomycetes inhibited the standard strain M tuberculosisH37Rv whereas the SHRE sensitive and SHRE resistant Mtuberculosis isolates were inhibited by the extracts of 23(4259) actinomycetes (Table 2)

The diversity of antagonistic activity exhibited by theactinomycetes is given in Table 3 In total 43 (7963)actinomycetes showed antagonistic activity against one ormore of the 11 pathogens tested Eleven (2037) actino-mycetes failed to inhibit any of the test pathogens Anothereleven isolates (2037) were active against one or morenumber of M tuberculosis isolates Gram positive bacteriaand Gram negative bacteria Eight (1481) isolates wereactive only against Gram positive bacteria and M tubercu-losis isolates whereas one each exclusively inhibited Grampositive bacteria (Y1) Gram negative bacteria (M17) fungi(CSA19) Gram positive and Gram negative bacteria (Y32)and M tuberculosis and Gram negative bacteria (M7) Onlytwo isolates K8 and TA36 inhibited both M tuberculosisisolates and C albicans without showing activity against anyother bacterial pathogens The only strain CSA15 isolatedfrom coffee plantation soil showed activity against all theM tuberculosis isolates Gram positive and Gram negativebacteria and fungi Thus diverse antagonistic activity wasexhibited by the actinomycetes isolated from all the rareecosystems investigated in this study

33 Antagonistic Activity against Nonmycobacterial Patho-gens In agar plug method 27 isolates that is 50 of theactinomycete isolates showed inhibition against at least oneor more number of nonmycobacterial pathogens testedMaximum of 17 (3148) and 16 (2962) isolates were activeagainst S aureusMTCC96 and B subtilisMTCC441 respec-tively Among the Gram negative bacteria P vulgaris wasinhibited by 11 (2037) actinomycetes None of the isolateswere found to be active against P aeruginosa Four (555)actinomycetes inhibited the growth of C albicans (Table 2)Ten out of 18 soluble pigment producers were found to exhibitantibacterial activity The crude ethyl acetate extracts from16 selected actinomycete isolates showed activity against thebacterial pathogens and M tuberculosis strains tested Themean value of activity is presented in Table 4

4 Discussion

Pathogenic microorganisms have evolved sophisticatedmechanisms to inactivate antibiotics and rendered an urgentneed for new antibiotics that would target the emergingmultidrug resistance [18] Consequently search for novelsources of potent antibiotics is desperately needed todevelop potent drugs Microbial resources have made anincredible contribution to the antibiotic drug discovery anddevelopment process over the last seven decades [19] Inparticular actinomycetes are the most important source ofbioactive natural compounds with a long track record ofproducing novel molecules [20] The present study reportedthe bioactive potential of actinomycetes isolated from lessexplored ecosystems against M tuberculosis and othernonmycobacterial pathogens

According to Berdy [7] great number of antibiotic com-pounds exhibit exclusive activities against Gram positivebacteria while only 15 are active against Gram negativebacteria In the present study 50 of the actinomycetes

4 International Scholarly Research Notices

Table2Num

bero

factinom

ycetes

exhibitin

gactiv

ityagainstn

onmycob

acteria

lfung

aland

mycob

acteria

lpatho

gens

Ecosystems

Num

bero

factin

omycetes

Num

bero

fantagon

isticiso

latesa

gainst

Non

mycob

acteria

lpatho

gens

Mtub

erculosis

Fung

i

Saureus

MTC

C96

Bsubtilis

MTC

C44

1E

coli

Styphi

Pvulga

risK

pneu-

moniae

Paeruginosa

MTB

H37Rv

MTB

SHRE

sensitive

MTB

SHRE

resistant

Calbicans

Thar

DesertRa

jasthan

54

3mdash

mdash3

mdashmdash

55

5mdash

Rubb

erforest

Kerala

61

1mdash

mdash1

mdashmdash

23

11

Coff

eeplantatio

nKe

rala

123

11

22

2mdash

55

52

Thiru

kurung

udiTamil

Nadu

73

mdashmdash

mdash2

mdashmdash

34

21

YercaudHillsTamilNadu

94

4mdash

11

1mdash

41

4mdash

Thottabetta

HillsTamil

Nadu

3mdash

3mdash

mdashmdash

1mdash

22

3mdash

Mun

narH

illsTamilNadu

5mdash

1mdash

mdash3

mdashmdash

33

3mdash

Siruvani

HillsTamilNadu

72

31

1mdash

mdashmdash

mdashTo

talIsolates

5417

162

411

3mdash

2423

234

Percentage

100

3148

2962

37

742037

555

000

4444

4259

4259

555

International Scholarly Research Notices 5

Table3Diversityof

antagonisticactiv

ityexhibitedby

actin

omycetes

isolatedfro

mrare

ecosystems

Actin

omycetes

activ

eonlyagainst

Actin

omycetes

from

different

ecosystems

Totalactive

isolates

Percentage

Thar

desert

Rubb

erforest

Coff

eeplantatio

nTh

iruku

rung

udi

Yercaudhills

Thottabetta

Mun

narh

ills

Siruvani

hills

Mtub

erculosis

D18

K11K3

8CS

A2CS

A4

CSA20

TA4TA

27Y3

Y7Y13

Y23Y2

5mdash

M2M3

S816

2962

G+bacteria

mdashmdash

mdashmdash

Y1mdash

mdashmdash

118

5Gminusbacteria

mdashmdash

mdashmdash

mdashmdash

M17

mdash1

185

Fung

imdash

mdashCS

A9

mdashmdash

mdashmdash

mdash1

185

MTB

G+andGminus

bacteriaand

fung

imdash

mdashCS

A15

mdashmdash

mdashmdash

mdash1

185

MTB

andG+and

Gminusbacteria

D6D13D

16K3

CSA24

TA3TA

22Y10

T2M8

S211

2037

MTB

andG+

bacteria

D25

mdashCS

A19

TA38

Y8T6

T7

mdashS4S5

81481

MTB

andGminus

bacteria

mdashmdash

mdashmdash

mdashmdash

M7

mdash1

185

G+andGminusbacteria

mdashmdash

mdashmdash

Y32

mdashmdash

mdash1

185

G+andGminusbacteria

andfung

imdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

MTB

andfung

imdash

K8mdash

TA36

mdashmdash

mdashmdash

2370

Totaln

umbero

factiv

eisolates

54

76

93

54

437963

Noactiv

itymdash

K7K

10

CSA7CS

A8

CSA11

CSA12

CSA18

TA34

mdashmdash

mdashS21S26S28

112037

Totalisolates

56

127

93

57

54100

6 International Scholarly Research Notices

Table4Ac

tivity

ofselected

actin

omycetes

againstb

acteria

fun

giand

Mtub

erculosis

isolates

Actin

omycetes

Saureus

MTC

C96

Bsubtilis

MTC

C441

Ecoli

Styphi

Pvulga

risK

pneumoniae

Paeruginosa

Calbicans

Mtub

erculosis

H37Rv

SHRE

sensitive

Mtub

erculosis

SHRE

resistant

Mtub

erculosis

D6

123plusmn05

13plusmn000

mdashmdash

156plusmn052

mdashmdash

mdash696plusmn25

7464plusmn255

6242plusmn278

D13

132plusmn06

mdashmdash

mdash122plusmn034

mdashmdash

mdash9513plusmn21

9365plusmn253

8342plusmn365

D16

115plusmn05

105plusmn05

mdashmdash

8plusmn000

mdashmdash

mdash4986plusmn21

7531plusmn361

6506plusmn258

D18

mdashmdash

mdashmdash

mdashmdash

mdashmdash

8641plusmn

187379plusmn346

9504plusmn316

D25

211plusmn02

226plusmn059

mdashmdash

mdashmdash

mdashmdash

8710plusmn26

7581plusmn

229

8071plusmn

324

K38

mdashmdash

mdashmdash

mdashmdash

mdashmdash

7705plusmn15

6812plusmn243

7546plusmn380

CSA15

105plusmn05

83plusmn040

103plusmn026

121plusmn05

108plusmn041

12plusmn00

mdash106plusmn03

8598plusmn09

9472plusmn366

6737plusmn274

CSA20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

7372plusmn29

7489plusmn243

7940plusmn293

TA3

11plusmn000

mdashmdash

mdash78

6plusmn011

mdashmdash

mdash650plusmn229

9156plusmn351

5347plusmn222

TA27

mdashmdash

mdashmdash

mdashmdash

mdashmdash

8129plusmn32

9492plusmn306

8039plusmn317

T6mdash

223plusmn055

mdashmdash

mdashmdash

mdashmdash

7926plusmn04

862plusmn236

9621plusmn

332

T7mdash

205plusmn050

mdashmdash

mdashmdash

mdashmdash

9158plusmn45

8332plusmn350

9344plusmn350

M7

mdashmdash

mdashmdash

93plusmn026

mdashmdash

mdash673plusmn233

9638plusmn12

86966plusmn416

M8

mdash104plusmn043

mdashmdash

14plusmn000

mdashmdash

mdash568plusmn202

6631plusmn12

15811plusmn18

4S2

mdash15plusmn000

7plusmn000

72plusmn03

mdashmdash

mdashmdash

9348plusmn30

8316plusmn283

91093plusmn455

S4121plusmn02

216plusmn10

2mdash

mdashmdash

mdashmdash

mdash9616plusmn31

8862plusmn301

6732

6plusmn231

International Scholarly Research Notices 7

isolated from rare ecosystems showed activity against non-mycobacterial pathogens whereas 72 of the isolates arefound to inhibit sensitive andor resistant strains of Mtuberculosis Similar to earlier reports [21 22] more numberof actinomycetes showed inhibited Gram positive bacteriacompared to Gram negative bacteria

Wide variation in the percentage of active isolates andin their activity spectra has been reported from differentecosystems [6 22 23] Eleven different patterns of activitieswere recorded against 11 test organismsThe key observationsmade in this analysis include the following (i) antagonisticisolates are distributed in all the ecosystems studied (ii) themaximum number of isolates (2962) is exclusively activeagainst M tuberculosis followed by 2037 of the isolatesinhibiting Gram positive and Gram negative bacteria andM tuberculosis The broad spectrum of activity detected insome of the Streptomyces isolates in this study could bedue to different antimicrobial compounds produced by theisolates each one with a species- or group-specific activity[22] andor to the presence of more than one compound withbroad spectrum of action like the antibiotic meroparamycinwhich is active against Gram positive bacteria yeasts andfilamentous fungi [24] among others These findings clearlyevidenced that antagonistic actinomycetes isolated from rareecosystems are potential sources for compounds showingantibacterial and antituberculous activity

Certain issues are associated with primary screening insolid medium and liquid culture based secondary screeningThere can be loss of bioactive metabolite production insecondary screening while using a mediumwhich is differentfrom the one used for primary screening Certain actino-mycete strains produce antibiotic compounds only in solidmedium but fail to produce the same in liquid medium [25ndash27] Moreover the whole organism based primary screeningmethods like cross streakcross spot methods are not suitablefor highly biohazardous organisms like M tuberculosis Toaddress all these issues agar plugs which contain the extracel-lular bioactive compounds were tested against nonmycobac-terial pathogens The crude extract from the same mediumwas tested against M tuberculosis Most of the secondarymetabolites including antibiotics are extracellular in natureand extracellular products of actinomycetes exhibit potentantimicrobial activities [28ndash31] Similar observations weremade in the present study

There are number of mycobacterial drug susceptibilityassays used for the screening of natural products describedover the period of time [32] Screening methods usingLJ or 7H11 agar require large amounts of crude extractsor purified compounds to be incorporated in the mediaIt also requires about three or more weeks of incubationto produce results Conventional testing using LJ slants isunsuitable for screening uncharacterised novel compoundswhose heat stability is unknown LRP assay used in thisstudy is a rapid liquid culture based and less laboriousmethod for high throughput screening of a large number ofcompounds for antimycobacterial activity [33] As a brothbased method LRP assay qualifies to be ideal for screeningsuch novel compounds The need for only small quantityof the extractscompounds to obtain results within 3 days

qualifies the test further Natural products from variousnatural sources like plants actinomycetes and fungi [16 3435] and synthetic compounds [36] had been screened forantitubercular activity by adopting LRP assay

Antibiotics isolated and characterised from strains thatshow broad spectrum activity can be used to treat variedmicrobial infections The availability of such drugs acrossthe counter and wide usage of the same may lead to theemergence and spread of drug resistance among the existingpathogens [37] Selection of antibiotics that are exclusivelyacting against M tuberculosis andor limited number ofnonmycobacterial pathogens may circumvent this problem

5 Conclusion

Findings of the present work concluded that less exploredecosystems investigated in this study are the potentialresource for bioactive actinomycetes However in this studyonly the crude extracts were evaluated and found to beactive againstMycobacterium tuberculosis and other nonmy-cobacterial pathogens In general crude extracts are complexmixture of compounds So additional research like bioassayguided fractionation and characterization will be needed tovalidate whether a single useful compound can be foundand it is also needed to determine the MIC and meaningfultoxicity and specificity studies In particular isolation andcharacterization of active molecule from the potential strainslike D25 CSA15 will pave the way for the developmentof promising antibiotics against M tuberculosis and othernonmycobacterial pathogens

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] BMukhopadhyay andN K Ganguly ldquoTuberculosis research inIndiardquo Current Science vol 105 no 5 pp 594ndash596 2013

[2] R S Kashyap A R Nayak A A Husain et al ldquoTuberculosis inIndia the continuing challengerdquo Current Science vol 105 no 5pp 597ndash606 2013

[3] A M Ginsberg ldquoDrugs in development for tuberculosisrdquoDrugs vol 70 no 17 pp 2201ndash2214 2010

[4] M V N de Souza ldquoPromising drugs against tuberculosisrdquoRecent Patents on Anti-Infective Drug Discovery vol 1 no 1 pp33ndash44 2006

[5] E J Ashforth C Fu X Liu et al ldquoBioprospecting for antitu-berculosis leads from microbial metabolitesrdquo Natural ProductReports vol 27 no 11 pp 1709ndash1719 2010

[6] M George A Anjumol G George and A A M HathaldquoDistribution and bioactive potential of soil actinomycetes fromdifferent ecological habitatsrdquo African Journal of MicrobiologicalResearch vol 6 no 10 pp 2265ndash2271 2012

[7] J Berdy ldquoThoughts and facts about antibiotics where we arenow and where we are headingrdquo Journal of Antibiotics vol 65no 8 pp 385ndash395 2012

8 International Scholarly Research Notices

[8] L A Maldonado W Fenical P R Jensen et al ldquoSalinis-pora arenicola gen nov sp nov and Salinispora tropica spnov obligate marine actinomycetes belonging to the familyMicromonosporaceaerdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 5 pp 1759ndash1766 2005

[9] W N Hozzein and M Goodfellow ldquoStreptomyces synnemato-formans sp nov a novel actinomycete isolated from a sanddune soil in Egyptrdquo International Journal of Systematic andEvolutionary Microbiology vol 57 no 9 pp 2009ndash2013 2007

[10] J Nachtigall A Kulik S Helaly et al ldquoAtacamycins A-C 22-membered antitumor macrolactones produced by Streptomycessp C38rdquo Journal of Antibiotics vol 64 no 12 pp 775ndash780 2011

[11] Q Wang F Song X Xiao et al ldquoAbyssomicins from theSouth China Sea deep-sea sediment verrucosispora sp naturalthioethermichael addition adducts as antitubercular prodrugsrdquoAngewandte ChemiemdashInternational Edition vol 52 no 4 pp1231ndash1234 2013

[12] D P Labeda and M C Shearer ldquoIsolation of actinomycetes forbiotechnological applicationsrdquo in Isolation of BiotechnologicalOrganisms from Nature D P Labeda Ed vol 19 pp 1ndash19McGraw-Hill New York NY USA 1990

[13] E B Shirling and D Gottileb ldquoMethods for characterizationof Streptomyces speciesrdquo International Journal of SystematicBacteriology vol 16 no 3 pp 313ndash340 1966

[14] R Balagurunathan M Radhakrishnan and S T Somasun-daram ldquoL-glutaminase producing actinomycetes from marinesediments-selective isolation semi quantitative assay and char-acterization of potential strainrdquo Australian Journal of Basic andApplied Sciences vol 4 no 5 pp 698ndash705 2010

[15] G P Eccleston P R Brooks and D I Kurtboke ldquoThe occur-rence of bioactive micromonosporae in aquatic habitats of theSunshine Coast in Australiardquo Marine Drugs vol 6 no 2 pp243ndash261 2008

[16] M Radhakrishnan S Suganya R Balagurunathan and VKumar ldquoPreliminary screening for antibacterial and antimy-cobacterial activity of actinomycetes from less explored ecosys-temsrdquoWorld Journal of Microbiology and Biotechnology vol 26no 3 pp 561ndash566 2010

[17] V Kumar S Balaji N S Gomathi et al ldquoPhage cocktail tocontrol the exponential growth of normal flora in processedsputum specimens grown overnight in liquid medium for rapidTB diagnosisrdquo Journal of Microbiological Methods vol 68 no 3pp 536ndash542 2007

[18] M S Butler M A Blaskovich and M A Cooper ldquoAntibioticsin the clinical pipeline in 2013rdquo Journal of Antibiotics vol 66no 10 pp 571ndash591 2013

[19] A L Demain and S Sanchez ldquoMicrobial drug discovery 80years of progressrdquo Journal of Antibiotics vol 62 no 1 pp 5ndash162009

[20] P A Jose and S R D Jebakumar ldquoUnexplored hypersalinehabitats are sources of novel actinomycetesrdquo Frontiers in Micro-biology vol 5 article 242 3 pages 2014

[21] M Radhakrishnan S Balaji and R Balagurunathan ldquoTher-motolerant actinomycetes from Himalayan mountainmdashantag-onistic potential characterization and identification of selectedstrainsrdquoMalaysian Applied Biology vol 36 pp 59ndash65 2007

[22] S P Salamoni M B Mann F S Campos A C Franco J CGermani and S T van der Sand ldquoPreliminary characterizationof some Streptomyces species isolated from a composting

process and their antimicrobial potentialrdquo World Journal ofMicrobiology and Biotechnology vol 26 no 10 pp 1847ndash18562010

[23] N Sahin and A Ugur ldquoInvestigation of the antimicrobialactivity of some Streptomycetes isolatesrdquo Turkish Journal ofBiology vol 27 pp 79ndash84 2003

[24] M Y El-Naggar S A El-Assar and S M Abdul-GawadldquoMeroparamycin production by newly isolated Streptomycessp strain MAR01 taxonomy fermentation purification andstructural elucidationrdquo Journal of Microbiology vol 44 no 4pp 432ndash438 2006

[25] S B Ilic S S Konstantinovic Z B Todorovic et al ldquoCharacter-ization and antimicrobial activity of the bioactivemetabolites instreptomycete isolatesrdquoMicrobiology vol 76 no 4 pp 421ndash4282007

[26] M Anibou A Chait A Zyad M Taourirt Y Ouhdouchand A Benherref ldquoActinomycetes from Moroccan habitatsisolation and screening for cytotoxic activitiesrdquoWorld Journal ofMicrobiology and Biotechnology vol 24 no 10 pp 2019ndash20252008

[27] S Radhika S Bharathi M Radhakrishnan and R Balagu-runathan ldquoBioprospecting of fresh water actinobacteria iso-lation characterization and antagonistic potential of selectedactinobacterialrdquo Journal of Pharmacy Research vol 4 pp 2584ndash2586 2011

[28] M Radhakrishnan D Saravanan R Balagurunathan and VKumar ldquoFungal bioproespecting from sundarban mangroveforest with special reference to antibacterial and antimycobac-terial activityrdquo International Journal of PharmTech Research vol3 no 2 pp 719ndash723 2011

[29] S K Augustine S P Bhavsar and B P Kapadnis ldquoA non-polyene antifungal antibiotic from Streptomyces albidoflavusPU23rdquo Journal of Biosciences vol 30 no 2 pp 201ndash211 2005

[30] MV Arasu VDuraipandiyan P Agastian and S IgnacimuthuldquoAntimicrobial activity of Streptomyces spp ERI-26 recoveredfrom Western Ghats of Tamil Nadurdquo Journal de MycologieMedicale vol 18 no 3 pp 147ndash153 2008

[31] D Mohanraj S Bharathi M Radhakrishnan and R Bal-agurunathan ldquoBioprospecting of actinobacteria from Yelagirihills with special reference to antibacterial activityrdquo Journal ofChemical and Pharmaceutical Research vol 3 no 3 pp 439ndash446 2011

[32] J G B Sanchez and V V Kouznetsov ldquoAntimycobacterialsusceptibility testing methods for natural products researchrdquoBrazilian Journal of Microbiology vol 41 no 2 pp 270ndash2772010

[33] W R Jacobs Jr R G Barletta R Udani et al ldquoRapid assessmentof drug susceptibilities ofMycobacterium tuberculosis by meansof luciferase reporter phagesrdquo Science vol 260 no 5109 pp819ndash822 1993

[34] S Prabuseenivasan and V Kumar ldquoAntimycobacterial activityof selected plant essential oilsrdquo in Proceedings of the NationalSeminar onMicrobial Biotechnology R Balagurunathan andMRadhakrishnan Eds p 206 Tamil Nadu India 2006

[35] A Molly J James C S Misra L D M Sahadevan TArunkumar and VThankamani ldquoAntimycobacterial activity ofthe plant extracts of Alstonia scholarisrdquo International Journal ofCurrent Pharmaceutical Research vol 4 pp 40ndash42 2012

International Scholarly Research Notices 9

[36] V Raparti T Chitre K Bothara et al ldquoNovel 4-(morpholin-4-yl)-N1015840-(arylidene)benzohydrazides synthesis antimycobac-terial activity and QSAR investigationsrdquo European Journal ofMedicinal Chemistry vol 44 no 10 pp 3954ndash3960 2009

[37] A S Coco M A Horst and A S Gambler ldquoTrends in broad-spectrum antibiotic prescribing for children with acute otitismedia in the United States 1998ndash2004rdquo BMC Pediatrics vol 9article 41 2009

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