Ramkhamhaeng International Journal of Science and Technology (2018) 1(1):1-7 ORIGINAL ARTICLE Isolation, identification and antimicrobial activities of actinobacteria 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 bacteria, 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), Streptomyces (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 taxonomy 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