Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

ORIGINAL ARTICLE

Isolation, identification and antimicrobial activities of associated with fire ant, Solenopsis geminata

Kittitad Rordkhrora, Kawinnat Buaruanga, Paranee Sripreechasakb, Somboon Tanasupawatc, Wongsakorn Phongsopitanuna*

a Department of Biology, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand, 10240 b Department of Biotechnology, Faculty of Science, Burapha University, Chonburi, Thailand, 20131. c Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand, 10330.

*Corresponding author: [email protected], [email protected]

Received: 31 January 2018 / Accepted: 27 March 2018 / Published online: 30 April 2018

Abstract. Actinobacteria, Gram-stain positive with high decade, there are several novel compounds G+C content , have been well known as the isolated from actinobacteria associated with antibiotic producer. To find out the new habitat for searching new actinobacteria, we isolated the social insects including amycomycins A-B actinobacteria from the fire ant, Solenopsis geminata, (Guo et al. 2012), pseudonocardones A-C collected in Thailand. Totally, 6 actinobacteria were (Carr et al. 2012a), fasamycins C-E (Qin et al. isolated and identified using 16S rRNA gene, as 2017), formicamycins A-M (Qin et al. 2017), (2 isolates) and Nocardia (4 isolates) macrotermycins A-D (Beemelmanns et al. species. Both Streptomyces isolates showed 7 antimicrobial activities against tested microorganisms 2017) , microtermolides A-B(Carr et al. used in this study. Moreover, Nocardia isolate SE2 2012b), sceliphorolactam (Oh et al. 2011), and showed low 16S rRNA gene similarity to those known termisoflavones A-C (Kang et al. 2016). actinobacterial species and represented the undescribed In this study, the actinobacteria from the species. fire ant, Solenopsis geminata, collected in Thailand were isolated and the antimicrobial Keywords: Actinobacteria, Fire ant, Solenopsis geminata, Antimicrobial activities, Diversity. activities from the isolates were screened. We have attempted to find a novel source for 1. Introduction isolating novel actinobacteria to solve the re- isolation strain problem The new habitat to Microorganisms, especially Actinobac- discoveri the new actinobacterial strains are teria, Gram-stain positive with high% of G+C expected. content bacteria, are a primary resource of critical bioactive compounds. However, it has 2. Materials and Methods recently become challenging to find the novel compounds because of the re-isolation of the 2.1 Sample collection and Isolation of antibiotic-producing actinobacteria (Matsu- actinobacteria moto and Takahashi 2017). The ant samples, Solenopsis geminata, Insect-bacterial symbioses have been were collected in Chonburi Province, well known since the discovery of the Thailand. Individual ants were collected in symbiosis association between fungus- killing jar saturated with acetone. Then, ant growing ants and Actinobacteria (Currie et al. samples were washed with sterile water and 1999). The insects, especially social insects, further used for actinobacterial isolation. are often used the antibiotic-producing Actinobacteria were isolated from ants using bacterial symbionts to protect their nests standard serial dilution methods. Briefly, five (Beemelmanns et al. 2016). Consequently, ants were ground using a pestle. Then, 0.5 ml these social insects become promising sources of sterile distilled water was added to the for the natural product discovery. In the past sample and vortex for 1 minute. The sample 1

Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

solution was spread on humic acid vitamin ISP2 agar plates and incubated at 30oC for 14 agar (HV agar) supplemented with nalidixic days. Then, the tested microorganisms, (50 g ml-1) acid and cycloheximide (25 g including Bacillus subtilis ATCC 6633, ml-1) (Hayakawa and Nonomura 1987) and Kocuria rhizophila ATCC 4341, Staphylo- incubated at 30oC for 14 days. The coccus aureus ATCC 25923, Pseudomonas actinobacterial colonies were collected and aeruginosa ATCC 27853, Escherichia coli purified on ISP2 agar (Shirling and Gottlieb ATCC 25922 and Candida albicans ATCC 1966). The pure cultures were preserved in 10231, were inoculated on the plates by a glycerol solution (15% w/v) at -20 oC. single streak with 90o angles to the 2.2 Characterization of actinobacteria actinobacteria and incubated at 37 oC for 24 2.2.1 Cultural and Morphological hour. Finally, the inhibition area was recorded. characteristics Cultural characteristics of the isolates 3. Results were determined on the culture gown ISP2 agar media at 30 oC for 14 days. The Six actinobacteria were isolated from fire morphology of the isolates was observed using ant samples. Based on morphology and 16S light microscopy (Shirling and Gottlieb 1966). rRNA gene analysis, the isolates could be 2.2.2 16S rRNA gene and phylogenetic clearly classified into two groups. analysis Group 1 including SE1, SE2, SE7, and SE13 DNA was extracted from the showed yellowish white to pale orange yellow actinobacterial mycelia, obtained from the color on aerial mycelia. The morphological culture grown in yeast-dextrose broth at 30 oC observation revealed that the substrate mycelia for 4-7 days, using the method described by of these isolates were fragmented (Table 1). Tamaoka (1944). The 16S rRNA gene ampli- The BLAST search of 16S rRNA gene showed fication was carried out using primers 20F (5′- that isolates SE1, SE7 and SE13 were closely GAGTTTGATCCTGGCTCAG-3′) and 1500R related to Nocardia thailandica NBRC (5′-GTTACCTTGTTACGACTT-3′) 100428T with similarity of 99.86, 99.93% and (Suriyachadkun et al. 2009). PCR products 100%, respectively. Meanwhile, isolates SE2 were purified using Gel/PCR kit (Geneaid). was closely related to Nocardia pseudo- Nucleotide sequencing of PCR products was brasiliensis NBRC 108224T with similarity of carried out using universal primers (Lane 98.00%. Therefore, the member in this group 1994) (Macrogen, Seoul, Korea). BLASTN was identified as Nocardia (Table 2). analysis of the 16S rRNA sequences was Group II including SE4 and SE11. Both performed using EzBioCloud server (https:// isolates produced long aerial mycelia. The www.ezbiocloud.net) (Yoon et al. 2017). The spiral spore chain was observed on SE11 but sequences of all actinobacterial isolates were not SE4. Based on cultural characteristic on aligned with selected sequences obtained from ISP2 agar, SE4 produced yellowish white the GenBank/EMBLDDBJ database using aerial mass and dark grayish yellow substrate CLUSTAL W (Thompson et al. 1997). The mycelia while SE11 produced pale green maximum-likelihood (ML) (Felsenstein 1981) aerial mass and dark grayish yellow substrate phylogenetic tree was constructed using mycelia. The 16S rRNA gene analysis MEGA 7.0 (Kumar et al. 2016). All gaps were revealed that isolate SE4 and SE11 are closely eliminated before using for tree calculation. related to Streptomyces roseofulvus The confidence values of tree nodes were NBRC13194T and Streptomyces nogalater evaluated using the bootstrap resampling JCM 4799T with similarity of 99.65% and method based on 1,000 replications 99.79%, respectively. Therefore, these isolates (Felsenstein 1985). were members of the genus Streptomyces. 2.3 Screening of antimicrobial activity Based on Figure 1, isolates SE1, SE2, SE7 Antimicrobial activity screening was and SE13 shared the clade with members of determined using cross streak method. The the genus Nocardia. Meanwhile, isolates SE4 actinobacteria were streaked on one side of the and SE11 shared the clade with members of 2

Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

the genus Streptomyces. The phylogenetic tree 98.7% of 16S rRNA gene similarity (ML) is clearly confirmed the BLAST results recommended by Stackebrandt & Ebers for that these actinobacteria were the members of testing the genomic uniqueness of a novel the genus Streptomyces and Nocardia. isolates (Stackbrandt and Eber 2006). Hence, The results of antimicrobial activities SE2 represents the candidate of novel screening showed that none of Nocardia Nocardia species status. However, additional isolates in this study showed antimicrobial studies by using polyphasic and the activities against the tested microorganism. In comparative genome analysis are required to the other hand, both Streptomyces SE4 and prove this hypothesis. SE11 showed some antimicrobial activities. In this study, only two actinobacterial SE4 exhibited anti-Gram positive bacteria genera were isolated from the target fire ant against K. rhizophila ATCC 4341 and S. samples. Typically, the most abundant aureus ATCC 25923, but not against B. actinobacteria in the soil are Streptomyces subtilis ATCC 6633,others Gram-negative species. However, four from six bacteria and yeast. For SE11, it displayed actinobacterial isolates obtained from this activity against Candida albicans ATCC study were Nocardia spp. Some members of 10231, but not against any other tested the genus Nocardia can cause Nocardiosis, the bacteria (Table 2). Nocardia infection, but some species are non- pathogenic. In the previous report, N. 4. Discussion thailandica and N. pseudobrasiliensis were firstly isolated from clinical specimen (Ruimy This study is the first report of the culture- et al. 1996; Kageyama et al. 2004). The re- dependent isolation of actinobacteria from the isolation of these two Nocardia species fire ant, Solenopsis geminata, in Thailand. including one candidate new Nocardia species However, the culture-dependent methods are from fire ant should be mentioned in the role known to underestimate the true diversity of of Nocardia associated with ant. microbes in the sample. With respect to the Unfortunately, in this investigation, we culture-independent method, the previous collected ant samples from only one ant study of the bacterial diversity in Solenopsis colony. Therefore, the comparison between geminata in the USA by using 16S amplicon colonies will be further performed 454 pyrosequencing found that the major Both Streptomyces isolates obtained in bacterial group of So. geminata worker was this study exhibited antimicrobial activities Spiroplasma (Ishak et al. 2011). Moreover, the against the tested microorganisms. Normally, small amount of the actinobacteria members S. roseofulvus and S. nogalater have been were found without any detection of the reported as the producers for frenolicin B Nocardia species in those fire ant samples (Iwai et al. 1987) and nogalamycin (Ylihonko studied in the USA. Thus, the culture- et al. 1996), respectively,It is possible that the dependent methods should be useful when we antimicrobial activities of SE4 and SE11are have the goal to isolate specific culturable derived from these compounds. microbes. In conclusion, the results from this study In the past decade, several new confirm that the phrase “new habitat new actinobacterial species, for example, microbe” still works. Microbispora camponoti (Han et al. 2016), Micromonospora polyrhachis (Xiang et al. Acknowledgements 2014), Nocardia lasii (Liu et al. 2016), Streptomyces formicae (Bai et al. 2016), and This research was supported by and Streptomyces polyrhachii (Yu et a. 2013), Thailand Research Fung to WP (MRG61) and were isolated from ant species. In this study, grant for International Research Integration: the isolate SE2 showed 98.00% similarity of Research Pyramid, Ratchadaphiseksomphot 16S rRNA gene to those known Nocardia Endowment Fund (No. GCURP_58_01_ species. This value is lower than the value of 33_01), Chulalongkorn University. 3

Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

SE2 (LC429298) 79 Nocardia takedensis NBRC 100417T (BAGG01000024)

83 T Nocardia pseudobrasiliensis NBRC 108224 (BDBS01000093) 66 T Nocardia rayongensis RY45-4 (AB889540) Nocardia asteroides NBRC 15531T (BAFO01000006) 62 SE7 (LC429297) Nocardia thailandica NBRC 100428T (BAGK01000053) 100 99 SE1 (LC429295)

SE13 (LC429296) 93 Nocardia shimofusensis NBRC 100134T (BDBT01000060) 93 Corynebacterium diphtheriae NCTC 11397T (X84248) 94 Mycobacterium tuberculosis H37RVT (X58890) 60 Pseudonocardia thermophila NRRL B-1978T (EF588218)

Micromonospora chalcea DSM 43026T (X92594)

Frankia alni ACN14AT (KX674376) 74 77 Geodermatophilus africanus DSM 45422T (HE654550) T 100 Microbispora rosea IFO14044 (D86936) Microtetraspora glauca DSM 43311T (X97891)

T 96 Nocardiopsis dassonvillei DSM 43111 (X97886)

74 Thermomonospora curvata IFO 15933T (D86945) 59 T 99 Actinomadura madurae DSM 43067 (X97889) T 77 Kitasatosporia setae KM-6054 (U93332) Streptacidiphilus albus DSM 41753T (AF074415) Streptomyces albus DSM 40313T (AJ621602)

99 SE4 (LC429300) 99 Streptomyces roseofulvus NBRC 13194T (AB184327) 92 54 T Streptomyces roseolus NBBRC 12816 (AB184168) Streptomyces filamentosus NBRC 12767T (AB184130) Streptomyces achromogenes subsp. rubradiris NBRC 14000T(AB184561)

66 SE11 (LC429299)

94 Streptomyces nogalater JCM4799T (AB045886)

T 60 Streptomyces lavenduligriseus NRRL ISP-5487 (JOBD01000085) 83 Streptomyces eurythermus ATCC 14975T (D63870) T Micrococcus luteus DSM 20030 (AJ536198) Actinomyces bovis ATCC13683T (X81061) 78 Bifidobacterium bifidum KCTC 3202T (U25952) Bacillus subtilis DSM10T (AJ276351)

0.050

Figure 1. Phylogenetic tree of actinobacterial isolates and type strains/type species of the Phylum Actinobacteria. Bacillus subtilis DSM10T was used as the out group. The number of branch nodes indicate bootstrap percentage derived from 1,000 replication (only values > 50% are shown). Bar, 0.050 substitutions per nucleotide position.

4

Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

Table 1. Cultural and morphological characteristics of actinobacterial isolates Isolate Cultural characteristic on ISP2 agar Morphology no. Growth Aerial mycelia Substrate Colony color Soluble color mycelia color pigment SE1 Good Pale orange Light orange Moderate orange - Fragmented substrate yellow yellow yellow mycelia SE2 Good Pale orange Strong orange Moderate orange - Fragmented substrate yellow yellow yellow mycelia SE4 Good Yellowish Dark grayish Dark grayish - Long aerial mycelia white yellow yellow SE7 Good Yellowish Light orange Light orange - Fragmented substrate white yellow yellow mycelia SE11 Good Pale green Dark grayish Colorless Moderate Spiral spore chain on yellow olive aerial mycelia brown SE13 Good Yellowish Light orange Strong yellow - Fragmented substrate white yellow mycelia

Table 2. 16S rRNA gene analysis of the actinobacterial isolates and their antimicrobial activities Isolate Top-Hit taxon Similarity Accession no. Inhibition zone (mm)* no. (%) S B K E P C SE1 Nocardia thailandica NBRC 99.86 LC429295 ------100428T SE2 Nocardia pseudobrasiliensis 98.00 LC429298 ------NBRC 108224T SE4 Streptomyces roseofulvus NBRC 99.65 LC429300 42 - 26 - - - 13194T SE7 Nocardia thailandica NBRC 99.93 LC429297 ------100428T SE11 Streptomyces nogalater JCM 99.79 LC429299 - - - - - 6 4799T SE13 Nocardia thailandica NBRC 100 LC429296 ------100428T * -, no activity; S = Staphylococcus aureus ATCC 25923; B = Bacillus subtilis ATCC 6633; K = Kocuria rhizophila ATCC 4341; E = Escherichia coli ATCC 25922; P = Pseudomonas aeruginosa ATCC 27853; C = Candida albicans ATCC 10231

5

Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

References japonicas Mayr. Antonie Van Leeu Bai L, Liu c, Guo L, Piao C, Li Z, Li J et al. 109:215-223 (2016) Streptomyces formicae sp. nov., Hayakawa M, Nonomura H (1987) Humic a novel actinomycete isolated from the acid-vitamin agar, a new medium for head of Camponotus japonicas Mayr. the selective isolation of soil Antonie Van Leeu 109:253-261. actinomycetes. J Ferment Technol Beemelmanns C, Guo H, Rischer M, Poulsen 65:501-509 M (2016) Natural products from Ishak HD, Plowes R, Sen R, Kellner K, microbes associated with insect. Meyer E, Estrada DA et al. (2011) BEILSTEIN J Org Chem 12:314-317 Bacterial diversity in Solenopsis Beemelmanns C, Ramadhar TR, Kim KH, invicta and Solenopsis geminate and Kalssen JL, Cao S, Wyche TP et al. colonies characterized by 16S (2017) Macrotermycins A-D, glycosy- amplicon 454 pyrosequencing. Microb lated macrolactams from a termite- Evol 61:821831 associated Amycolatopsis sp. M39. Org Iwai Y, Kora A, Takahashi Y, Kayashi Y, Lett 19:1000-1003 Awaya J, Masuma R et al (1987) Carr G, Derbyshire ER, Caldera E, Currie Production of deoxyfrenolicin and a CR Clardy J (2012a) Antibiotic and new antibiotic, frenolicin B by antimalarial quinones from fungus- Streptomyces roseofulvus strain AM- growing ant-associated Pseudono- 3867. J Antibiot 31:959-965 cardia sp. J Nat Prod 75:1806-1809 Kageyama A, Poonwan N, Yazawa K, Carr G, Pousen M, Klassen JL, You Y, Suzuki S, Kroppenstedt RM, Mikami Wyche TP, Bugni TS et al. (2012b) Y (2004) Nocardia vermiculata sp. Microtermolides A and B from nov. and Nocardia thailandica sp. termite-associated Streptomyces sp. nov. isolated from clinical specimens. and structural revision of vinylamycin. Actinomycetologica 18:27-33 Org Lett 14:2822-2825 Kang HR, Lee D, Benndorf R, Jung WH, Currie CR, Scott JA, Summerbell RC, Beemelmanns C, Kang KS et al. Malloch D (1999) Fungus-growing (2016) Termisoflavones A-C, ants use antibiotic-producing bacteria isoflavonoid glycosides from termite- to control garden parasites. Nature associated Sterptomyces sp. RB81. J 398:701-704 Nat Prod 79:3072-3078 Felsenstein J (1981) Evolutionary trees from Kumar S, Stecher G, Tamura K (2016) DNA sequences: a maximum MEGA7: molecular evolutionary likelihood approach. J Mol Evol genetics analysis version 7.0 for biffer 17:368-376 datasets. Mol Biol Evol 33:1870-1874 Felsenstein J (1985) Confidence limits on Lane DJ (1994) 16S/23S rRNA sequencing. phylogenies: an approach using the In: Stackebrandt E, Goodfellow M bootstrap. Evolution 39:783-791 (eds) Nucleic Acid Techniques in Guo ZK, Liu SB, Jiao RH, Wang T, Tan X, Bacterial Systematics. Wiley, Ge HM (2012) Angucyclines from an Chichester, pp 115-175 insect-derived actinobacterium Liu C, Bai L, Ye L, Zhao J, Yan K, Xiang W Amycolatopsis sp. HCa1 and their et al. (2016) Nocardia lasii sp. nov., a cytotoxic activity Bioor Med Chem novel actinomycete isolated from the Lett 22:7490-7493 cuticle of an ant (Lasius fuliginosus L). Han C, Liu C, Zhao J, Guo L, Lu C, Li J et Int J Syst Evol Microbiol 109:1513- al. (2016) Microbispora camponoti sp. 1520 nov., a novel actinomycete isolated Matsumoto A, Takahashi Y (2017) from the cutical of Camponotus Endophytic actinomycetes: promising

6

Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7

source of novel bioactive compounds. J O’Donnel AG(eds) Chemical Methods Antibiot 70:517-519 in Prokaryotic Systematics. Wiley, Oh D-C, Poulsen M, Currie CR, Clardy J Chichester, pp 463-470 (2011) Sceliphrolactam, a polyene Thompson JD, Gibson TJ, Plewniak F, macrocyclic lactam from a wasp- Jeanmougin F Higgins DG (1997) The associated Streptomyces sp. Org Lett CLUSTAL_X windows interface: 13:752-755 flexible strategies for multiple Qin Z, Munnoch JT, Devine R, Holmes NA, sequence alignment aided by quality Seipke RF, Wilkonson KA et al. analysis tools. Nucleic Acids Res (2017) Formicamycins, antibacterial 25:4876-4882 polyketides produced by Streptomyces Xiang W, Yu C, Liu C, Zhao J, Yang L, Xie formicae isolated from African B (2014) Micromonospora polyrhachis Tetraponera plant-ants. Chem Sci sp. nov., an actinomycete isolated from 8:3218 edible Chinese black ant (Polyrhachis Ruimy r, Riegle P, Carlotti A, Boiron P, vinica Roger). Int J Syst Evol Bernardin G, Montel H et al. (1996) Microbiol 64:495-500 Nocardia pseudobrasiliensis sp. nov., a Ylihonko K, Tuikkanen J, Jusssila S, Cong new species of Nocardia which groups L, Mantsala P (1996) A gene cluster bacterial strains previously identified involved in nogalamycin biosynthesis as Nocardia brasiliensis and associated from Streptomyces nogalator: with invasive diseases. Int J Syst sequence analysis and complemen- Bacteriol 46:259-264 tation of early-block mutations in the Shirling EB, Gottlieb D (1966) Methods for anthracycline pathway. Mol Gen Genet characterization of Streptomyces 251:113-120 species. Int J Syst Bacteriol 16:313- Yoon SH, Ha SM, Kown S, Lim J, Kim Y et 340 al. (2017) Introducing EzBio-Cloud: a Stackebrandt E, Ebers J (2006) Taxonomic taxonomically united database of 16S parameters revisited: tarnished gold rRNA gene sequence and whole- stands. Microbiol Today 33:152-155 genome assemblies. Int J Syst Evol Suriyachadkun C, Chunhametha S, Thawai Microbiol 67:1613-1617 C, Tamura T, Potacharoen W, Yu C, Liu C, Wang X, Zhao J, Yang L, Gao Kirtikara K et al. (2009) Planotetra- R et al. (2013) Streptomyces spora thailandica sp. nov., isolated polyrhachii sp. nov., a novel from soil in Thailand. Int J Syst Evol actinomycete isolated from an edible Microbiol 59:992-997 Chinese black ant (Polyrhachis vicina Tamaoka J (1944) Determination of DNA Roger). Antonie Van Leeu 104:1013- base composition. In: Goodfellow M, 1019

7