Identification of Streptomyces spp. from garbage dump soils in Surabaya, Indonesia

Background and Aim: Streptomyces is a well-known agent of secondary metabolite production. This study aimed to identify Streptomyces spp. from garbage dump soils in Surabaya based on the 16S rRNA gene sequence. Moreover, the structure of new chemical compounds used for treating infectious diseases in humans, animals, and plants was elucidated. Materials and Methods: We isolated Streptomyces spp. from garbage dump soils in Surabaya. In this study, all isolates were characterized according to phenotype, and they were also confirmed by 16S rRNA gene sequence analysis using real-time polymerase chain reaction. Multiple sequence alignment and molecular phylogeny analyses were conducted using the MEGA 5.0 software, and then the TREE VIEW program was used to display the phylogenetic tree. The level of DNA similarity was also evaluated using the basic local alignment search tool (BLAST) program and then compared with nucleotide sequences stored in the GenBank database using National Center for Biotechnology Information BLAST. Results: The eight Streptomyces spp. showed different nucleotide sequence lengths in gel electrophoresis and photography, which is in accordance with the results observed in the phylogenetic tree. New types of Streptomyces spp., Sp-C, Sp-D, Sp-Ep, Sp-G, and Sp-I, were found from the waste heap in Surabaya. Of these, Sp-Ep was very closely related to Streptomyces indonesiasis and Streptomyces nashvillensis. Sp-F was identified as Streptomyces levis strain NRRL B-24299, and Sp-C was identified as Synodontis filamentosus. Sp-D was related to Sida javensis and Staphylococcus roseus. Sp-G was related to Streptomyces roseoviridis strain NBRC 12911 and Streptomyces thermocarboxydovorans strain AT52. Sp-I was related to Streptomyces cangkringensis and Streptomyces asiaticus. Finally, Sp-A was related to Sansevieria laurentii strain LMG 19959. Conclusion: Based on the phylogenetic tree, new strains of Streptomyces isolate, namely, Sp-D, Sp-Ep, Sp-G, and Sp-I, were found in the garbage dump soils of Surabaya. This new strain can produce antibiotics to be used as an alternative to antibiotics; however, further research is needed to confirm the activity.

Various biodegradative and biotechnological screening processes, based on the diversity of actinomycetes, especially Streptomyces, are applied in the pharmaceutical industry. Moreover, several new antibiotics with numerous variations have been produced using Streptomyces spp., which exceed those produced using other genera of actinomycetes. A large number of species or strains of Streptomyces have been demonstrated to produce new antibiotics existing in nature. More than 6000 compounds have been obtained from Streptomyces spp., which are commercially available as antibiotic, antiparasitic, antifungal, and anticancer agents. One species of Streptomyces can produce two to three natural antibiotics. This is accomplished by isolating and characterizing tens of thousands of these compounds. The majority of them have been developed into drugs that can be used to treat various types of diseases in humans, animals, and plants [2,5,6].
Streptomyces spp. have been identified primarily using conventional classification methods based on their morphological and phenotypic characteristics. The impact on the taxonomy of Streptomyces increased over the past few decades due to the use of molecular biology methods, such as 16S rRNA gene sequencing and BOX-polymerase chain reaction (PCR) fingerprinting [7][8][9].
Copyright: Kurnijasanti and Sudjarwo. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
In the present study, we used 16S rRNA gene sequencing to classify Streptomyces isolates from garbage dump soils in Surabaya, Indonesia.

Ethical approval
This study did not use any experimental animals. Hence, ethical approval did not require in this study.

Study period and location
This research was conducted from January 2021 to August 2021. Isolation and identification of Streptomyces were carried out at the Tropical Diseases Center, Airlangga University.

Isolation of Streptomyces spp. from garbage dump soil samples
The garbage dump soil sample collected from Surabaya, Indonesia, was used for the isolation of Streptomyces spp. Briefly, 1 g of the soil sample was transferred into a flask containing 10 mL distilled water. Then, it was filtered through a two-layered muslin cloth. The sample was diluted to 10 -3 , 10 -4 , and 10 -5 concentrations. Next, 0.2 mL of each dilution was placed on starch agar medium (starch 9.0 g, l-asparagine 9.0 g, ammonium sulfate 2.0 g, Tris 2.0 g, sodium chloride 1.0 g, dipotassium sulfate 0.5 g, magnesium sulfate 0.2 g, calcium chloride 0.1 g, trace solution 1 mL, potassium dihydrogen phosphate 0.5 g, and agar 15 g, all of which were dissolved in 1 L distilled water at pH 7.0) plates, supplemented with the antifungal agent nystatin (50 µg mL -1 ), and incubated for 7 days at 35±20°C. Plates with approximately 200 colonies were selected. Single colonies were streaked on the same medium to purify selected colonies.

Total DNA isolation
Khattab et al. [10] reported that molecular and bioinformatics analyses were conducted to identify Streptomyces strains. Genomic DNA was extracted using the Corbin method with several modifications, according to a previously described protocol [10,11]. Briefly, one colony was cultured in 50 mL of liquid ISP4 medium at 28°C in a shaking incubator for 18-24 h. Then, the culture was centrifuged at 5000 rpm for 3 min, and the resulting supernatant was discarded. Then, Streptomyces were collected, by being suspended in Solution I containing 1 mM ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate 10 mM Tris (pH 7.4), and 0.1 mg/mL proteinase K. Furthermore, the streptomyces suspension was added to Solution II containing 0.8 M NaCl and 1% CTAB for 1 h at 37°C, it will be added to the lysate, and then incubated for 20 min at 65°C. The sample was extracted using chloroform: isoamyl alcohol with the same volume (24:1). The nucleic acid is precipitated in the aqueous phase with isopropanol and then purified using 70% ethanol.

Amplification and sequencing of 16S rRNA gene by PCR
The primers Strep F; 5-AGAGTTTGAT CCTGKGTCAG-3 and Strep R; 5-AAGGGAG GTGATCCAKKGKGA-3 were used in PCR amplification of the 16S rRNA gene against Streptomyces strains [12][13][14]. Each primer of the PCR mixture in 50 I polymerase buffer contained 30 pmol, 100 ng of chromosomal DNA, 200 M dNTPs, and 2.5 U of Taq polymerase. The primary denaturation temperature for PCR amplification was 94°C for 1 min, followed by 94°C for 1 min, and the annealing temperature was 57°C for 60 s. The extension step consisted of 35 cycles of 72°C for 60 s. The final extension was performed at 72°C for 5 min. Then agarose gel electrophoresis was used to analyze the PCR reaction mixture, which is a size marker. In addition, it is indicated by the use of Nucleic Acid Gel Electrophoresis and Blotting (Thermo Scientific™ Fermentas GeneRuler DNA Ladder Mix, USA) 1 kb. The remaining mixture was purified using QIA rapid PCR purification reagent (Qiagen, USA). The Terminator Cycle Sequencing kit was used to obtain the 16S rRNA gene sequence of both strands (ABI Prism 310 Genetic Analyzer, Applied Biosystems, USA).

Gel electrophoresis and photography
To separate the PCR amplification products, the mini-gel set ( Bio-Rad, USA) was used to process 1% w/v ultrapure agarose powder in 1× TBE buffer (pH 8.3) at 100 V for 60-70 min. The gel was stained with ethidium bromide (0.5 g/mL) and then analyzed using BioDocAnalyze (Biometra, Germany). A molecular weight marker of 250 bp was used.

Sequence similarities and phylogenetic analysis
Multiple sequence alignment and molecular phylogeny analyses were performed using the MEGA 5.0 software (www.megasoftware.net). The TREE VIEW program was used to display the phylogenetic tree [15][16][17]. The level of DNA similarity was evaluated using the basic local alignment search tool (BLAST) program (www.ncbi.nlm.nih.gov/ blst) by comparing with nucleotide sequences stored in the GenBank database using National Center for Biotechnology Information (NCBI) Blast [18,19].

Identification of Streptomyces isolates
This study applied a numerical taxonomy using the 16S rRNA-based Streptomyces spp. program for species identification. This was done as recommended in the international key [9,13]. The 16S rRNA gene was used as a reference for bacterial identification because this gene is the most resistant to change or evolution. Table-1 shows the results of the gene sequencing of 16S rRNA using BLAST, wherein the eight strains were confirmed as Streptomyces spp. The analysis was based on partial 16S rRNA gene sequencing and the nucleotide sequence data stored in the GenBank database ( NCBI database).

Results
The nucleotide sequence homology of Streptomyces spp. from the garbage dump soils of Surabaya showed a sequence similarity of 95-99% with the GenBank database sequences. The similarity values of the nucleotide sequences were as follows: 95% for Sp-A, 97% for Sp-C and Sp-D, 96% for Sp-Ea, 99% for Sp-Ep and Sp-F, and 98% for Sp-G and Sp-I. Table-1  The identification of Streptomyces spp. was based on the 16S rDNA sequence data, which provided the best information about Streptomyces and can be used to identify several new types of Streptomyces strains. The phylogenetic tree shown in Figure-2 was derived using the neighbor-joining method from the distance matrix. The majority of sequences clustered into groups in the phylogenetic analysis. However, the results revealed the presence of different types of Streptomyces 16S rDNA sequences, suggesting several new types of Streptomyces species.
The nucleotide sequence of the 16S rRNA gene from Streptomyces spp. was used as the basis for constructing the phylogenetic tree, wherein the sequencing results were obtained using the MEGA version 5.0 program. Next, the eight Streptomyces spp. from the garbage dump soil isolates were compared with five local Indonesian isolates of Streptomyces spp. and 12 Streptomyces isolates from the GenBank database.   Figure-2 shows the comparison of sequencing results of the different Streptomyces isolates and the relationship between the isolates and their closest phylogenetic neighbors. The different phylogenetic lines were formed from several sequences, whereas the groups in the Streptomyces 16S rRNA gene tree contained those from other sequence groups. The phylogenetic tree analysis revealed the presence of a new type of Streptomyces spp., namely, Sp-Ep, which was still related to Streptomyces indonesiasis and Streptomyces nashvillensis. Moreover, researchers observed that Streptomyces olivoreticuli was related to Streptomyces yogyakartensis, which was termed as Sp-Ea. Another type identified in this study was Streptomyces levis strain NRRL B-24299, which was termed as Sp-F. Synodontis filamentosus was termed as Sp-C. Another new type of Streptomyces strain closely related to Sida javensis and Staphylococcus roseus was termed as Sp-D. The type Sp-G was related to Streptomyces roseoviridis strain NBRC 12911 and Streptomyces thermocarboxydovorans strain AT52. The next new type Sp-I was related to Streptomyces asiaticus and Streptomyces cangkringensis. Finally, the type Sp-A was related to Streptomyces LMG 19959. In general, all these isolates belong to the same genus, namely, Streptomyces, but they are of different types.
As shown in Figure-2, the phylogenetic relationships based on the order of 16S rRNA between the eight Streptomyces are interrelated between one species and another. Furthermore, the neighbor-joining method using MEGA X software (www.megasoftware.net) was used as the basis for evolutionary analysis. 0.005 nucleotide substitutions per alignment position were represented by stems, and the number above that branch was called the bootstrap value.

Discussion
In the present study, we used 16S rRNA gene sequencing to classify Streptomyces isolates from garbage dump soils in Surabaya, Indonesia and identified new, specific strains that can produce antibiotics to be used as alternative drugs. Streptomyces represent an important source of bioactive compounds, which are widely used commercially to produce antibiotic, antiparasitic, antifungal, and anticancer agents [2,4,6]. Furthermore, continuing the search for new bioactive compounds is important because of the increasing number of antibiotic-resistant bacteria every year. However, huge challenges exist in this regard because identifying new secondary metabolites is extremely difficult, which thus requires the isolation, characterization, and screening of new members of the genus Streptomyces. Moreover, several newly confirmed bioactive compounds are derived from Streptomyces from unexplored habitats, which might be extremely rich sources of antibiotics. Therefore, we isolated Streptomyces spp. from the garbage disposal soils of Surabaya, Indonesia.
The 16S rDNA gene was amplified using primers to identify Streptomyces isolates, followed by PCR for molecular identification, which is a sensitive and specific detection method for Streptomyces. The 16S rDNA target gene was used for the selected PCR primer for detecting the eight Streptomyces isolates. The BLAST was used to compare the Streptomyces 16S rRNA gene sequences and those in public databases, consistent with that recommended in the NCBI website [6,12,19]. This was done to determine the similarity between sequences in the GenBank database.
In the phylogenetic tree construction for Sp-A, Sp-C, Sp-D, Sp-Ea, Sp-Ep, Sp-F, Sp-G, and Sp-I strains, the 16S rRNA gene sequences with high similarity were used in this study. The sequencing results showed that the strains belonged to the genus Streptomyces. They were used to compare several strains described validly with local Indonesian isolates that were selected as outgroups. These isolates were closely related to several strains, type Sp-Ep identified in this study was closely related to S. indonesiasis and S. nashvillensis. Sp-Ea was related to S. olivoreticuli and S. yogyakartensis. Sp-F was related to S. levis strain NRRL B-24 299. Sp-C was related to S. filamentosus. Sp-D was related to S. javensis and S. roseus. Sp-G was closely related to S. roseoviridis strain NBRC12911 and S. thermocarboxydovorans strain AT52. Meanwhile, another new species Sp-I was closely related to S. cangkringensis and S. asiaticus. Finally, Sp-A was found to be related to S. laurentii strain LMG 19959. All isolates identified in this study belonged to the same genus Streptomyces and were distinguished by their type. Complete information is presented in the phylogenetic tree of Streptomyces spp. (Figure-2).

Conclusion
The phylogenetic tree analysis of Streptomyces spp. revealed the presence of the new types of Streptomyces species in Surabaya garbage dump soils: Sp-D, Sp-Ep, Sp-G, and Sp-I. This new strain can produce antibiotics to be used as an alternative to antibiotics; however, further research is needed to confirm the activity.