Exploring antimycobacterial potential and profiling secondary metabolite gene clusters in the whole genome of Bacillus isolated from dogfruit (Archidendron pauciflorum)

Abstract

Bacillus spp. associated with tropical medicinal plants are potential sources of secondary metabolites possessing antimycobacterial properties. This study evaluated the antimycobacterial activity of endophytic Bacillus species against Mycobacterium smegmatis and analyzed putative secondary metabolite gene clusters (SMGCs) in the complete genome sequence of the selected isolate. Among the four isolates tested, colony and metabolite extract from Bacillus sp. strain DJ4 strongly inhibited the growth of M. smegmatis, a surrogate bacterium for Mycobacterium tuberculosis (Mtb). The extract was categorized as bactericidal, since the minimum inhibitory concentration (MIC) and MBC of the bacterial extract were 31.25 μg/ml and 125 μg/ml, respectively. The most effective inhibition of biofilm formation and eradication of M. smegmatis cells’ biofilm was shown by the 2 × MIC extract treatment. Eighteen volatile compounds (VOCs) were identified by gas chromatography-mass spectrometry analysis. Some VOCs found in the extract have been reported to act as antibacterial agents. Whole-genome analysis revealed that Bacillus sp. strain DJ4 is similar to Bacillus velezensis strain KCTC 13012. Eight SMGCs were identified in the bacterial genomes. In conclusion, the present study indicates that endophytic Bacillus species, especially Bacillus sp. strain DJ4, are a new source of antimycobacterial compounds. This discovery may allow further exploration of secondary metabolites and genomic features of this endophytic bacterium to open up great prospects in the pharmaceutical industry to combat Mtb infection.

Full text

© 2024 Jepri Agung Priyanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License
(https://creativecommons.org/licenses/by/4.0/).
*Corresponding Author
Jepri Agung Priyanto, Division of Microbiology, Department of Biology,
Faculty of Mathematics and Natural Sciences, IPB University, Bogor,
Indonesia.
E-mail: jepriyanto @ apps.ipb.ac.id
INTRODUCTION
Tuberculosis (TB) disease, caused by Mycobacterium
tuberculosis (Mtb), is the most frequent single infectious illness
leading to global mortality [1]. Nevertheless, anti-TB drugs are
more limited compared to agents existing for other bacterial
infections [2]. Despite TB being declared as a global health crisis
by WHO in 1993, only three new anti-TB drugs were approved
and introduced to the market from 2012 to 2019: namely
bedaquiline (approved by the Food and Drug Administration
US in 2012), delamanid (approved by the European Medicines
Agency Europe in 2014), and pretomanid (approved by the
Food and Drug Administration US in 2019) [3,4]. However, the
prevalence of TB drug resistance increases challenges in nding
novel anti-TB drugs that are effective in shortening TB treatment
[5]. Discovering new anti-TB agents involves time-intensive
research due to the slow growth of Mtb. Another limitation in
developing anti-TB agents is the necessity of a biosafety level
3 laboratory for conducting studies related to Mtb. Utilizing
non-pathogenic and fast-growing Mycobacterium smegmatis
for anti-TB investigation has exhibited several successes in
past research. Mycobacterium smegmatis strains are commonly
susceptible to TB medicines, such as isoniazid, rifampicin, and
ethambutol [6]. It has also been well-studied that M. smegmatis
Journal of Applied Pharmaceutical Science Vol. 0(00), pp 001-012, xxx, 2024
Available online at http://www.japsonline.com
ISSN 2231-3354
Exploring antimycobacterial potential and profiling secondary
metabolite gene clusters in the whole genome of Bacillus isolated
from dogfruit (Archidendron pauciflorum)
Jepri Agung Priyanto1*, Muhammad Eka Prastya2, Egiyanti Nur Widhia Hening1, Rika Indri Astuti1
1Division of Microbiology, Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, Indonesia.
2Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research, and Innovation Agency (BRIN), Serpong, South
Tengerang, Indonesia
ARTICLE HISTORY
Received on: 05/06/2024
Accepted on: 14/09/2024
Available Online: XX
Key words:
Endophyte, Bacillus, Biofilm,
Genome, Mycobacterium
smegmatis, Secondary
metabolite gene cluster
ABSTRACT
Bacillus spp. associated with tropical medicinal plants are potential sources of secondary metabolites possessing
antimycobacterial properties. This study evaluated the antimycobacterial activity of endophytic Bacillus species
against Mycobacterium smegmatis and analyzed putative secondary metabolite gene clusters (SMGCs) in the
complete genome sequence of the selected isolate. Among the four isolates tested, colony and metabolite extract
from Bacillus sp. strain DJ4 strongly inhibited the growth of M. smegmatis, a surrogate bacterium for Mycobacterium
tuberculosis (Mtb). The extract was categorized as bactericidal, since the minimum inhibitory concentration (MIC)
and MBC of the bacterial extract were 31.25 µg/ml and 125 µg/ml, respectively. The most effective inhibition of
biolm formation and eradication of M. smegmatis cells’ biolm was shown by the 2 × MIC extract treatment.
Eighteen volatile compounds (VOCs) were identied by gas chromatography-mass spectrometry analysis. Some
VOCs found in the extract have been reported to act as antibacterial agents. Whole-genome analysis revealed that
Bacillus sp. strain DJ4 is similar to Bacillus velezensis strain KCTC 13012. Eight SMGCs were identied in the
bacterial genomes. In conclusion, the present study indicates that endophytic Bacillus species, especially Bacillus
sp. strain DJ4, are a new source of antimycobacterial compounds. This discovery may allow further exploration
of secondary metabolites and genomic features of this endophytic bacterium to open up great prospects in the
pharmaceutical industry to combat Mtb infection.
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DOI: 10.7324/JAPS.2025.204561

002 Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012
on the inhibition of biolm formation and eradication of
Mycobacterium cell biolms of Mycobacterium strains. This
is an important insight because Mycobacterium is a biolm-
forming bacterium, and the biolm structure promotes the
persistence of the bacterium in response to antimicrobial
agents. In addition, genome proling of Bacillus is still
underexplored, although it is benecial for identifying novel
SMGCs and predicting possible biosynthetic pathways related
to antimycobacterial compound production. Therefore,
the present study aimed to evaluate the antimycobacterial
properties of four endophytic Bacillus spp. from A.
pauciorum and their inhibitory effects on M. smegmatis
biolm formation and cell biolm eradication. Additionally,
whole-genome analysis was conducted to identify secondary
metabolite gene clusters (SMGCs) within the genome of the
most promising endophyte isolate. The composition of volatile
compounds (VOCs) in the extract was determined through
analysis using gas chromatography-mass spectrometry (GC-
MS). From this study, we have successfully explained the
potential of endophytic Bacillus as an anti-TB agent. The
data were proven from the results of in vitro analysis through
antibacterial assays supported with antibiolm tests. At the
same time, we also managed to report genomic aspects by
providing the whole genome sequence of the most potential
Bacillus and identifying its SMGCs. The most potential
Bacillus extract has also been proled using GC-MS.
These results are important reports that could incorporate
the analysis of the potential of endophyte bacterial active
compounds as new anti-TB agents.
MATERIALS AND METHODS
Source of endophyte isolates
Four endophytic bacteria were isolated from A.
pauciorum, namely Bacillus sp. strain DJ4, B. subtilis strain
DJ7, Bacillus velezensis strain DJ9, and Bacillus megaterium
strain AJ5 [12]. These isolates were stored at the Laboratory of
Microbiology, Department of Biology, Faculty of Mathematics
and Natural Sciences, IPB University, Indonesia. The 16S rRNA
sequences of these isolates could be accessed under the NCBI
is over 90% genetically identical to Mtb, shares common genes
in stress response, and exhibits similarity in cell wall structure
and metabolism [6,7]. Therefore, M. smegmatis serves as a
suitable model for evaluating drug candidates against Mtb.
Endophytic microbes are promising sources of
metabolites. These microbes live in plant tissues and play
important roles in enhancing plant growth and protecting
the host against pathogens [8]. Insights from ethnobotanical
knowledge have proven valuable in identifying potential
host plant reservoirs for endophytic microbes. Southeast
Asia’s native plant, dogfruit (Archidendron pauciorum), is
locally used as a vegetable and is exploited for its therapeutic
functions to cure several diseases, such as diarrhea, headaches,
fevers, colds, coughs, and stomach-aches [9]. Exploiting
the endophytic bacteria from this plant may provide a new
alternative source of antimycobacterial agents. In vitro study
supported by a genome mining approach could be an effective
way to screen and evaluate endophytic bacteria as sources of
secondary metabolites. The development of next-generation
sequencing and powerful computational tools in the big-data
era has provided researchers with a useful genomic database
to easily identify diverse and novel secondary metabolite gene
clusters (SMGCs) in the entire bacterial genome, which is
benecial for drug discovery [10].
In our previous studies, four endophytic Bacillus spp.
isolated from A. pauciorum exhibited antibacterial activity
against antibiotic-sensitive strains (Staphylococcus aureus
ATCC6538, Bacillus subtilis ATCC19659, Pseudomonas
aeruginosa ATCC15442, and Escherichia coli ATCC8739),
along with antibiotic-resistant strains (P. aeruginosa M19, B.
subtilis M18, E. coli M4, and Klebsiella pneumoniae M19)
[11,12]. However, the antimycobacterial activity of these
isolates is yet to be investigated. Of note, numerous previous
studies have shown that Bacillus spp. isolated from various
environmental samples exhibit remarkable antimycobacterial
activity. Some Bacillus strains isolated from the soil and
water samples, as well as the endophyte Salicornia brachiata
displayed antimycobacterial potency against Mycobacterium
strains [13,14]. However, none of these studies have evaluated
the effect of secondary metabolites derived from Bacillus
Table 1. Colony and cell characteristics of the four endophytic Bacillus spp. isolated from A. pauciorum.
Characteristics Bacterial strains
Bacillus sp. strain DJ4 B. subtilis strain DJ7 B. velezensis strain DJ9 B. megaterium strain AJ5
Colony morphology
Shape Circular Circular Circular Circular
Margin Entire Entire Entire Entire
Elevation Flat Flat Convex Convex
Optic Opaque Opaque Opaque Opaque
Pigmentation Pearl white Light ivory Pale belge Dark ivory
Cell morphology
Shape Bacilli Bacilli Bacilli Bacilli
Arrangement Monobacilli Monobacilli Diplobacilli Diplobacilli
Gram Positive Positive Positive Positive
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accession numbers: PP178167.1, OR511995.1, OP164675.1,
and OP164672.1, respectively.
Colony and cell morphology characterization
Four endophytic isolates were cultured in nutrient
agar (NA) medium (Oxoid) and incubated for 24 hours. Colony
characteristics, including shape, margin, elevation, optic, and
pigmentation, were observed. Approximately 18-hour-old
cultures were stained using the Gram staining technique to
characterize their cell characteristics, such as shape, arrangement,
and Gram reaction, and observed under a light microscope
(Olympus CX31) with 1,000 × resolution. Additionally, the cell
morphology of each isolate was also observed using a scanning
electron microscope (SEM). The pellets from bacterial cultures
aged 24 hours were rinsed using 100 µl of sterile distilled water.
Afterward, 10 µl of the suspension was applied onto the surface
of a single-polished Silicon Wafer (Sigma) and left to incubate
for 18 hours. Subsequently, the SEM specimen was coated with
sputter-gold particles (Hitachi®, Tokyo, Japan) and observed
using an SEM-JEOL JSM-IT200 (JEOL, South Korea) at a
resolution of 5,000 ×, with a working distance of 5 µm and an
accelerating voltage of 10 kV.
Screening of antimycobacterial activity
Preliminary screening for antimycobacterial
activity was performed using the streak plate method against
Figure 1. Colony and cell morphology of the four endophytic Bacillus spp. isolated from A. pauciflorum. Cell morphology under SEM: (A1) B. megaterium strain
AJ5, (B1) Bacillus sp. strain DJ4, (C1) B. subtilis strain DJ7, (D1) B. velezensis strain DJ9. Cell morphology under light microscope after stained using Gram
technique: (A2) B. megaterium strain AJ5, (B2) Bacillus sp. strain DJ4, (C2) B. subtilis strain DJ7, (D2) B. velezensis strain DJ9. Colony morphology: (A3) B.
megaterium strain AJ5, (B3) Bacillus sp. strain DJ4, (C3) B. subtilis strain DJ7, (D3) B. velezensis strain DJ9.
Table 2. Antimycobacterial activity of endophytic Bacillus spp. against M. smegmatis.
No. Isolates Diameter of inhibition zone (mm)
Endophytic bacteria colony Endophytic bacteria extract
1Bacillus megaterium strain AJ5 9 ± 0.81a8.6 ± 0.47b
2Bacillus sp. Strain DJ4 29.33 ± 0.47d17.7 ± 0.47e
3Bacillus subtilis strain DJ7 10 ± 0b8.3 ± 0.47b
4Bacillus velezensis strain DJ9 28 ± 0.81c16 ± 0d
5 Tetracycline Not tested 15 ± 0.81c
6 DMSO Not tested 0 ± 0a
Note: Distinct superscript letters within the identical column denote statistically significant variances (p-value of each treatment group is 0.000).
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004 Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012
M. smegmatis ATCC700084 (collection of the Research Center
for Pharmaceutical Ingredients and Traditional Medicine,
National Research, and Innovation Agency (BRIN), South
Tangerang, Indonesia). Approximately 1 ml of M. smegmatis
inoculum (1 × 108 CFU/ml) was added into 100 ml of Mueller
Hinton Agar medium (Himedia) and poured into a petri dish.
After the medium had solidied, the overnight endophyte
colony was spot inoculated onto the surface of the medium and
cultivated for 24 hours at ± 37°C. The diameter of the inhibition
zone was expressed in millimeters [12].
Metabolites extraction
Each endophytic Bacillus strain was cultured overnight
in a nutrient broth (NB) medium (Oxoid). The culture (1% v/v)
was seeded to 1 l of NB medium and incubated for 3 days at
± 28°C with agitation at 120 rpm to optimize extracellular
secondary metabolite production. The cultures were extracted
by mixing an equal volume of ethyl acetate and shaking for
20 minutes. The upper layer was then dried at 50°C using an
evaporator. The extract was re-dissolved in 1% DMSO (Merck)
for antimycobacterial and antibiolm analyses [12].
Antimycobacterial test of endophytic extracts
The antimycobacterial activity of each endophytic
extract (1 mg/ml) was analyzed using the disk diffusion method
as described previously. Approximately 200 µg/ml tetracycline
(Sigma-Aldrich) and 1 % DMSO (Merck) were used as positive
and negative controls, respectively [15].
Evaluation of the minimum inhibitory concentration (MIC)
and the minimum bactericidal concentration (MBC)
The extract from each endophytic isolate underwent
further assays to evaluate the MIC using a microdilution test
[15]. Briey, microdilution was performed using Mueller
Hinton (MH) Broth (Himedia) containing extract (100 µl)
ranging from 1,000 µg/ml to 1.95 µg/ml in 96 sterile well plates
(Biologix). Subsequently, the M. smegmatis suspension (100
µl) at a density of 0.5 McFarland (1 × 108 CFU/ml, Thermo
Fisher Scientic) was added into the wells (nal bacterial
concentration of 5 × 107 CFU/ml) and incubated overnight at
37°C. The MIC was dened as the lowest extract concentration
that suppressed the visible growth of M. smegmatis. The MBC
was determined by plating 100 µl suspension from the well
with no growth of M. smegmatis. The lowest concentration of
endophytic extract demonstrating complete killing of the target
bacteria was considered the MBC. One percent of DMSO served
as the negative control, while tetracycline (Sigma-Aldrich) was
employed as the positive control. The MBC/MIC ratio was
analyzed to classify the antimycobacterial properties of the
endophytic extracts as either bacteriostatic or bactericidal. If the
ratio was ≤ 4, the extract was categorized as bactericidal, but if
the ratio exceeded 4, the extract was dened as bacteriostatic.
Antibiofilm analysis
The four endophytic extracts underwent antibiolm
analysis using a microtiter plate test [15]. In brief, 100 µl
of M. smegmatis cells at a density of 1 × 108 CFU/ml were
inoculated into brain heart infusion (BHI) medium (Merck) in
the 96-well plates containing various extract concentrations (2
× MIC, 1 × MIC, ½ × MIC, ¼ × MIC) and incubated overnight.
The medium was then removed from each well, and the
wells were washed twice with phosphate buffer saline (PBS,
Sigma-Aldrich). The formed biolm was stained with 200 µl
of 0.1% crystal violet (Merck) for 30 minutes at 37°C. The
excess staining solution was removed by washing once with
PBS. Subsequently, the stained biolm was eluted in 200 µl
of DMSO (99%). The optical density of the suspension was
measured at 595 nm using an ELISA reader (Thermo Scientic
Varioskan Flash-Thermo Fischer). Control plates without any
Figure 2. Antimycobacterial activity of endophytic bacterial colony (A1) B.
megaterium strain AJ5, (B1) Bacillus sp. strain DJ4, (C1) B. subtilis strain
DJ7, (D1) B. velezensis strain DJ9, and endophytic bacterial extracts (A2) B.
megaterium strain AJ5, (B2) Bacillus sp. strain DJ4, (C2) B. subtilis strain DJ7,
(D2) B. velezensis strain DJ9, compared to (E) tetracycline, and (F) 1% DMSO.
Table 3. The MIC, MBC, and MBC/MIC ratio of the endophytic Bacillus species against Mycobacterium smegmatis.
No. Endophytic extracts MIC and MBC (µg/ml)
MIC MBC MBC/MIC Category
1Bacillus megaterium strain AJ5 125 500 4 Bactericidal
2Bacillus sp. Strain DJ4 31.25 125 4 Bactericidal
3Bacillus subtilis strain DJ7 125 1,000 8 Bacteriostatic
4Bacillus velezensis strain DJ9 31.25 250 8 Bacteriostatic
5 Tetracycline 7.81 15.62 2 Bactericidal
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treatment were used as reference. The percentage of inhibition
of biolm formation was determined in comparison to that of
the control. The experimental data were expressed as average
± standard deviation from triplicates. The biolm structure
of each treatment was observed using an SEM. The biolm
matrix was collected and applied onto the surface of a single-
polished Silicon Wafer (Sigma), and then incubated for 18
hours. Subsequently, the SEM specimens underwent coating
with sputter-gold particles (Hitachi®, Tokyo, Japan), and
examination was conducted using a SEM-JEOL JSM-IT200
(JEOL, South Korea) at a resolution of 5,000 ×, with a working
distance of 5 µm and an accelerating voltage of 10 kV.
Biofilm cells eradication assay
To examine the biolm cells eradication effect of
endophytic extracts on preformed cell biolm (a ve-day
biolm), a methyl tetrazolium test was conducted following the
procedure outlined by Wintachai et al. [16]. In the experiment,
200 µl of M. smegmatis culture (1 × 108 CFU/ml) was introduced
into 96-well plates containing BHI medium (Merck) and left for 5
days at 37°C to allow for mature biolm formation. The medium
was replaced daily (every 24 hours) with fresh BHI medium
supplemented with 0.25% glucose. Additionally, the endophytic
extracts were added at different concentrations (2 × MIC, 1 ×
MIC, ½ × MIC, ¼ × MIC) and incubated for 24 hours at 37°C.
The medium was removed from the well, and the cell biolm
was stained with 10 µl of 5 mg/ml methyl tetrazolium solution
(Merck), and then incubated for 4 hours at 37°C. Subsequently,
the formazan crystals were dissolved with 200 µl of 99% DMSO.
Cell biolms of M. smegmatis that did not undergo any treatment
were utilized as controls. Optical density was measured at 595
nm using an ELISA reader (Varioskan Flash-Thermo Fischer,
Thermo Scientic). The experimental data were presented as
average ± standard deviation from three replications.
Analysis of VOCs composition
The composition of VOCs in the endophyte extract
was analyzed using a GC-MS system (Agilent Technologies
Figure 3. The effect of endophytic Bacillus spp. extracts on (A) the biofilm-forming capacity of M. smegmatis, and (B) in eradicating M. smegmatis cell biofilm.
Dissimilar letters following the bars in each treatment mean statistically significant difference (p-value of each treatment group is 0.000).
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006 Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012
Statistical analysis
The experimental data obtained from the antibacterial
assay, determination of MIC and MBC, antibiolm analysis,
and biolm eradication assays were conducted in triplicate.
Subsequently, the data underwent one-way analysis of variance,
6890N Inert C, USA) according to the manufacturer’s
instructions. Mass spectra and chromatograms were processed
using the MSD ChemStation Data Analysis software (G1701EA
E.02.02.1431).
Whole-genome sequencing and detection of secondary
metabolic gene clusters
The bacterial cells from the 24-hour-old culture
underwent centrifugation at 15,000 rpm for 1 minute. Genomic
DNA isolation was conducted using the Presto™ Mini gDNA
Bacteria Kit (Geneaid) following the kit’s protocol. The bacterial
genome sequence was acquired utilizing Oxford Nanopore
Technology. De novo assembly was executed employing the
Flye (v.2.8.3). Subsequently, mapping and genome polishing
were iteratively performed four times polishing using
Minimap2 (v.2.24–r1122) and Racon (v.1.5.0). The assembled
sequence and genome completeness of closely related species
were assessed using dfast-qc (v.0.4.2) and BUSCO (v.5.4.4).
The quality assessment of the assembled sequence was
carried out with Quast software (v5.0.2). Visualization of the
bacterial genome was accomplished using Circos (v.0.69–8).
AntiSMASH bacterial version v5.1.2 was employed to annotate
SMGCs within the bacterial genome (https://antismash.
secondarymetabolites.org) [17].
Figure 4. Biofilm structure of M. smegmatis after treated with (A) DMSO,
(B) medium only, and extract derived from (C) Bacillus sp. strain DJ4, (D) B.
megaterium strain AJ5, (E) B. subtilis strain DJ7, (F) B. velezensis strain DJ9,
at the concentration of 2 × MIC.
Table 4. VOCs prole of the metabolite extract from Bacillus sp. strain DJ4.
No Compounds Retention
times (mins)
Quantity
(%)
Similarity
(%)
Bioactivity Other sources References
1 2,4-di-tert-butylphenol 14.749 4.05 97 Antibacterial, cytotoxic,
Antifungal, antioxidant
Endophytic
Streptomyces sp. KCA1;
Lactococcus sp.;
[18,19]
2 1-Octadecene 18.039 0.44 97 − − −
3 Cyclo(L-prolyl-L-valine) 18.430 3.40 96 − − −
4 7,9-di-tert-butyl-1-oxaspiro(4,5)
deca-6,9-diene-2,8-dione
19.261 0.41 91 Alpha amylase inhibitor Cestrum octurnum [20]
5 Dibutyl phthalate 19.740 2.60 78 Antibacterial soil Streptomyces
albidoflavus 321.2
[21]
6 Cycloeicosane 20.055 0.96 99 − − −
7 Palmitic acid 20.522 0.32 99 − − −
8 9,12-Octadecadienoic acid
(Z,Z)- methyl ester
21.026 0.29 92 − − −
9 Methyl stearate 21.316 0.25 99 − − −
10 Tributyl acetylcitrate 22.374 1.73 90 − − −
11 Phthalic acid 23.345 0.46 93 − − −
12 Pyrrolo[1,2-a]pyrazine-
1,4-dione, hexahydro-3-
(phenylmethyl)
23.761 1.84 98 Antibacterial, antioxidant,
antifungal
Bacillus tequilensis
MSI45; Streptomyces sp.
VITPK9
[22,23]
13 Oxacyclopentadecan-2-one 24.328 0.36 90 − − −
14 Lauric acid 25.021 6.66 93 Antimicrobial coconut oil [24]
15 1-Hexacosene 25.223 0.71 99 − − −
16 Squalene 26.836 0.73 98 Antimicrobial and
cytotoxic
Stichopus hermanni [25]
17 Dodecanamide 27.075 0.34 96 − − −
18 Dl-alpha-tocopherol 29.533 0.92 98 antioxidant palm [26]
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entire, and opaque, respectively (Table 1). These isolates
also have bacilli in shape, and are stained purple using Gram
staining procedure. The cell arrangement of Bacillus sp. strain
DJ4 and B. subtilis strain DJ7 were monobacilli, whereas the
cell arrangement of B. velezensis strain DJ9 and B. megaterium
strain AJ5 were diplobacilli (Fig. 1).
Antimycobacterial activity of Bacillus species isolated from
A. pauciflorum
The four endophytic Bacillus spp. exhibited varied
antimycobacterial activities against M. smegmatis. Inhibition
zone formation was determined using endophytic bacterial
colonies and their metabolite extracts (Table 2). Among the
four endophytic isolates, Bacillus sp. strain DJ4 consistently
displayed the strongest antimycobacterial activity, showcased
by its colony and extract with inhibition zone diameters of
29.33 ± 0.47 mm and 17.7 ± 0.47 mm, respectively. Overall,
the endophytic bacterial colonies demonstrated larger inhibition
zone diameters against M. smegmatis compared to their
metabolite extracts. The inhibition zone was also observed with
tetracycline as the positive control (diameter: 15 ± 0.81 mm)
and absent in 1% DMSO as the negative control (Fig. 2).
The MIC and MBC of the four endophytic extracts
were determined using a microdilution assay. The MIC ranged
from 31.25 µg/ml to 125 µg/ml, while the MBC ranged from
125 µg/ml to 1,000 µg/ml (Table 3). However, the MIC
followed by further statistical analysis through Tukey’s test
using Statistical Product and Service Solutions software version
29.0. The level of statistical signicance was established at p <
0.05 to determine signicance.
RESULTS
Colony and cell characteristics
The four isolates of endophytic Bacillus spp. used
in this study had different colony characteristics, especially
in terms of elevation and pigmentation, but the four isolates
had the same shape, margins, and optics, which were circular,
Figure 5. (A) Annotated genome of Bacillus sp. strain DJ4. Figure Legend (from outer): contig (blue), genes (grey), pseudogenes (blue), coding sequences (CDS;
black), rRNA (green), tRNA (purple), depth (depth >50 = green; depth <50 = red), gc content (gc content >50% = purple; gc content <50% = brown); (B) Putative
SMGCs from the Bacillus sp. strain DJ4 genome with the antiSMASH database.
Table 5. Genomic features of Bacillus sp. strain DJ4.
Genomic features
Size of the genome assembly (bp) 4,016,867
GC content (%) 46.39
Number of contigs 3
Largest contig (bp) 1,945,594
Mean read length (bp) 9,605.3
Read length N50 (bp) 10,774
Coding sequences (CDSs) 3,909
Genome completeness (%) 99.1
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008 Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012
Table 6. Putative SMGCs present in the genome of Bacillus sp. strain DJ4.
Cluster Length (bp) Type Most Similar Known Gene Cluster Similarity (%) MIBiG ID References
Cluster 1 93,789 Polyketide Difficidin from Bacillus velezensis FZB42 100 BGC0000176 [27]
Cluster 2 51,794 Nonribosomal peptide Bacillibactin from Bacillus subtilis 168 100 BGC0000309 [28]
Cluster 3 7,298 Other* Bacilysin from Bacillus velezensis FZB42 100 BGC0001184 [29]
Cluster 4 135,850 Nonribosomal peptide Fengycin from Bacillus velezensis FZB42 100 BGC0001095 [30]
Cluster 5 96,451 Hybrid polyketide +
Nonribosomal peptide
Bacillaene from Bacillus velezensis FZB42 100 BGC0001089 [31]
Cluster 6 88,218 Polyketide Macrolactin H from Bacillus velezensis FZB42 100 BGC0000181 [32]
Cluster 7 22,117 Linear azole (in)-
containing peptides
(LAP)
Plantazolicin from Bacillus velezensis FZB42 91 BGC0000569 [33]
Cluster 8 41,884 Nonribosomal peptide
+ lipopeptide
Surfactin from Bacillus velezensis FZB42 78 BGC0000433 [34]
*A cluster harboring a protein related to secondary metabolites that does not align with any other classification.
and MBC of endophytic extracts are still higher than that of
tetracycline, which had MIC and MBC of 7.81 µg/ml and
15.62 µg/ml, respectively. Furthermore, based on their MBC/
MIC ratios, two bacterial extracts derived from B. megaterium
strain AJ5 and Bacillus sp. strain DJ4 were categorized as
bactericidal, whereas extracts from B. subtilis strain DJ7 and
B. velezensis strain DJ9 were categorized as bacteriostatic. The
remarkable antimycobacterial activity of the endophytic extract
was demonstrated by Bacillus sp. strain DJ4 with the lowest
MIC and MBC of 31.25 µg/ml and 125 µg/ml, respectively.
Mycobacterium smegmatis biofilm inhibition and biofilm cell
eradication activities
The endophytic extracts reduced the biolm-forming
capability of M. smegmatis. The biolm-forming capacity of
M. smegmatis decreased in a dose-dependent manner with
increasing extract concentration (Fig. 3A). The greatest
inhibition of biolm formation was observed for all extracts at
2 × MIC. At this extract concentration, biolm formation was
inhibited by 49.87 %–71.50 %. Extracts from Bacillus sp. strain
DJ4 demonstrated remarkable antibiolm activity, with the
highest inhibition percentage of biolm formation reaching up
to 71.50%. The four endophytic extracts also exhibited biolm
cell eradication effects on the preformed M. smegmatis biolms.
Similar to the prevention of biolm formation, the biolm cell
eradication effect of endophytic extracts was dose-dependent
(Fig. 3B). The higher the endophytic extract applied, the greater
the percentage of biolm cell eradication effect was achieved.
Consequently, 2 × MIC of endophytic extracts showed the
highest cell eradication percentage, ranging from 47.99% to
60.53%. The most signicant cell eradication effect was also
demonstrated by Bacillus sp. strain DJ4-derived extract, with a
biolm cell eradication percentage of 60.53%.
Furthermore, SEM images revealed a pronounced
accumulation of the exopolysaccharide matrix in both the
DMSO (Fig. 4A), and medium-only treatment (Fig. 4B).
Conversely, a notable decrease in the exopolysaccharide
matrix was observed in the treatments with endophytic extracts
derived from Bacillus sp. strain DJ4 (Fig. 4C), B. megaterium
strain AJ5 (Fig. 4D), B. subtilis strain DJ7 (Fig. 4E), and B.
velezensis strain DJ9 (Fig. 4F). Following endophytic extract
treatments, the exopolysaccharide layer of M. smegmatis was
thinner, less compact and separated into small pieces, indicating
that the endophytic extract treatments inhibited the production
of exopolysaccharide and the formation of the biolm matrix.
Notably, the sample treated with extract derived from Bacillus
sp. strain DJ4 exhibited the thinnest biolm structure compared
to untreated control, DMSO, and other endophytic extract
treatments.
VOCs composition of extract from Bacillus sp. strain DJ4
GC-MS analysis identied 58 VOCs peaks in the
extract from Bacillus sp. strain DJ4. Eighteen compounds were
found in the bacterial extract by matching with the spectra of
the recognized compounds (Table 4). These compounds include
2,4-di-tert-butylphenol (4.05%), 1-octadecene (0.44%), cyclo
(L-prolyl-L-valine) (3.40%), 9-di-tert-butyl-1-oxaspiro(4,5)
deca-6,9-diene-2,8-dione (0.41%), dibutyl phthalate
(2.60%), cycloeicosane (0.96%), palmitic acid (0.32%),
9,12-octadecadienoic acid (Z,Z)-methyl ester (0.29%), methyl
stearate (0.25%), tributyl acetyl citrate (1.73%), phthalic acid
(0.46%), pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-
(phenylmethyl) (1.84%), oxacyclopentadecan-2-one (0.36%),
lauric acid (6.66%), 1-hexacosene (0.71%), squalene (0.73%),
dodecanamide (0.34%), dl-alpha-tocopherol (0.92%), and other
unknown compounds.
Whole genome sequence and SMGCs of the most potential
isolate
Based on the complete genome sequence, Bacillus sp.
strain DJ4 exhibited a close relation (97.55%) to B. velezensis
strain KCTC 13012 (accession no: GCA_001267695.1). The
genome assembly size of this bacterium was 4,016,867 bp,
having a 46.3% GC content (Fig. 5A). The genome assembly
reached a completion rate of 99.1%, with the largest contig
length at 1,945,594 bp and a read length N50 of 10,774 bp.
Genome annotation via NCBI PGAP identied 3,909 coding
sequences within the bacterial genome (Table 5). The complete
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Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012 009
genome sequence of this bacterium is accessible in the NCBI
GenBank database under accession number CP144358.1.
The presence of SMGCs within the genome of
Bacillus sp. strain DJ4 was predicted using antiSMASH.
Annotation results revealed eight SMGCs, encompassing gene
clusters involved in the production of non-ribosomal peptides,
polyketides, linear azole (in)e-containing peptides (LAP),
hybrid non-ribosomal peptides and polyketides, hybrid non-
ribosomal peptides and lipopeptides, as well as an unknown
compound. Some genes shared their similarity with biosynthesis
clusters of known compounds, such as difcidin, bacillibactin,
bacilysin, fengycin, bacillaene, macrolactin H, plantazolicin,
and surfactin, with similarities ranging from 78% to 100% (Fig.
5B). Interestingly, all these gene clusters were identied across
the Bacillus group, encompassing B. velezensis and B. subtilis
(Table 6).
DISCUSSION
Bacillus species isolated from the internal tissues
of medicinal plants are considered promising sources of
compounds with valuable pharmacological functions,
particularly as antimicrobial agents [35]. Various chemical
substances produced by endophytic bacteria may contribute
to their antimicrobial activity. In this study, antimycobacterial
properties of four endophytic Bacillus from A. pauciorum,
namely Bacillus sp. strain DJ4, B. subtilis strain DJ7, B.
megaterium strain AJ5, and B. velezensis strain DJ9, have
been evaluated against M. smegmatis, a surrogate bacterium
commonly used in preliminary study of antituberculosis
candidates.
The four isolates had different colony and cell
characteristics, particularly different in colony pigmentation,
and elevation, as well as their cell arrangement. All isolates
also belonged to Bacilli Gram-positive bacteria according to
Gram staining. Interestingly, all endophytic colonies showed
antagonistic activity against M. smegmatis, as indicated by
the formation of a clear zone around the endophytic colony.
Endophytic bacteria produce antibacterial compounds that
are excreted extracellularly near the colony. Consequently,
M. smegmatis growth in this area was inhibited. Secondary
metabolites from the four endophytic bacteria were extracted
and assessed using a disc diffusion test. The results showed
that their metabolites also showed antimycobacterial activities
with different degrees of signicance. Extract from Bacillus sp.
strain DJ4 displayed the strongest antimycobacterial activity
with the largest inhibition zone of 17.7 ± 0.47 mm, followed
by B. velezensis strain DJ9, B. megaterium strain AJ5, and B.
subtilis strain DJ7. The diverse antimycobacterial effects of the
endophytic isolates indicate the diverse chemical constituents
produced by these endophytic bacteria.
The MIC of the four endophyte extracts was
investigated to dene the lowest extract concentration capable
of inhibiting the visible growth of M. smegmatis, along with the
MBC, as the lowest concentration that could kill M. smegmatis
cells completely. The MIC of the extracts varied from 31.25
µg/ml to 125 µg/ml, and MBC from 125 µg/ml to 1,000 µg/ml,
indicating that M. smegmatis has different sensitivity to each
endophytic extract tested. However, the extracts with MIC less
than 1,000 µg/ml are considered to have noteworthy antibacterial
properties [36]. In this case, extract from Bacillus sp. strain DJ4
demonstrated the strongest antimycobacterial activity because
the extract had the lowest MIC and MBC (MIC: 31.25 µg/ml;
MBC: 125 µg/ml) compared to that of other extracts. Extracts
from Bacillus sp. strain DJ4 and B. megaterium strain AJ5
were categorized as bactericidal (MBC/MIC ratio: 4), whereas
extracts from B. subtilis strain DJ7 and B. velezensis strain DJ9
were categorized as bacteriostatic (MBC/MIC ratio: 8). These
results indicate that endophytic Bacillus spp. isolated from A.
pauciorum have potential as antimycobacterial agents. Several
studies have investigated the antimycobacterial mechanisms
of natural compounds against Mycobacterium. Well-known
mechanisms include destructing bacterial cell membranes [37],
blocking specic metabolic pathways [38], inhibiting cell wall
synthesis [39], inhibiting ribosome function [40], inhibiting
efux pumps [41], and inhibiting DNA replication [42].
Biolm formation stands as the primary virulence
factor in Mycobacteria. TB treatment spans an extended period
(around 6–9 months) due to Mtb cell’s ability to survive within
human lungs, forming biolms for shielding against the host’s
immune response and various antibiotics [43]. Consequently,
the infection persists, showing high persistence and tolerance
to available anti-TB drugs. Hence, targeting the inhibition
and destruction of biolms holds promise for enhancing TB
treatment success. Encouragingly, the four endophytic extracts
exhibited inhibitory effects on M. smegmatis biolm formation
in vitro. Notably, metabolites extracted from Bacillus sp. strain
DJ4 displayed a signicant effect. At a concentration of 2 ×
MIC, the extract inhibited biolm formation by up to 71.50%
after 24 hours of treatment. The effectiveness increased with
higher extract concentrations, suggesting a need for a higher
concentration than that required to suppress the bacterium’s
growth. However, the same bacterial extract showed lower
effectiveness in eradicating M. smegmatis cells within the
biolm, evidenced by a lower percentage (60.53%) of cell
eradication. This indicates increased resistance of the target
bacterium to the endophytic extract after biolm formation.
Hence, a combination of antibacterial agents might be necessary
to eliminate biolms and bolster TB chemotherapy efcacy.
VOCs produced by the most potent bacteria (Bacillus
sp. strain DJ4) were identied using GC-MS analysis.
Surprisingly, some substances with a high similarity to the spectra
database (≥90%) found in the extract are known to possess
pharmaceutical properties (e.g., antimicrobial, antioxidant,
alpha-amylase inhibitor, and cytotoxic) (Table 5). Other
compounds not listed in Table 5 showed low similarity (<90%),
indicating that VOCs might be unidentied or new compounds.
The VOCs identied in the extract are also be produced by other
microbial sources, such as endophytic Streptomyces sp. KCA1,
Lactococcus sp., soil Streptomyces albidoavus 321.2, Bacillus
tequilensis MSI45, and Streptomyces sp. VITPK9, by plants
(such as Cestrum octurnum, and coconut oil), and by animals
(such as Stichopus hermanni) (Table 4). VOCs derived from
Bacillus and other natural sources exert antimicrobial action,
possibly by destroying the bacterial cell membrane, damaging
DNA, interfering with protein structure and function, and
downregulating the expression of genes involved in pathogen
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010 Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012
virulence [44,45]. Additionally, particular VOCs, such as
eugenol inhibit biolm formation by interfering with quorum-
sensing signals and controlling transmembrane transport [46].
VOCs also have an eradicating effect on established biolms
due to their ability to diffuse and destabilize the polysaccharide
matrix in mature biolms [47]. Therefore, VOCs found in the
extract may contribute to the antimycobacterial and antibiolm
activities of the Bacillus sp. DJ4 derived extract.
Bacillus sp. strain DJ4 was deemed the most potent
isolate based on its antibacterial and antibiolm activities. In
a prior study, the bacterium showed a close relation (similarity
100%) to Bacillus amyloliquefaciens strain B29 through the
16S rRNA sequence [12]. However, the 16S rRNA-based
identication has limitations as the sequence is not sufcient
for species-level bacterial identication and intra-strain
differentiation due to its short size (~1,300 bp) compared to
the general 4–6 million bp size of complete bacterial genomes
[48]. Therefore, a more comprehensive bacterial identication
requires sequencing the complete bacterial genome. In this
study, the complete genome of this bacterium was sequenced.
According to the average nucleotide identity from dfast_qc,
the bacterium was closely related (97.55% similarity) to B.
velezensis strain KCTC 13012. Bacillus velezensis strain KCTC
13012 was suggested as a relative of B. amyloliquefaciens
strain KCTC 13012, a well-known producer of broad-spectrum
antibiotics [49].
Secondary metabolic gene clusters within the genome
of Bacillus sp. strain DJ4 were annotated by AntiSMASH.
Polyketide, non-ribosomal peptide, LAP, and lipopeptide-
coding genes were found in the bacterial genome. The clusters
shared high similarity (similarity: 78%–100%) with known
products, such as difcidin (100%), bacillibactin (100%),
bacilysin (100%), fengycin (100%), bacillaene (100%),
macrolactin H (100%), plantazolicin (91%), and surfactin
(78%), which were synthesized by B. velezensis and B. subtilis
(Table 6). Interestingly, bacillibactin produced by endophytic
B. subtilis NPROOT3 has previously been reported as having
antimycobacterial activity against M. smegmatis MTCC6 [14].
Additionally, all similar gene products showed antibacterial
properties against a broad spectrum of bacteria (Table 5).
Moreover, the bacterium may also carry genes contributed
in the biosynthesis of VOCs because the products of these
genes have been detected in bacterial extracts. However,
these genes could not be identied through this methodology
because of the limited genome database of VOCs-related genes
in bacteria. Bacillus VOCs are mostly derived from amino
acid degradation, sulfur reduction, terpene synthesis, glucose
oxidation, and lactate fermentation [50]. Further studies are
necessary to investigate whether the secondary metabolic gene
clusters found in the bacterium are expressed under controlled
laboratory conditions.
CONCLUSION
The current study showcases the potential of the four
endophytic Bacillus species, especially Bacillus sp. strain DJ4
isolated from A. pauciorum, as promising sources of novel
antimycobacterial substances. Extract from this bacterium
showed signicant inhibition of growth, biolm formation, and
eradication of M. smegmatis cell biolm. The identication of
eight secondary metabolic gene clusters within the bacterial
genome suggests their potential contribution to the production
of antimycobacterial compounds. These ndings offer
compelling evidence for the antimycobacterial capacity of
endophytic Bacillus and highlight its genomic features, which
hold promise for drug discovery. This opens avenues for
uncovering new antimycobacterial agents from endophytic
bacteria, fostering opportunities within the pharmaceutical
industry. Future investigations should prioritize isolating
active constituents and optimizing metabolite production
using diverse biological, chemical, and molecular techniques.
Additionally, in-depth exploration of the functionality
of the identied secondary metabolic gene clusters in
Bacillus sp. strain DJ4 through mutagenesis or heterologous
overexpression, coupled with understanding the biosynthesis
pathways of antimycobacterial compounds, will enhance the
exploration and harnessing of novel compounds from this
endophytic bacterium.
ACKNOWLEDGMENTS
All authors acknowledge Dr. Tjandrawati Mozef
(BRIN, Indonesia) for providing Mycobacterium smegmatis
ATCC700084 used in this study.
AUTHOR CONTRIBUTIONS
All authors made substantial contributions to
conception and design, acquisition of data, or analysis and
interpretation of data; took part in drafting the article or revising
it critically for important intellectual content; agreed to submit
to the current journal; gave nal approval of the version to be
published; and agree to be accountable for all aspects of the
work. All the authors are eligible to be an author as per the
international committee of medical journal editors (ICMJE)
requirements/guidelines.
FINANCIAL SUPPORT
Funding for this research was provided by the
Directorate of Research and Innovation at IPB University,
through the Young Lecturer Research Program (Penelitian
Dosen Muda) 2023 (grant number: 11425/IT3/PT.01.03/
P/B/2023) awarded to Jepri Agung Priyanto.
CONFLICT OF INTEREST
The authors report no nancial or any other conicts
of interest in this work.
ETHICAL APPROVALS
This is an observational study did not involve human
or animal participants. Ethics approval was not required for this
study.
DATA AVAILABILITY
All experimental data collected in this research are
provided in this paper.
PUBLISHER’S NOTE
All claims expressed in this article are solely those
of the authors and do not necessarily represent those of the
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Priyanto et al. / Journal of Applied Pharmaceutical Science 0 (00); 2024: 001-012 011
publisher, the editors and the reviewers. This journal remains
neutral with regard to jurisdictional claims in published
institutional afliation.
USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED
TECHNOLOGY
The authors declares that they have not used articial
intelligence (AI)-tools for writing and editing of the manuscript,
and no images were manipulated using AI.
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How to cite this article:
Priyanto JA, Prastya ME, Hening ENW, Astuti RI. Exploring
antimycobacterial potential and proling secondary
metabolite gene clusters in the whole genome of Bacillus
isolated from dogfruit (Archidendron pauciorum). J Appl
Online First
Pharm Sci. 2024. http://doi.org/10.7324/JAPS.2025.204561