Chiang Mai J. Sci. 2012; 39(3) 373

Chiang Mai J. Sci. 2012; 39(3) : 373-388http://it.science.cmu.ac.th/ejournal/Contributed Paper

Actinomycetes from Tropical Limestone CavesNanthavut Niyomvong [a], Wasu Pathom-aree [b], Arinthip Thamchaipenet [c, d] and KannikaDuangmal* [a,d][a] Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.[b] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.[c] Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.[d] Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, Chatuchak,

Bangkok 10900, Thailand.*Author for correspondence; e-mail: fscikkd@ku.ac.th

Received: 4 November 2011Accepted: 28 May 2012

ABSTRACT The cultivable actinomycetes from tropical limestone caverns at Khao No-Khao

Kaeo karst, Thailand, were investigated. In total, 276 actinomycetes were isolated from 10 soilsamples using different selective isolation procedures. The predominant actinomycetes fromall samples were members of the genus Streptomyces (94.6%) as they containedLL-diaminopimelic acid (A2pm) in their whole cell hydrolysates. The remaining 15 isolates(5.4%), rich in meso-A2pm in whole cell hydrolysates were characterized by 16S rRNA genesequencing. They were shown to represent strains from 8 different genera namely Actinomadura,Actinoplanes, Gordonia, Microbispora, Micromonospora, Nocardia, Nonomuraea and Saccharopolyspora.A good agreement was found between the results based on morphology, presence ofmeso-A2pm and 16S rRNA gene phylogeny. Microwave treatment of soil samples was foundto promote the isolation of rare actinomycetes. Successful isolation of members of thegenus Actinomadura and Saccharopolyspora from caves was reported for the first time. This studyconfirms significant diversity of cultivable actinomycetes from neglected habitats such aslimestone caves in Thailand.

Keywords: actinomycetes, limestone cave, selective isolation, 16S rRNA gene

1. INTRODUCTION Members of the actinomycetes,

especially streptomycetes, have long beenrecognized as prolific producers of usefulbioactive compounds [1]. The search for novelantibiotics and other bioactive microbialmetabolites is important for the fight againstnew and emerging pathogens [2, 3, 4].Isolation of actinomycetes from uniquenatural habitat is of interest to avoid re-

isolation of strains that produce knownbioactive metabolites and usually lead tohighly diversified actinomycete communities.There is an increasing awareness of thepotential value of rare actinomycetes (non-streptomycete actinomycetes) as sources ofnew metabolites. Neglected habitats areproving to be a particularly good source ofnovel actinomycetes that produce new

374 Chiang Mai J. Sci. 2012; 39(3)

bioactive compounds, as exemplified bystudies on actinomycetes isolated fromdesert soil [5, 6], lichens [7] and marinehabitats [8, 9, 10]. Therefore, actinomyceteshave been intensively surveyed in differentecological area and underexplored environments in various parts of the world [11, 12,13].

Cave is one of habitat attractingmicrobiologists’ interest during the pastdecade [14, 15] with a combination ofunique conditions including high humidity,relatively low and stable temperature [16].Many microbiologists believe that microbescollected from pristine sites that wereunexplored or rarely visited by humanswere likely to be novel taxa or strainswhich produce unique beneficial chemicalcompounds. Limestone karsts are sedimentaryrocks which consist primarily of calciumcarbonate. In Southeast Asia, karsts coveran area of around 400,000 square kilometers,with geological ages ranging from theCambrian to the Quaternary [17]. The highspecies diversity on karsts made from avarious ecological afforded by complexterrains, fissured cliffs and caves, and variableclimatic conditions [18].

In an early work, great diversity ofcultivable actinomycetes was found in theAltamira cave, Cantabria, Spain [14, 16],Tito Bustillo, Spain [16, 19] and Grotta deiCervi, Italy [20]. Many novel species havebeen discovered from cave environments,such as Actinocorallia cavernae [21], Agromycessubbeticus [22], Amycolatopsis halotolerans [23],Nocardia altamirensis [24], Nocardia speluncae [25],including novel genera such as Knoellia [26]and Hoyosella [27]. However, the studies ofactinomycetes diversity in caves of Thailandare still rare [28, 29].

The primary aim of this present studywas to determine the diversity of cultivablerare actinomycetes from limestone caves in

Khao No-Khao Kaeo karst area ofThailand. The efficiency of various selectiveisolation procedures employed was alsodiscussed.

2. MATERIALS AND METHODS2.1 Samples Collection

Ten soil samples were collected from4 unnamed caves located in Khao No-KhaoKaeo karst, Nakhon Sawan province,Thailand (15° 56' N and 99° 52' E) inMarch 2007. The upper 5 cm layer of cavesoils were collected in sterile plastic bagsand transported to the laboratory. Soilswere air dried at room temperature for7-10 days before grind with mortarand sieved. The pH of each soil samplewas determined following the proceduredescribed by Reed and Cummings [30] usinga glass electrode pH meter and expressedas an average of triplicate readings.

2.2 Selective Isolation and Enumerationof Actinomycetes

One gram of dried soil was asepticallydiluted in 9 ml of sterile 0.85% NaClsolution. The resultant 10-1 soil suspensionwas agitated on a shaker at 150 rpm at roomtemperature and subjected to the followingpretreatment regimes unless otherwiseindicated:

a) Wet heat: Soil suspension was heatedin a water bath at 50°C for 6 min and cooledat room temperature [31].

b) Dry heat: One gram of dried soilsample was heated at 120°C in hot air ovenfor 1 h [32].

c) Dry heat and phenol: One gram ofheated soil sample (at 120°C for 1 h) wasdiluted in 9 ml of sterile 0.85% NaClsolution. The resultant soil suspension (1 ml)was added to 9 ml of 1.5% phenol solution[32]. The mixture was then incubated atroom temperature for 30 min.

Chiang Mai J. Sci. 2012; 39(3) 375

d) Electromagnetic wave: Thispretreatment was adapted from Bulina etal. [33]. Soil suspension was irradiated withthe microwave oven at frequency of 2460MHz and a power setting at 100 watt for45 sec.

e) No treatment: Soil suspension wasdiluted without any prior pretreatment.

The pretreated samples were furtherdiluted down to 10-4 using 0.85% NaClsolution. One hundred microliters of anappropriate dilution were spread over thesurface of starch casein (SC) agar plates [34]for pretreatment regime a-d and Humicacid-Vitamin (HV) agar [35] for regime dand e. All media were supplemented withnalidixic acid (25 μg/ml) and ketoconazole(100 μg/ml) to inhibit the growth ofbacteria and fungi, respectively. The pH ofall media was adjusted to 7.

Numbers of presumptive actinomycetecolonies on each plate were counted afterincubation of the inoculated plates at 30°Cfor 2 weeks and expressed as mean colonyforming units (c.f.u.) per gram dry weight soilof three plates. The putative actinomycetecolonies were randomly selected andpurified by streak plate method on GYEagar [1% glucose (w/v), 1% yeast extract(w/v) and 1.5% agar (w/v)] until pureculture was obtained. Purified isolates weremaintained on GYE and oatmeal agar (ISP3) slopes [36]. Stock cultures were preparedby transferring mycelium and spores ofpurified isolates into 20% glycerol solutionand kept at -20°C for long term preservation[37].

2.3 Colour Grouping and Characterisa-tion of the Isolates

Putative actinomycete isolates wereinoculated onto oatmeal agar [36] andpeptone-yeast extract-iron agar plates (ISP 7)[36] and incubated at 30°C for 14 and 4 days,

respectively. The oatmeal agar plates wereexamined for aerial spore mass colour,substrate mycelium colour and diffusiblepigments using NBS/IBCC color chart[38]. The peptone-yeast extract-iron agarplates were recorded for melanin pigmentproduction.

The presence of isomer of diaminopimelic acid (A2pm) in whole cell hydrolysateswere determined according to the methodsdescribed by Becker et al. [39] and Hasegawaet al. [40] using biomass obtained from aculture grown on ISP 2 agar [36]. The sporechain morphology of selected LL-A2pmcontaining isolates grown on oatmeal agarplates was examined by light microscopy.Spore-chain morphology and sporeornamentation of representative meso-A2pm containing strains were observedusing both light microscopy and scanningelectron microscopy (JEOL JSM 5600 LV).

2.4 16S rRNA gene Amplification andAnalysis

Total genomic DNA from 15 isolatesthat contained meso-A2pm was extracted usingstandard method [41]. The 16S rRNA genewas amplified by PCR with the universalbacterial primer 27f (5’AGAGTTTGATCMTGGCTCAG 3') and 1525r (5' AAGGAGGTGWTCCARCC 3' [42]. Amplificationwere performed using GeneAmp PCRSystem 9700, according to the followingprofile: initial denaturation at 95°C for3 min followed by 30 cycles of 95°C for1 min, 55°C for 1 min and 72°C for 1 min;these cycles were followed by a final extensionat 72°C for 5 min. The PCR products werepurified and sequenced (Macrogen, Korea)using universal primers [42]. The resultant16S rRNA gene sequences were alignedagainst corresponding sequences of the typestrains of actinomycete species, retrievedfrom EMBL/GenBank database, using the

376 Chiang Mai J. Sci. 2012; 39(3)

CLUSTAL_X [43] and PHYDIT programs(http://plaza.snu.ac.kr/~jchun/phydit/).An unrooted phylogenetic tree was inferredusing the neighbour-joining and maximum-parsimony tree-making algorithms [44]. Thetree topology was evaluated by bootstrapanalyses of the neighbour-joining databased on 1000 resamplings. All analyses werecarried out using TREECON software [45].

3. RESULTS AND DISCUSSION3.1 Characteristics of Cave and Cave Soils

Khao No-Khao Kaeo karst is an areawhich formed 440 million years ago, situated282 m above medium sea level on the westof Banpotpisai district, Nakhon SawanProvince, Thailand. All caves are not touristattraction sites and free from humanactivities. Samples were light brown incolour with bat guano in sample A1 and A2;fine soil with stone in sample B1-B3, sandysoil in sample C1-C3 and fine soil withslug shell in sample D1 and D2. The average

pH of all soil samples was between 5.5 and7.6 (Table 1). Most samples were weak acid(5.5 - 6.5), except sample B2, B3, C1 andC3 which were neutral (6.5 - 7.6). The slightlyacidic pH may be due to animal secretionsuch as bat guano. Mineralization processesof such animal inputs resulted in theproduction of nitric acid and sulphuric acidhave been reported [46]. The alkali pH of8.29 was reported from soil collected fromPhanangkoi cave in northern Thailand [28].

3.2 Selective Isolation and Enumerationof Actinomycetes

A total of 276 actinomycete strains wereisolated from 10 cave soil samples withthe use of various methods. In general,actinomycetes were isolated from allsamples. The highest number of isolateswas from sample A2 (78 isolates) with thelowest numbers from sample C3 (3 isolates)(Table 1). The total actinomycete counts wereranged from 0 - 8.5 × 104 c.f.u. per gram, a

Table 1. Soil pH and total actinomycetes counts in soil samples from different pretreatmentmethods (c.f.u./g dry soil).

1.2 × 103

5.8 × 104

3.0 × 102

7.2 × 104

8.5 × 104

1.7 × 104

4.0 × 102

8.5 × 102

1.4 × 103

9.5 × 102

b

c

d

Sample pH a) Wet heat(SC)

b) Dry heat(SC)

c) Dryheat andphenol(SC)

d) Electromagnetic wave

(SC) (HV)e) Notreatment(HV)

Totalnumberofisolates

A1A2B1B2B3C1C2C3D1D2

6.46.25.87.67.56.95.56.85.76.5

3.6 × 103

1.2 × 103

ND2.0 × 103

1.6 × 103

5.0 × 103

NDND1.0 × 102

ND

ND3.0 × 102

ND4.1 × 103

1.3 × 103

1.6 × 103

NDNDNDND

1.2 × 103

2.3 × 103

4.0 × 102

8.4 × 103

5.2 × 104

6.4 × 104

4.0 × 102

4.0 × 103

1.0 × 102

3.4 × 103

6.0 × 103

1.2 × 104

3.0 × 102

7.2 × 104

7.7 × 104

1.4 × 104

2.0 × 102

3.9 × 104

8.0 × 102

6.0 × 103

1.2 × 103

2.8 × 103

2.1 × 103

3.5 × 103

6.0 × 104

2.0 × 104

2.0 × 103

7.0 × 103

1.2 × 103

3.0 × 103

57847662346344

276

N.D. - Not detected, no any colonies on the agar plates which incubated at 30°C for up to4 weeks.

Chiang Mai J. Sci. 2012; 39(3) 377

slightly higher count than the earliestreport in Thailand by Laorpaksa et al. [47](4.4 - 7.0 × 103 c.f.u. per gram), which maybe a result from the use of selectivepretreatments in this study. Several selectiveisolation procedures were employed inorder to isolate actinomycetes from cavesoil samples collected from Khao No-KhaoKaeo limestone karst. It has been shownpreviously that the treatment of samplesmay lead to growth inhibition of somegenera and stimulated germination of othergenera [11, 33]. Both moist and dry-heatpretreatment regimens have long been usedto select for various actinomycete groups[48]. Moist-heat by heating the soil dilutionin a water bath at 45, 50 or 70°C has beenused to eliminate the spreading of bacterialcolonies on actinomycete isolation plates.They are extremely useful standardprocedures for routine isolation [48]. Inthis experiment, the general selectiveisolation procedure for actinomycetes byspreading on starch casein agar and humicacid vitamin agar showed the numbers ofactinomycete isolates in each cave soilsample ranged from 3.0 × 102 to 8.5 × 104

c.f.u. per gram dry soil sample (Table 1).Dry heat treatment either at 120°C for 1hr (procedure b) or dry heat at 120°C for1 hr and 1.5% phenol (procedure c) showedthe most specific and effectivein reducing the number of undesirablebacteria. However, these treatments alsoresulted in a reduction in numbers of viableactinomycetes recovered. For example, therewas no colony appeared on the agar platesfrom sample B1, C2, C3 and D2 whenprocedure b was used as pretreatment. Similarresult was also observed with pretreatmentprocedure c for sample A1, B1, C2, D1 andD2, even after the plates were incubated at30°C for up to four weeks.

The efficiency of microwave treatment

was evident in this study as most non-streptomycetes actinomycetes were obtainedusing this regime. Pretreatment of soil sampleswith 2460 MHz microwaves at 80 W for30 sec resulted in a significant increase(threefold on average) in the proportion ofrare actinomycetes isolated [33]. The analysisof whole cell hydrolysates revealed that mostnon-streptomycetes (meso-A2pm containingstrains) were recovered from isolationassociated with procedure d, which usedmicrowave irradiation at 2460 MHz, settingat 100 watt for 45 sec, before spreading onHV agar. Twelve isolates of actinomycetes,which are rich in meso-A2pm, were recoveredfrom this procedure. The remaining 4 non-streptomycete actinomycetes were alsoisolated from HV agar seeded with un-treatedsamples. HV agar was reported to supportgood sporulation of rare actinomycetes whichoffers an advantage for rapid morphologicalidentification [35, 49] with long-workingdistance objective lens. This media wasdesigned for isolation and enumeration ofsoil actinomycetes [35]. It has been usedsuccessfully in recovery of various rare andnovel actinomycete taxa from environments[28, 29, 50, 51, 52].

3.3 Colour Grouping and Chemotaxono-mic Characterization

A total of 276 actinomycetes withdifferent colony types were recovered fromcave soil samples with the use of variousmethods. Chemotaxonomic analysis of wholecell hydrolysates revealed the presence ofthe LL-A2pm isomer in 261 isolates (94.6%)and meso-A2pm isomer in 15 isolates (5.4%).The LL-A2pm containing isolates formedbranched substrate mycelia with aerialmycelia that formed chains of spores. Theywere assigned to the genus Streptomyces [53].These isolates could be assigned to 10 multi-membered colour groups based on

378 Chiang Mai J. Sci. 2012; 39(3)

observation of spore color on oatmeal agar.The largest taxon, dominated by grey sporecolour group, encompassed 39.5%, followingby white (33.8%) and brown (16.1%) sporecolour group. The assignment of these isolatesto various colour groups indicated highdegree of diversity. Colour grouping hasbeen shown to provide a valuable index oftaxonomic diversity of streptomycetes innatural habitats [6, 54]. On the basis of theirmorphological characteristics and analysis ofdiaminopimelic acid isomer, all cave soilsamples were dominated by representativeof the genus Streptomyces, a result in goodagreement with those from earlier surveys[20, 29, 55, 56, 57].

The 15 remaining meso-A2pm containingactinomycetes, assigned to non-streptomyceteactinomycetes or rare actinomycetes weresubjected to 16S rRNA gene sequencing.Morphological examined by light microscopyand scanning electron microscopy (Figure 1)was also supported the results receivedfrom the detection of diaminopimelic acidtype in cell hydrolysates. Cylindrical sporangiawere well developed on the substrate

mycelium of Actinoplanes sp. NN272 (Figure1a). Actinomadura sp. NN242 formedspiral spore chains with smooth sporeornamentation (Figure 1b). Gordonia sp.NN234 grew as short rods that occur inshort chains (Figure 1c). Nocardia sp.NN256 formed substrate mycelium thatfragments into rod-shaped (Figure 1d). Itis evident that these strains presentedmorphological properties typical of theirassigned genera.

3.4 Molecular Taxonomic of meso-A2pmContaining Actinomycetes

All 16S rRNA gene sequencing studieswere focused on non-streptomycetes isolateswhich were preliminary identified on thebasis of morphological characteristics andchemotaxonomic analysis. Fifteen isolateswhich contained meso-A2pm in whole cellhydrolysates were selected for moleculartaxonomical studied for nearly completed16S rRNA gene sequence analysis. The resultingsequences, 1366 nt to 1428 nt, depending onthe isolate (except for strain NN274, 977 nt)were determined and compared with the

Figure 1.Scanning electron micrographs of spore ornamentation of representative isolatesfrom Khao No-Khao Kaeo limestone karst. (a) Actinoplanes sp. NN272 grown on ISP 4 [36]for 21 days; (b) Actinomadura sp. NN242 grown on ISP 3 for 21 days; (c) Gordonia sp. NN234grown on GYE agar for 5 days; (d) Nocardia sp. NN256 grown on ISP 2 for 7 days. Barintervals is 1 μm.

Chiang Mai J. Sci. 2012; 39(3) 379

sequences available in the GenBankdatabases using BLASTN. The 16S rRNAgene sequences revealed that they weremembers of the following 8 genera:Actinomadura (2 isolates, familyThermomonosporaceae), Actinoplanes (2isolates, family Micromonosporaceae),Gordonia (2 isolates, family Nocardiaceae),Microbispora (2 isolates, familyStreptosporangiaceae), Micromonospora (3isolates, family Micromonosporaceae),Nocardia (1 isolate, family Nocardiaceae),Nonomuraea (2 isolates, familyStreptosporangiaceae) and Saccharopolyspora(1 isolate, family Pseudonocardiaceae), with97.7 - 99.8% similarity to their nearestneighboring strains. The percentages of 16SrRNA gene sequence identity of these isolatesto the closest type strain are presented inTable 2. Phylogenetic tree constructed basedon nearly complete 16S rRNA genesequences confirmed their affiliation to 5actinomycete families Micromonosporaceae,

Nocardiaceae, Pseudonocardiaceae,Streptosporangiaceae andThermomonosporaceae and supported byhigh bootstrap values (Figure 2).

Two Actinomadura strains NN236and NN242 were isolated from sample B2and D2, respectively. Both strains weremost closely related to A. napierensissharing a 16S rRNA gene similarity of 99.03and 98.96%, respectively. They clusteredwith A. meyerae in the 16S rRNA genetree but formed well separated subcladesupport by 99% bootstrap value (Figure2). Members of genus Actinomadura werewidely distributed in soils but have notbeen reported from the caves before.However, other members of familyThermomonosporaceae, genera Actinocoralliaand Spirillospora were reported from cavesin Northern Thailand [28, 29]. In addition,a new species of Actinocorallia cavernaeisolated from cave in Korea has beendescribed [21].

Table 2. Analysis of meso-A2pm containing isolates based on nearly completed sequence ofthe 16S rRNA gene with the most identity to the closest strain from GenBank database.

Genus Strain Source(treatment)

Closest type strain Similarity (%)

Actinomadura

Actinoplanes

Gordonia

Microbispora

Micromonospora

NocardiaNonomureae

Saccharopolyspora

NN236NN242NN265NN272NN234NN279NN274NN277NN275NN264NN271NN256NN273NN278NN257

B2 (e, HV)D2 (e, HV)A2 (d, HV)A2 (e, HV)C2 (e, HV)C2 (d, HV)A1 (d, HV)A1 (d, HV)D2 (d, HV)D2 (d, HV)A2 (d, HV)B3 (d, HV)B2 (d, HV)B2 (d, HV)B3 (d, HV)

Actinomadura napierensisActinomadura napierensisActinoplanes xinjiangensisActinoplanes xinjiangensisGordonia terraeGordonia terraeMicrobispora mesophilaMicrobispora mesophilaMicromonospora siamensisMicromonospora siamensisMicromonospora chalceaNocardia asteroidesNonomuraea roseoviolaceaNonomuraea candidaSaccharopolyspora phatthalungensis

99.0398.9698.2798.1499.8099.7997.7497.9099.098.9799.5799.0898.4999.2698.57

380 Chiang Mai J. Sci. 2012; 39(3)

Figure 2. Neighbor-joining tree showing the position of various genera of the selectedactinomycetes isolated from the Khao No-Khao Kaeo limestone karst amongst its phylogeneticneighbors based on almost complete 16S rRNA gene sequences. Asterisks indicate branchesof the tree that was also found using maximum-parsimony tree making algorithm. Numbersat the nodes show the percentage bootstrap values. The bar represents 0.1 substitutions pernucleotide position.

Chiang Mai J. Sci. 2012; 39(3) 381

Strains NN265 and NN272, werefound to present only in sample A2. A 16SrRNA gene tree showed that they formeda separate line of descent from their nearestActinoplanes neighbours (Figure 2), therelationship strongly supported by 100%bootstrap value. Sequence similaritycalculated based on neighbour joininganalysis indicated that they were closest toA. xinjiangensis, sharing 98.27 and 98.14%16S rRNA gene similarity values,respectively. These observations suggestedthat these 2 strains may representa new species. Representatives from othergenera in family Micromonosporacea previouslyreported from caves were Dactylosporangium[55] and Micromonospora [29].

The 3 Micromonospora strains, anothermember of family Micromonosporaceae, werealso isolated from sample A2 (strain NN271)and sample D2 (strains NN264 and NN275).Strain NN271 was closely related to M. chalceawith 99.57% similarity. Strains NN264 andNN275, both shared the highest 16S rRNAgene similarity values of 98.97% and 99%with M. siamensis, respectively. Both strainswere likely to be a M. siamensis consideringtheir high 16S rRNA gene similarity values.M. siamensis was recently proposed for strainoriginated from Thai peat swamp forest ofThailand [58]. The finding of this species incave soil suggested it is widely distributedin terrestrial habitats. The isolation ofMicromonospora strains were also reportedfrom caves in northern Thailand [29] andKorea [55]. In addition, one new genus infamily Micromonosporaceae was proposed asCatelliglobosispora koreensis for strain isolatedfrom gold mine cave in Korea [59, 60].

Only one Nocardia strain NN256 wasisolated from sample B3. It was clusteredtogether with Nocardia asteroides andN. neocaledoniensis with 99.08 and 98.8% levelof 16S rRNA gene similarity, respectively.

Another member of the family Nocardiaceae,is genus Gordonia represented by strains NN234and NN279. Both were isolated from sampleC2. They were located adjacent to G. terraein the phylogenetic tree and showed highestlevel of similarity of 99.8% (Figure 2).Members of the family Nocardiaceae arecommon and widely distributed in aquaticand terrestrial environments; they areprobably involved in the turnover of organicmatter in natural habitats.

Family Streptosporangiaceae wasrepresented by members of the genusMicrobispora and Nonomuraea. StrainsNN274 and NN277, isolated from sampleA1, fell within the evolutionary radiationoccupied by genus Microbispora (Figure 2).They are most closely related to M.mesophila with relatively low 16S rRNAsimilarity values of 97.3% and 97.6%,respectively. The highest 16S rDNAsimilarity between the Microbispora typestrains was with M. corallina NBRC 16416T

and M. siamensis DMKUA 245T. Althoughthese organisms have a relatively highsequence similarity (98.4%) they have a lowDNA-DNA relatedness value [61], a valuewell below the recommended cut-off pointfor the delineation of bacterial species [62].Therefore, strains NN274 and NN277 maymerit recognition as new validly describedMicrobispora species subjected to polyphasictaxonomic characterization.

Two Nonomuraea strains NN273 andNN278 were isolated from sample B2.They were closely related to N. roseoviolaceaand N. candida, respectively. Strain NN273shared the highest 16S rRNA gene similarityof 98.4% with N. roseoviolacea whereas thoseof strain NN278 and N. candida were 99.49%.Members of this genus were also found ina recent study by Nakaew et al. [29] includinga hitherto unknown subline. Of note is thatgenus Nonomuraea so far has been reported

382 Chiang Mai J. Sci. 2012; 39(3)

only from Thai caves. No previous studiesreported the presence of members of thisparticular genus from other caves either byculture dependent or culture independentapproaches. It is still unclear about thenature of its distribution and function incave habitats.

Strain NN257 was identified as memberof the genus Saccharopolyspora with S.phatthalungensis as its nearest neighboursharing a 16S rRNA similarity value of 98.57%.It formed a separated subclade with itsnearest neighbours in the 16S rRNA gene tree(Figure 2). Members of genus Saccharopolysporawere mostly isolated from soil. Recently,new species isolated from lake sedimentand marine sponge were also reported.Members of genus Saccharopolyspora havepreviously only been reported from Altamiracave, Spain by culture independent surveyusing DGGE analysis. This is the firsttime that cultured representative of genusSaccharopolyspora was isolated from caves.Members of this genus are of particularinterest as they are known producers ofindustrially important antibiotics such as themacrolide antibiotic erythromycin producedby Saccharopolyspora erythraea. Worldwideannual sales of erythromycin and itsderivatives are worth billions of dollars [63].Other family Pseudonocardiaceae membersincluded Amycolatopsis [16, 20]; Pseudonocardia[29] and Saccharomonospora [55] were previouslyreported from caves. Indeed, 2 new speciesof Amycolatopsis, A. halotolerans and A. jejuensis[23] and one new species of Pseudonocardia,P. kongjuensis [64] were also described.

Generally, a 16S rRNA gene similarityof below 97% between a newly isolatedstrain and validly described species warrantrecognition as a new species [65]. It has beenshown that a type strain of actinomyceteswith almost identical 16S rRNA genesequences have DNA-DNA relatedness

values well below the 70% cut-off pointrecommended by Wayne et al. [62] for thedelineation of strains that belong to thesame genomic species. For example, the typestrains of Nonomuraea dietzia and N. roseolahave identical 16S rRNA gene sequencebut showed less than 31% of DNA-DNArelatedness value [66]. The 16S rRNAsequence data show that several strainsisolated from this study belong to putativelynew species as they are well separated fromtheir known nearest phylogenetic neighbours(Figure 2). Though detailed polyphasictaxonomic characterization of these strainsare required to clarify their status.

Early works on cultivable caveactinomycetes were limited to only caveslocated in Italy and Spain. Soil samplescollected from Altamira [14, 16], Tito Bustilloand Llon n caves in Spain [16, 19] andGrotta dei Cervi, Italy [20] resulted in theisolation of 18 genera in 12 families.Streptomyces were dominated in allcaves. Rare genera such as Amycolatopsis,Microbacterium, Nocardia and Rhodococcuswere also widely distributed in these caves.In Asia, there were only reports from China,Korea and Thailand.

A recent study by Nakaew et al. [29]also reported the isolation of severalrare actinomycete genera (Actinocorallia,Catellatospora, Microbispora, Micromonospora,Nonomuraea, Pseudonocardia, Saccharothrixand Spirillospora) from Phatup Cave ForestPark and Phanangkhoi cave in northernThailand, many of which were also foundin the present study. However, Phatup andPhanangkhoi caves are situated in a touristattraction area which is open to public access.The impact of human introduction maycontribute to the diversity of actinomycetesfound from the site. Indeed, a correlationbetween the number of visitors and diversityof bacteria has been reported, the higher

Chiang Mai J. Sci. 2012; 39(3) 383

the number of visitors the higher the diversityof isolated strains, as exemplified by the dataobtained from Altamira, Tito Bustillo andLlon n caves [16]. Caves with restricted visitsin northern Spain (Llon n and La Garmacaves) also yielded lower diversity [19].Nevertheless, the results from Nakaew et al.[29] and our study extended the range ofcultivable actinomycetes from Thai caves to14 genera including some that never beenrecovered from cave habitats before such asNonomuraea and Saccharopolyspora. Thesefindings strongly suggested that actinomycetesare an indigenous population of microbialcommunity inhabited in caves. The notionthat is well supported by data from cultureindependent studies.

In conclusion, large numbers of actinomycetes were isolated from Khao No-KhaoKaeo limestone karst using various establishedisolation procedures. All samples weredominated by Streptomyces species. However,diverse rare genera were also present.HV agar supplemented with nalidixic acidand ketoconazole in combination withmicrowave irradiation pretreatment of soilsamples has been demonstrated to permitthe recovery of rare actinomycetes fromlimestone cave soils compared with otherpretreatments. A 16S rRNA gene sequencesanalysis suggested that some of the isolatesmay represent novel species.

At the time of writing, 4 new genera and20 new species of actinomycetes from cavesrepresenting 11 families have been describedsince 1999 (gathered from InternationalJournal of Systematic and EvolutionaryMicrobiology; [67]). However, the resultsfrom previous culture independent studiesrevealed a greater diversity than conventionalcultural based studies [57, 68, 69, 70, 71,72]. An availability of pure cultures ofsuch diversity is important to better ourunderstanding on the physiology of these

bacteria and their functions in cave habitats.The discrepancy of actinobacterial diversitypreviously reported by culture dependentand culture independent studies includingour results highlighted an urgently need forimproved selective isolation procedures toassess the true diversity of actinomycetes incaves for bioprospecting. The presentfindings not only provide further evidencethat a variety of actinomycetes are commonin caves but show implications of thisdiversity for biotechnological exploitationas actinomycetes are well known as themost prolific producer of bioactivecompounds [1, 3, 73].

ACKNOWLEDGEMENTSThis work was supported by the Higher

Education Research Promotion and NationalResearch University Project of Thailand,Office of the Higher Education Commissionand Kasetsart University Research andDevelopment Institute (KURDI).

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