Potential Antimicrobe Producer of Endophytic Bacteria from Yellow Root Plant (Arcangelisia flava (L.)) Originated from Enggano Island

Exploration studies of endophytic bacteria from Arcangelisia flava (L.) and their potential have not much been conducted. This research aims to explore and characterize the antimicrobial activity of endophytic bacteria in A. flava against pathogenic bacteria. This research consists of several steps including the isolation of bacteria, screening of the antimicrobial activity assay using the dual cross streak method, molecular identification through 16s rDNA analysis, and characterization of bioactive compound production through PKS-NRPS gene detection and GC-MS analysis. There are 29 endophytic bacteria that were successfully isolated from A. flava. The antimicrobial activity showed that there are four potential isolates AKEBG21, AKEBG23, AKEBG25, and AKEBG28 that can inhibit the growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The 16S rDNA sequence analysis showed that these isolates are identified as Bacillus cereus. These four isolates are identified as able to produce the bioactive compounds through the detection of polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS)-encoding genes. B. cereus AKEBG23 has the highest inhibition against pathogenic bacteria, and according to the GC-MS analysis, five major compounds are allegedly involved in its antimicrobial activity such as butylated hydroxytoluene (BHT), diisooctyl phthalate, E-15-heptadecenal, 1-heneicosanol, and E-14-hexadecenal. This result suggested that B. cereus AKEBG23 as the endophytic bacterium from A. flava has a beneficial role as well as the plant itself. The bacterium produces several bioactive compounds that are allegedly involved in its antimicrobial activity against pathogenic bacteria.


Introduction
Enggano Island is one of the outer islands in Indonesia which administratively belongs to the Bengkulu Province ( Figure 1). As one of the outer islands, Enggano has a high foral diversity in several ecosystems such as mangrove, beach, riparian, natural forest, and swamp ecosystems. Te high foral diversity in Enggano included the medicinal plants utilized by local people as the traditional medicine inherited from the former generation [1]. Arcangelisia fava (L.) is one of the medicinal plants found in Enggano. Te local people named "kayu kuning" (yellow wood) or "akar kuning" (yellow root), which generally consists of three diferent species such as Arcangelisia fava (L.), Fibraurea tinctoria and Coscinium fenestratum [2]. A. fava is widely distributed in Indonesia, such as in Sumatera, Kalimantan, Sulawesi, Java, and Maluku [3].
Te local people use the A. fava plants to treat several diseases such as malaria, dysentery, and fever [4,5]. Previous research has also shown the potency of A. fava as the producer of an antioxidant, antidiabetic, and antimicrobial compound such as berberine [6][7][8]. Previous research also reported that the alkaloid extract from A. fava. has the potential to treat skin-related fungal infections caused by Candida albicans and Trichophyton mentagrophytes [6].
Te potential of A. fava as a medicinal plant is not only found in the direct usage of the plant metabolite, but also its endophytic microbes reside in it. Several endophytic microbes isolated from A. fava leaves produce the antimicrobial compound. Previous research reported that coelomycetes AFKR-18, the endophytic fungi isolated from A. fava, produce pachybasin as the major antimicrobial and antifungal compound against Escherichia coli, Bacillus subtilis, Micrococcus luteus, Candida albicans, and Aspergillus favus [9]. Coelomycetes also produce phloroglucinol which has strong antimicrobial activity against E. coli [10]. Instead of fungi, a recent study also reported that some endophytic bacteria from A. fava have antimicrobial activity, such as Bacillus cereus AKEBG28, which has antimicrobial activity against Escherichia coli and Staphylococcus aureus [11].
According to these research works, the endophytic microbe of A. fava has the potential as the antimicrobial compound producer. Te data of endophytic microbes from A. fava from Enggano Island are not well studied, especially related to the benefcial compound produced by the microbes. Terefore, the objectives of this research are to characterize the endophytic bacteria isolated from A. fava originated from Enggano Island as the potential antimicrobe and conduct the profling of the bioactive compound produced by the bacteria.

Sample Collection.
Te A. fava samples were collected from Enggano Island (Figure 1). Te plant was collected and divided into 3 parts that are roots, stems, and leaves, and each sample was stored and will be used as a source for microbial isolation.

Isolation and Purifcation of Bacteria.
Isolation of endophytic bacteria was conducted using the serial dilution method from 10 −1 to 10 −8 . Te plant tissue was surfacesterilized, crushed, and diluted in a sterile saline solution. As much as 0.1 mL of each dilution was inoculated to nutrient agar (NA) medium using the spread plate method and incubated at 30°C for 24-48 hr. Te grown colony was then gradually purifed by inoculating to the new NA medium.

Antimicrobial Assay.
Screening of potential antimicrobial isolates was performed using the cross streak method [12] with a few modifcations against four pathogenic microbes, namely, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Te isolate was cultured overnight in tryptic soy broth (TSB) and adjusted using the McFarland turbidity standard of 0.5 so that the cells reach 1.5 × 10 8 CFU. As much as 1% (v/v) of pathogenic microbes were inoculated into 100 mL of tryptic soy agar (TSA) at ±40°C, homogenated, and poured onto a sterile Petri dish. Te endophytic bacteria were then streaked in the TSA medium containing pathogenic microbes and incubated at 30°C for 48 hr. Te clear zone around the potential endophytic bacteria indicates the antimicrobial activity. Te diameter of the zone was measured to determine the strength of the antimicrobial nature of each isolate. Te further antimicrobial assay was performed for the selected potential isolates with a similar method with three replicates. Te assay was carried out by using a pure bacterial colony, fltrate of the medium, cell pellets, and ethyl acetate extract. As for the fltrate and ethyl acetate extract, the antimicrobial assay used was the disk difusion method [13,14].

Identifcation of Bacteria.
Te sequencing of 16S rDNA was used to identify the endophytic bacteria. Several selected isolates were grown in NA for 48 h for DNA extraction. Te DNA was extracted using a Geneaid Presto bacterial DNA Extraction Kit (Geneaid Biotech Ltd.; New Taipei City, TW) according to the standard protocol provided by the manufacturer. Pure genomic DNA was then amplifed for 16S rDNA using universal primer 63F/1387R [15] using the T100 Termal Cycler (Bio-Rad Laboratories; Hercules, CA, USA). Te PCR product was then sequenced using the Sanger sequencing method outsourced in a service laboratory. Te 16S rDNA sequences were analyzed using ChromasPro software (Version 1.7.7; Technelysium Pte Ltd.; South Brisbane, QLD, AU) for the quality checking and trimming process. Te assembled forward and reverse 16s rDNA sequences were compared to an online database in GeneBank through the basic local alignment tool for nucleotide (BLAST-N) [16]. Te neighbor-joining tree was generated using MEGA version 7.0 [17] using the bootstrap method, with 1000x replicates.

Extraction and Profling of the Bioactive Compound.
Profling of bioactive compounds was conducted through ethyl acetate extraction and then continued in gas chromatography-mass spectrophotometry (GC-MS) analysis. As much as 10 mL of 24 h potential bacterial isolates were inoculated into 1 L of the TSB medium, the suspension was incubated in a shaker incubator at 30°C for 72 h. Te extraction of the metabolite from the potential isolates was conducted by the addition of ethyl acetate 1 : 1 (v/v) and shaking for 20 min. Te upper layer of the suspension (solvent layer) was then separated and evaporated in a rotary evaporator at 42°C. Te evaporated extract obtained then weighted and diluted in 1 mL dimethyl sulfoxide 50% (DMSO) to determine the concentration. GC-MS analysis was performed using Shimadzu GC-MS-QP 2010 (Shimadzu Corporation; Tokyo, JPN) with RTX-5MS column. Te result of mass spectra was then compared to the National Institute of Standards and Technology (NIST) database version 11 (NIST; Gaithersburg, MD, USA).

Isolation and Screening of Potential Endophytic Bacteria.
Tere are 29 endophytic bacterial isolates successfully isolated from the roots, leaves, and stem of A. fava through the serial dilution method. Tese isolates have diferent morphological characteristics as mentioned in Table 1. 29 isolates were then roughly screened for antimicrobial activity using the cross-streak method against S. aureus, E. coli, P. aeruginosa, and C. albicans. Te potential antimicrobial isolates show a clear zone around the colony (Table 1).
According to the screening results, four potential isolates qualitatively inhibited the growth of S. aureus and E. coli, namely, AKEBG21, AKEBG23, AKEBG25, and AKEBG28. Tese all isolates are obtained from the stem of A.fava and have similar morphological characteristics such as palecolored colony, rod-shaped, and Gram-positive( Figure 2). Tese isolates will be used for the further antimicrobial assay.

Antimicrobial Activity of Selected Endophytic Bacteria.
Four selected isolates were tested for further antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Te isolates were grown in TSB overnight and centrifuged to concentrate the cells and separate the cells and its medium. Te assay was conducted for the pure isolates, pelleted cells, supernatant (medium), and ethyl acetate extract for the isolates. According to the assay, all isolates can inhibit the growth of E. coli and S. aureus either in the form of pure isolates, pelleted cells, supernatant (medium), and ethyl acetate extract. However, only the ethyl acetate extract of all isolates can inhibit P. aeruginosa with relatively higher activity than fltrates and pelleted cells ( Table 2).
Te isolate AKEBG23 has the highest and most stable antimicrobial activity among the four isolates against the pathogenic bacteria tested. Generally, the antimicrobial activity was found higher in pure isolates and ethyl acetate extract than in pelleted cells and supernatant. Previous research reported that the cell-free supernatant from bacterial isolates mostly had no antimicrobial activity against S. aureus and B. subtilis. However, the crude extract of those isolates had clear antimicrobial activity [19]. Conversely, Danilova et al. [20] reported that the supernatant of Lactobacillus plantarum had antimicrobial activity against food spoilage and pathogenic bacteria. It is allegedly due to the two kinds of antimicrobial compounds produced extracellularly by the bacteria. According to these former research works, it can be known that the antimicrobial compound can be found either in the cellfree supernatant or in the crude extract of the bacteria.

Identifcation of Selected Potential
Isolates. Te four potential isolates obtained were identifed through the sequencing of 16S rDNA. Te 16S rDNA sequences are compared with the gene bank online database using BLAST-N [16] with a search set restricted to the type material so that the DNA sequences used for identifcation are well curated and validly identifed. According to the phylogenetic tree International Journal of Microbiology analysis (Figure 3), all isolates are closely related to Bacillus cereus. It is also supported by the morphological characteristics (Table 1), which mentioned that isolates AKEBG21, AKEBG23, AKEBG25, and AKEBG28 are rod-shaped, Gram-positive bacteria which are the main morphological characteristics of Bacillus.

Amplifcation of PKS and NRPS-Encoding Gene.
Apart from the identifcation, detection of polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS)-encoding genes was also conducted to determine the potential of each isolate to produce the bioactive compound. Te detection of PKS and NRPS-encoding genes was carried out using the specifc primer pair degKS2F.gc/degKSR5.gc for PKS which produce 700 bp amplicon and degNRPS-1F.i/ degNRPS-4R.i for NRPS which produces 1000 bp amplicon [18].
According to the amplifcation, the four selected isolates have PKS and NRPS genes which indicate the potential to produce the bioactive compound. Te sequence analysis    Figure 3: Neighbor-joining tree of four potential bacterial isolates with the highest antimicrobial activity. Te tree was tested using the bootstrap method with 1000x replicates.  [21]. Te NRPS gene analysis shows that all amplifed NRPS fragments were closely related to the Bacillus NRPS gene with various similarities (Table 4). Previous research has showed that Bacillus subtilis and Bacillus fexus with antimicrobial activity against Vibrio species were also harboring PKS and NRPS genes [22]. Polyketide synthase (PKS) and nonribosomal peptide synthetase are the enzymes with multidomains which are responsible for bioactive compound production in many microorganisms, specifcally bacteria, actinobacteria, and fungi. Moreover, the NRPS gene has a signifcant role with PKS in bioactive compound biosynthesis which is composed of peptides such as polypeptides and lipopeptides [23].

3.5.
Profling of the Active Compound. As the isolate with the highest antimicrobial activity, the GC-MS profle of B. cereus AKEBG23 ethyl acetate extract shows that there are 16 bioactive compounds identifed in the crude extract ( Figure 4). Tese bioactive compounds were allegedly involved directly and indirectly in the antimicrobial activity of B. cereus AKEBG23 against four pathogenic bacteria. Tere are fve major compounds associated with antimicrobial activity, namely, butylated hydroxytoluene, diisooctyl phthalate, E-15-heptadecenal, 1-heneicosanol, and E-14hexadecenal (Table 5).
Butylated hydroxytoluene (BHT) is known as an antioxidant, which is commonly used as a standard antioxidant [51]. Tis compound was found as the highest detected compound in GC-MS analysis. Instead of controlling the oxidation process, the bioactivity of BHT is also reported as antimicrobial against Staphylococcus aureus [52]. Another research also reported that BHT was also found in the extract of green algae Scenedesmus obliquus with antimicrobial bioactivity [31].
Te second highest detected compound is diisooctyl phthalate which is mostly known as human-made pollutant phthalic acid esters (PAE). However, several research studies reported that PAE is produced by plants and microorganisms. PAE is also reported to have allelopathic, antimicrobial, and insecticidal activities which increase the competitiveness of the producer [53]. Diisooctyl phthalate is one of the PAE produced by fungi Fusarium oxysporum and Phoma herbarum and microalgae Nostoc sp.; it also has the antimicrobial activity against Staphylococcus aureus, Klebsiella pneumoniae, Trichophyton mentagrophytes, and Candida albicans [50,54]. Te reports related to diisooctyl phthalate produced by bacteria are limited; this can be a new report that fnds diisooctyl phthalate in bacteria, especially B. cereus.

Data Availability
Te GC-MS, PKS, and NRPS sequence data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.