Antibacterial and antibiofilm activities from soil Streptomyces spp. isolated from Muna Island, Indonesia against multidrug-resistant clinical isolates.

doi:10.21203/rs.3.rs-3811919/v1

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Antibacterial and antibiofilm activities from soil Streptomyces spp. isolated from Muna Island, Indonesia against multidrug-resistant clinical isolates.

https://doi.org/10.21203/rs.3.rs-3811919/v1

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The global increase in multidrug-resistant (MDR) bacterial infection has rapidly been gaining concern and leading for investigating new strategies to tackle this problem. In this study, the antibacterial potential of 25 soil actinomycetes strain has been evaluated by initial screening against MDR bacterial strains including Escherichia coli strain M19, Pseudomonas aeruginosa strain M19, Klebsiella pneumoniae strain M19, Bacillus subtilis strain M19, and Methicillin Resistant Staphylococcus aureus (MRSA). Among them, three actinomycetes isolates encoded APM-7, APM-11, and APM-21 exhibited strong and a broad antibacterial spectrum, hence there were selected for further study to extract its secondary metabolites following antibacterial, and antibiofilm assessment. The ethyl acetate extract of those three selected actinomycetes were evaluated for its antibacterial spectrum, and the minimum inhibitory concentration (MIC) ranged from 78 to 10000 µg/mL. Those extracts also displayed significant biofilm inhibition values ranging from 6.06 to 72.4%. Based on the results, APM-21 extract showed the best antibacterial and antibiofilm activities with the strongest values. Further, nucleotide sequencing of the 16S rRNA gene showed that these three potential strains APM-7, APM-11, and APM-21 to have identity with Streptomyces cyaneus, Streptomyces coerulescens, and Streptomyces panayensis, respectively. Moreover, based on Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) analysis, two antibacterial compounds namely rancimanycin III, and enteromycin were detected in all those three extracts. Interestingly, APM-21 extract also contains two prominent antibacterial substances including paramagnetoquinone C, and caerulomycin I, suggesting their contribution to the most potential activities recorded in this study. Ultimately, our study provides new insights into a promising candidate for use in an active compound combating strategy to prevent MDR bacterial strains infection.

Antibacterial

Antibiofilm

enteromycin

rancinamycin

Streptomyces spp

Multidrug resistance (MDR) bacteria considerably have a major impact on human well-being and is expected to increase global mortality and world economic turmoil. Among the diverse modulators of MDR strains, the consumption of weak medicines, low quality of antibiotics, poor infection control, and overuse of drugs related antibiotics are contributing to the emergence of MDR strains [1]. Hence, a comprehensive and multidisciplinary effort is needed to control its resistance by describing the role of antibiotics, the mechanism of action, and the origin of microorganisms [2]. Some previous reports showed that numerous MDR strains were arisen in Indonesian hospital. For example, Klebsiella pneumoniae MDR strains isolated from patients at Soeradji Tirtonegoro General Hospital, Central Java, were resistant to wide range of antibiotics, including ampicillin, amoxicillin, ceftriaxone, cefuroxime, cefazoline, nitrofurantoin, and fosfomycin [3]. In addition, Escherichia coli and K. pneumoniae strains isolated from patients at Dr. Zainoel Abidin, Aceh, Indonesia, were also resistant to beta-lactam antibiotics which results in failure of infection treatment [4]. Therefore, new alternative antibiotic therapy is needed to overcome these resistance cases.

At the same time, biofilms, defined as communities of microorganisms incorporated in a surface-adhered organic polymeric matrix, are important for bacterial virulence factors. It is estimated that more than 99% of all microorganisms reside in various ecosystems of biofilms formation which modulate stronger and more efficiently in dealing with the adverse environment compared to planktonic bacteria forms [5]. Biofilms are widely found in food processing, industry, water distribution, and medical sectors [6]. It is important to note that once the biofilm has formed, the bacteria are protected from immune defense, and chemicals such as antibiotics, detergents, and disinfectants. In severe conditions related to the inappropriate use of antibiotics has promote to strong adaptation of bacterial and selection of multidrug-resistant strains to form biofilm formation which could have negative impact for human health [7]. Elimination of biofilms requires degradation of the biofilm structure or discovery of antibiotics that can disrupt the biofilm matrix. Facing this phenomenon, investigations are needed on new antibacterials, and antibiofilm compounds that are safe and effective for replacement or rotation of the antibiotics used.

Actinomycetes are a group of filamentous Gram-positive bacteria that are known to be capable of producing various great antimicrobial compounds, such as streptomycin, actinomycin, macrolides, acetyl-griseoviridine, and desulphurized griseoviridine [8]. Actinomycetes are known to be isolated from various sources such as the aquatic environment, plants, animals, and soil. Interestingly, the most abundant source of actinomycetes is reported to be derived from soil. Each gram of soil contains about 18 × 10³ up to 2.9 × 10⁶ CFU/g of actinomycetes cells [9]. Isolation of actinomycetes from a unique environment is expected to obtain potential actinomycetes, especially as a producer of antibacterial compounds. The soil on Muna Island, Southeast Sulawesi, Indonesia has characteristics that are dry and calcareous, and has not been polluted by chemical pollutants, so that it can be used as a potential source of actinomycetes [10]. However, studies on the diversity and bioprospection of soil actinomycetes from this area have not been widely studied. The aim of the present work was to investigate the antibacterial and antibiofilm effects of soil Streptomyces spp. strains which isolated from Muna island, Indonesia against clinical MDR bacterial strain. Of note, no report regarding the antibiofilm activity against MDR clinical strains of soil Streptomyces spp. from Indonesia region is available. Therefore, this study belonged to the first report in relation to these interesting topics.

Isolation of soil actinomycetes

The soil samples were collected from Muna Island Southeast, Sulawesi, Indonesia [GPS: latitude 4°54'48.8"S, longitude 122°39'56.7"E]. These sample were acquired from a depth of 5–15 cm and was placed in sterile plastic bags, kept in an icebox, and then aseptically transferred to laboratory of Microbiology, IPB University, Indonesia to isolate targeted actinomycetes. As for isolation technique, we used standard serial dilution method for isolation of soil actinomycetes following method as described elsewhere with slight modification [11]. In short, a total of 1 gr soil samples were suspended in 9 mL of sterile 0.85% NaCl followed by vigorous vortex. Subsequently, 0.1 mL of serially diluted from 10⁴ to 10⁶ dilution was spread on the Humic Vitamin Agar (HVA) plated medium containing 100 µg/mL nystatin as for antifungal selective medium. Plates were then incubated at 28–29° C for 7 days prior to being observed. Single distinct actinomycetes colony was selected, purified, and maintained by subculturing in International Streptomyces Project 2 (ISP-2) medium (composition: 4 g/L yeast extract, 10 g/L malt extract, 4 g/L glucose, and 20 g/L agar, 1 L aquadest), and was preserved at 4°C for further experiment.

Screening for antibacterial activity by double layer method

Each isolate was re-cultured on ISP-2 agar medium, and incubated for 4 days at 28–30°C. On the other hand, we utilized 5 Multi Drugs Resistant (MDR) strains for targeted bacterial which were isolated from clinical patients in Dr. Kariyadi Hospital, Semarang, Central Java, Indonesia. Each bacterial target belongs to the gram-negative including Escherichia coli strain M4, Klebsiella pneumoniae strain M19, and Pseudomonas aeruginosa strain M19. As for gram positive bacterial target namely Bacillus subtilis strain M18, and methicillin-resistant Staphylococcus aureus (MRSA). Those bacterial targets were cultured in Nutrient Broth (NB) medium with shaking of 120 rpm, 37°C for 24 h. The test was designed by pouring Nutrient Agar (NA) medium into a petri dish. After the media had solidified, soon thereafter semi-solid NA medium was poured over it. Of note, this semi-solid NA medium had previously been inoculated with each bacterial target (OD₆₀₀ of 0.6) for 1% (v/v). After the semi-solid layer has solidified, the soil actinomycetes isolate culture (after 4 days incubation) were taken using a round glass rod (6 mm in diameter) and placed upside down on the semi-solid media (actinomycetes colonies was touched the surface of the semisolid agar), and the plates were incubated at 37°C for 24 h. The presence of antibacterial activity was indicated by the formation of an inhibition zone around actinomycetes colonies.

Fermentation and extraction of active metabolites

Three potential soil actinomycetes isolates which had antibacterial activity were cultured in ISP-2 liquid medium and incubated for 10 days at 28–30°C with a shaking at 120 rpm. Further, the culture of each soil actinomycete isolate was added with 1:1 (v/v) ethyl acetate solvent and followed by vigorous shaken at 150 rpm for 2 h. The solvent layer was then evaporated using a rotary evaporator at 45°C. The crude extract obtained is then stored at 4°C prior to being used [12].

Disc diffusion assay

In this study, we utilized the actinomycetes extract concentration for testing of 20 mg/mL. Briefly, each bacterial target that has been incubated for 24 h on NB media at 120 rpm, 37°C (OD₆₀₀ of 0.6) were inoculated 1.5% (v/v) into Mueller Hinton Agar (MHA) media, and then wait until it solidifies. An amount of 20 µL of actinomycetes extract was dropped on sterile disc (6 mm in diameter) and placed on the MHA plate surfaces. Petri dish was further incubated at 37°C for 24 h. The inhibition zone around the disc was observed and its diameter was calculated. 99% dimethyl sulfoxide (DMSO) and tetracycline (300 µg/mL) were applied as negative and positive controls, respectively [13].

MIC and MBC values calculation

Determination of MIC and MBC values was carried out using the micro-broth dilution test method on a 96-well microplate [14]. Briefly, row test on well A was filled with test sample, DMSO (negative control), and tetracycline (positive control) in Mueller-Hinton Broth (MHB) medium with a total volume of 200 µL. As for each of B-H well was filled with 100 µL of MHB medium. All in well A was then diluted twice starting from well A-H by taking 100 µL aliquots from well A then transferred to well B and so on. A total of 100 µL aliquots from the H well were discarded so that the volume of all wells became 100 µL. Subsequently, a total of 100 µL of the targeted bacterial suspension (0.5 McFarland or equivalent to 10⁸ CFU/mL) in 0.85% NaCl solution (w/v) was inoculated into each well, therefore the final volume of each well was 200 µL. The microplate was then shaked on 150 rpm at 37°C for 24 h. The clear well at the lowest sample concentration indicated the MIC value, while the MBC value was obtained by dipping 100 µL of the MIC value well culture and far above it in MHA media. MBC is defined as the concentration of the extract, which is able to kill all targeted bacteria, characterized by the absence of growth of bacterial colonies.

Antibiofilm assessment by crystal violet

Antibiofilm activity was carried out using crystal violet assay [13]. In short, individual wells of sterile microtiter 96 well plates were filled with 100 µL of Brain Heart Infusion (BHI) medium and inoculated with 100 µL of the extract at concentrations of ¼ × MIC, ½ × MIC, 1 × MIC, and 2 × MIC following with serially dilution until well H. Subsequently, each well was inoculated with bacterial target at Mc Farland standard 0.5 bacteria and incubated at 37°C for 2 h under shaking at 120 rpm. Further, the medium was removed, and washed with 0.85% NaCl. Each well was then flooded with 200 µL of 0.1% crystal violet staining followed with incubation for 30 min at 37°C. Absorbance indicating biofilm formation was calculated using ELISA Thermo Scientific Varioskan Flash (ThermoFischer) Elisa reader at a wavelength of 595 nm.

Eradication of cells biofilm

The eradication of cells biofilm was conducted using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Biofilm formation of each bacterial target produced in microtiter 96 well plates prior to be treated with sample. In this study, we used several concentrations of ¼ × MIC, ½ × MIC, 1 × MIC, and 2 × MIC. Briefly, 100 µL bacterial suspension in 0.85% NaCl (0.5 McFarland) was applied to microtiter 96 well plates and added with 100 µL of BHI medium followed by incubation for 5 days at 37°C with shaking on 120 rpm. The medium was periodically changed each 24 h with 200 µL BHI liquid medium (enriched with 0.25% glucose). After biofilm well produced, sample in each concentration supplemented to each plate and incubated again for 24 at the same conditions. Soon thereafter, the medium was eliminated and added with 10 µL of 5 mg/ml MTT solution (Roche) followed by incubation at 37°C for 3 h. An amount of 200 µL of 99% DMSO was applied to dissolve the insoluble formazan crystals and absorbance was detremined using ELISA reader at 595 nm [13].

Molecular identification of potential actinomycetes

Three potential actinomycetes isolates were identified based on the 16S rRNA gene sequence. The actinomycetes genomic DNA extraction procedure was performed using the PrestoTM Bacterial gDNA Mini Kit (Geneaid, Taiwan) according to the manufacturer’s protocol instructions. We used a set primer of 1387R (5'-GGG CGG WGT GTA CAA GGC-3'), and 63F (5'-CAG GCC TAA CAC ATG CAA GTC-3'). The 50 µL PCR reaction was prepared by mixing 5 µL forward primer (10 pmol), 5 µL reverse primer (10 pmol), 25 µL GoTaq Green® Master Mix 2X (Promega), 2 µL DNA template (concentration of 50–100 ng/µL), and 13 µL of nuclease-free water. PCR cycles were carried out for 35 cycles, with PCR conditions of pre-denaturation 94°C for 5 min, denaturation 94°C in 30 s, annealing 55°C in 45 s, elongation 72°C in 90 s, and post-PCR condition 72° C for 10 min [15]. PCR results were visualized by electrophoresis with an amplicon target of around 1,300 bp. Amplicons were then sequenced and analyzed using the BLAST nucleotide program (BLAST-N) at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree was constructed using MEGA (Molecular Evolutionary Genetic Analysis) software version 11.0 with the neighbor-joining tree method and a bootstrap value of 1000x.

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)

Three potential actinomycetes extract was identified its dominant compounds using Xevo G2- XS QTof (Quadrupole Time-of-Flight) mass spectrometry instrument (Waters, USA) via an electron spray interface (ESI). Separation of sample was conducted by stepwise gradients from 95% A and 5 B to 5% A and 95% B for 16 minutes (A contained 0.1% formic acid + distilled water; B contained acetonitrile + 0.1% formic acid). These chromatographic separation conditions were performed using an LC system in the form of Ultra Performance Liquid Chromatography (UPLC)/QTof MS analytical system (Waters). The MS conditions were set as follows: column temperature 40°C, cone voltage 30 V, mass range: 100–1200 Da, capillary 2kV, source temperature 120°C, cone gas flow 50 L/h, desolvation temperature 500°C, desolvation gas flow 1000 L/h, and collision energy (ramp: 10–40 eV). Further, mass spectrometry of each peak was identified using the type of electrospray ionization (ES) Xevo G2-S QTof (Waters) with Quadrupole Time-of-Flight mass spectrometry in positive ion mode. The identified mass and fragment ions were analyzed using the UNIFI software library incorporated to the instrument and the natural products atlas website services (https://www.npatlas.org/).

Statistical analysis

All the data obtained was analyzed in statistical analyses from three experiments and the results were reflected as the mean ± standard deviation. Statistical significance was investigated by one-way analysis or variance (ANOVA) followed by Multiple-Duncan tests which mean values considered statistically different if p < 0.05.

Screening for antibacterial activity

During the isolation process, we successfully isolated 25 actinomycetes isolates. Actinomycetes colonies showing distinct morphological characters were selected and maintained for further antibacterial screening (Fig. 1). Out of 25 total actinomycetes isolates, 21 isolates (84%) exhibited moderate to strong antibacterial activity against gram negative, and gram positive MDR bacterial strains (Table 1). Based on those results, we identified three isolates, namely APM-7, APM-11, and APM-21 showed the best antibacterial activity with the widest inhibition zone derived from its colony by dual culture method (Fig. 2). Therefore, those three isolates were then selected for extraction of its active compound.

Table 1

Antibacterial activity of 25 soil actinomycetes strains against multidrug-resistant clinical isolates
Actinomycetes isolate	Inhibition zone (mm)
Actinomycetes isolate	E. coli	P. aeruginosa	K. penumoniae		B. subtilis		S. aureus
APM 3	-	-	+	-		-
APM 5	+	+	+	-		++
APM 7	++	++	+	++		++
APM 8	-	-	+	-		-
APM 9	+	+	+	+		++
APM 10	-	-	+	-		-
APM 11	++	++	+	++		+++
APM 12	-	-	+	-		-
APM 13	-	-	+	-		-
APM 14	-	-	-	-		-
APM 15	++	++	+	++		+
APM 16	++	++	+	++		++
APM 17	-	-	+	-		-
APM 18	++	++	-	++		++
APM 20	-	-	-	-		-
APM 21	++	++	++	++		++
APM 23	+	+	+	++		+
APM 24	-	-	-	-		-
APM M2	-	-	+	-		-
APM M3	-	-	+	-		-
APM M4	++	++	++	-		-
APM M5	-	-	++	-		-
APM M6	-	-	++	-		-
APM M7	-	-	-	-		-
APM M8	-	+	++	+		++
Note: (-) no activity; (+++) highly active (inhibition zone:>13 mm); (++) moderately active (inhibition zone: 8–13 mm); (+) less active (inhibition zone: ≤8 mm).

Results from extraction process showed that APM-7 isolate produced the highest yield of its crude extract of 0.096% (Table 2). Further, based on disc diffusion antibacterial assay from those 3 selected extracts showed different values in inhibiting the growth against five bacterial targets ranging from 7.5 ± 0.5 to 15.0 ± 2.1 mm (Table 3). Among those three extracts tested, APM-7 extract exhibited the best activity against E. coli and K. pneumoniae. As for APM-11 extract showed the highest inhibition zone value against B. subtilis, and MRSA. On the other hand, P. aeruginosa was inhibited at the best value by APM-21 extract. Those 3 active extracts were then subjected to further antibacterial assessment to determine the MIC, and MBC values.

Table 2

Yield of crude extract from each selected actinomycetes.
Extract code	Culture volume (mL)	Weight (g)	Yield (% w/v)*
APM-7	1000	0.9635	0.096
APM-11	1000	0.4745	0.047
APM-21	1000	0.1045	0.010
*Note: w/v = weight/volume

Table 3

Antibacterial activity of selected soil actinomycetes crude extracts against multidrug-resistant clinical isolates by disc diffusion assay
Extract code	Inhibition zone (mm); (Average ± Standard deviation)
Extract code	E. coli	P. aeruginosa	K. pneumoniae	B. subtilis	S. aureus
APM 7	9.3 ± 0.4^c	9.3 ± 0.4^b	9.3 ± 0.4^b	8.0 ± 0.8^b	8.8 ± 0.6^b
APM 11	8.8 ± 0.2^c	9.2 ± 0.8^b	8.5 ± 0.4^b	15.0 ± 2.1^c	9.5 ± 0.4^b
APM 21	7.3 ± 0.4^b	9.5 ± 1.7^b	7.7 ± 0.8^b	7.5 ± 0.5^b	8.5 ± 1.5^b
Tetracycline	10.0 ± 0.3^d	9.7 ± 2.6^b	8.7 ± 3.2^b	16.7 ± 0.4^c	9.8 ± 1.2^b
DMSO	0 ± 0^a	0 ± 0^a	0 ± 0^a	0 ± 0^a	0 ± 0^a
Note: Extract used at 20 mg/mL, and tetracycline along with DMSO are applied at 300 µg/mL, and 1% (v/v), respectively; Different superscript letters in the same column indicated significantly different.

MIC and MBC determinations

The MIC of those three selected extracts was determined for the standard antibacterial study using a broth microdilution assay. The values varied between 78 and 10000 µg/mL which the lowest MIC value of 78 µg/mL was recorded from APM-21 extract against MRSA. On the other hand, the highest MIC (10000 µg/mL) was showed by APM-7 extract on K. pneumoniae. Intriguingly, APM-11 extract exhibited the lowest (MIC and MBC) values against 4 bacterial targets including E. coli (125 and 250 µg/mL), P. aeruginosa (250 and 500 µg/mL), K. pneumoniae (125 and 250 µg/mL), and B. subtilis (250 and 500 µg/mL) (Table 4). Of note, the lowest MIC and MBC values reflected the most active compound acting as antibacterial agents.

Table 4

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of selected soil actinomycetes crude extracts
Extract code	MIC and MBC values (µg/mL)
	E. coli		P. aeruginosa		K. Pneumoniae		B. subtilis		S. aureus
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
APM 7	625^c	1250^c	625^c	1250^c	10000^c	> 10000^d	2500^d	5000^d	156^c	312^c
APM 11	125^b	250^b	250^b	500^b	125^b	250^b	250^b	500^b	250^d	500^d
APM 21	625^c	1250^c	625^c	1250^c	2500^c	5000^c	625^c	1250^c	78^b	156^b
Tetracycline	8^a	16^a	8^a	16^a	31^a	62^a	4^a	8^a	4^a	8^a
Note: Different superscript letters in the same column indicated significantly different.

Antibiofilm ability

The results of the antibiofilm ability from these three selected extracts against all MDR bacterial strain demonstrated that all extracts significantly (p < 0.05) reduced biofilm formation in a concentration-dependent manner. The highest inhibition value was recorded on APM-21 extract (with concentration of 2×MIC) against MRSA with the inhibition value of 72.4% (Fig. 3C). Furthermore, in line with MIC and MBC values, we obtained that APM-11 extract on concentration of 2×MIC exhibited the highest inhibition activity against 4 bacterial targets with values of 66.29%, 62.14%, 70.27%, and 61.18% against E. coli, P. aeruginosa, K. pneumoniae, and B. subtilis, respectively (Fig. 3A-C). In contrast, the lowest reduction biofilm was recorded by APM-7 extract on concentration of ¼ × MIC with the inhibition values 6.02% and 6.20% against K. pneumoniae, and B. Subtilis biofilms, respectively (Fig. 3A).

Eradication of cells biofilm

The percentage of eradication cells biofilm in the presence of the three selected extracts is presented in Fig. 3D-F. There was a significant difference in the cell’s biofilm exposed to all three test samples at the concentration of 2×MIC when compared to the cell’s biofilm in ¼ × MIC treatment. The extract, APM-21 (2×MIC), reduced cells biofilm with highest value of 68% against MRSA. This corresponding extract also exhibited the best activity in reducing B. subtilis cell’s biofilm of 58.31% (Fig. 3E). As for APM-11 extract (2×MIC) eradicated the most cell’s biofilm of E. coli, and K. pneumoniae with values of 64.21%, and 65.92%, respectively. On the other hand, at the lowest concentration of ¼ × MIC, APM-7 extract had poor eradication of cell’s biofilm of 9.00%, and 7.82% against K. pneumoniae, and P. aeruginosa, respectively.

Conventional and molecular identification

Three selected actinomycetes were further identified based on conventional and molecular approach. Each isolate was grown on ISP-2 solid media and observed macroscopically and microscopically. Those potential isolates had different colony morphology, both in terms of diameter, shape, color, elevation, and colony boundaries (Table 5). In addition, microscopic observations showed that three isolates had a fibrous cell shape and were stained purple on Gram stain indicating gram positive (Fig. 4)

Table 5

Morphological characters of each selected actinomycetes.
Morphological characters	Isolates code
Morphological characters	APM-7	APM-11	APM-21
Colony diameter	± 0.5 mm	± 0.4 mm	± 0.7 mm
Shape	Circular	Irregular	Circular
Colony color	White	Yellow orange	Light brown
Elevation	Umbonate	Umbonate	Umbonate
Border	Lobate	Filamentous	Entire

Based on 16S rRNA gene analysis, those three potential isolates were identified as Streptomyces spp. with a high similarity value (> 98%) (Table 6). Further analysis indicated that APM-7, APM-11, and APM-21 isolates had a highly similarity with Streptomyces cyaneus strain NBRC 13346, Streptomyces coerulescens strain AS 4.1597, and Streptomyces panayensis strain 21, respectively. The phylogenetic tree that has been made showed that those three potential isolates and their homologous species belong to the same clade of Streptomyces spp. (Fig. 5).

Table 6

The identity of three selected isolates according to 16S rRNA sequence
Isolate code (accession number)	Closest relative species (accession number)	Query cover (%)	Similarity (%)
APM 7 (OR066165)	Streptomyces cyaneus strain NBRC 13346	100	98.85
APM 11 (OR066165)	Streptomyces coerulescens strain AS 4.1597	100	99.59
APM 21 (OR066165)	Streptomyces panayensis strain 21	100	99.25

LC-MS/MS analysis of 3 potential actinomycetes extracts

The LC-MS/MS analysis of three selected extracts revealed seven recognized putative compounds which were dominant in each extract (Fig. 6A-C). All those putative compounds were the result of analysis using the unify software incorporated with instruments combined with a comparison of MS fragmentation with library of the npatlas website (Supplementary Information S1). In addition, comparing the peaks' mass spectra in the unify and npatlas database aided in identifying them by relying on retention time, molecular formula, and molecular mass (Table 7). Interestingly, we identified 4 compounds which are presence in both APM-7 and APM-21 extracts namely rancinamycin III, enteromycin, bromoisorumbrin, and maremycin. As for APM-21 extract showed also having paramagnetoquinone C, and Caerulomycin I. On the other hand, APM-11 extract besides having rancinamycin III, and enteromycin, this extract also contains ashimide B and thiazohalostatin (Fig. 5B).

Table 7

LC-MS/MS profile from dominant compounds of selected actinomycetes strains crude extracts.
Retention time (minutes)	Compounds	Other sources	Bioactivity	References
APM-7 extract
2.56	Rancinamycin III	S. lincolnensis	Antibacterial	[33]
3.29	Enteromycin	Micromonospora sp.	Antibacterial	[34]
3.54	Unknown	-	-	-
4.24	7-deoxyechinosporin	S. pseudogriseolus; Amycolatopsis speibonae	Antibacterial, Antifungal	[35, 36]
7.43	Unknown	-	-	-
9.00	Bromoisorumbrin	Auxarthron umbrinum	Cytotoxicity	[37]
11.26	Maremycin	Streptomyces sp.	Antifungal	[38]
APM-11 extract
2.56	Rancinamycin III	S. lincolnensis	Antibacterial	[33]
3.29	Enteromycin	Micromonospora sp.	Antibacterial	[34]
5.61	Unknown	-	-	-
9.62	Unknown	-	-	-
9.87	Ashimide B	Streptomyces sp.	Cytotoxicity	[39]
10.36	Unknown	-	-	-
10.56	Thiazohalostatin	Actinomadura sp.	Cytoprotective	[40]
APM-21 extract
2.56	Rancinamycin III	S. lincolnensis	Antibacterial	[33]
3.29	Enteromycin	Micromonospora sp.	Antibacterial	[34]
3.54	Unknown	-	-	-
6.80	Paramagnetoquinone C	Actinoallomurus sp.	Antibacterial	[41]
9.00	Bromoisorumbrin	Auxarthron umbrinum	Cytotoxicity	[37]
9.80	Caerulomycin I	Actinoalloteichus cyanogriseus	Antibacterial	[42]
11.27	Maremycin	Streptomyces sp.	Antifungal	[38]

Researchers worldwide are currently searching intensively for novel, active, and broad-spectrum antibiotics from diverse microbial sources including actinomycetes which are prominent makers of active metabolites in soil habitats. Isolation of novel actinomycetes strains from extreme and unexplored areas is one of the strategies to unravel potential of new active metabolites [16]. From this perspective, Muna island is a region with natural views of karst hills with dry soil and calcareous rock [17]. Such a harsh environmental allow microbial colonization with the ability to synthesize unique antibacterial compounds. At the same time, since the emergence of multidrug-resistant bacteria and the ongoing effort to overcome infectious diseases, there is a high increasing demand for novel and effective antibiotics from various sources including actinomycetes [18]. Hence, our study was conducted to search for active metabolites against multidrug-resistant bacteria produced by soil actinomycetes.

The initial screening for antibacterial activity indicated that the 21 isolates had varying antibacterial activity (Fig. 2). The presence of antibacterial activity was showed by the formation of an inhibition zone around the actinomycetes colonies which indicated that bacterial cells targets is killed by active compounds produced by actinomycetes. We further identified 3 isolates encoding APM-7, APM-11, and APM-21 had the best ability to inhibit the growth of all five bacterial targets as indicated by the largest of the inhibition zone. Of note, the differences in the antibacterial activity of each isolate can be influenced by some factors such as compound structure, the concentration of active compounds, and the level of resistance of each targeted bacterial strain. In addition to corresponding results, those three potential actinomycetes isolates that can inhibit all bacterial target both Gram-negative and Gram-positive bacteria are thought to be able to produce broad-spectrum antibacterial compounds [19]. Therefore, those three actinomycetes isolates were selected for further intensive analysis to extract its active compounds following antibacterial and antibiofilm assessment.

Our findings of antibacterial activity from three selected actinomycetes extracts showed that the inhibition zone diameters ranged from 7.3 ± 0.4 to 9.5 ± 1.7 and from 7.5 ± 0.5 to 15.0 ± 2.1 mm against MDR gram-negative and positive bacterial strains, respectively (Table 3). These results were in line with previous investigations whereby actinomycetes isolated from soils exhibited considerable antibacterial activity against both gram-negative and positive bacteria [20]. In comparing with previous studies derived from Indonesian actinomycetes strain, our results showed stronger antibacterial activity based on disc diffusion method. Extract from Streptomyces sp. strain H11 isolated from soil mangrove exhibited inhibition zone of 5.8 ± 1.4 and 4.96 mm against E. coli and S. aureus, respectively [21]. However, another extract from actinomycetes isolated from soil rhizosphere exhibited stronger activity with the inhibition zone of 9.23 and 8.9 mm against E. coli and S. aureus, respectively [22]. Of note, some previous research was tested against non-MDR bacterial strains. In addition, the chemical and physical characteristics of soil, as well as the soil microbial taxa, differed depending on the environmental condition. Microbials must adapt and develop to withstand distinct stress circumstances and, as a result, have the potential to produce novel active compounds with specific bioactivities [23]. Therefore, our three potential actinomycetes from Muna island soil represent an unexplored source to unravel potential active compounds. Notably, a few reports have demonstrated antibacterial activity from actinomycetes active compounds against MDR bacterial strain [24], but they have not been reported previously derived from local Indonesian actinomycetes strains.

Furthermore, the antibacterial capacity of natural products could be classified based on the range of their MIC values. According to [25], an extract is classified as very active if it has MIC value (< 100 µg/mL), active (100⎯500 µg/mL), moderate (500⎯1000 µg/mL), and less active (> 1000 µg/mL). Our results showed that the APM-7 extract was categorized active against MRSA, and moderate activity on E. coli and P. aeruginosa. In addition, APM-11 extract has an active category against all bacterial targets. As for APM-21 extract was highly active against MRSA, and moderate category on E. coli, P. aeruginosa, and B. subtilis. Interestingly, we recorded that the lowest antibacterial MIC value which categorized as very active was found on the APM-21 extract against MRSA (78 µg/mL) (Table 4). This result was lower than that previous study derived from S. lienomycini BOGE18 extract (isolated from Egypt) with an MIC value of 250 µg/mL against similar MDR S. aureus strain WS12 clinical isolates [24]. Based on this report, our potential isolate APM-21 has stronger antibacterial activity as shown by lower MIC value. On the other hand, as comparing with another actinomycetes strain from Indonesia, namely S. koyangensis strain SHP 9 − 3 (MIC value of 7.81 µg/mL), our MIC value against S. aureus is slightly larger indicating weaker activity [26]. However, in previous study used non-MDR clinical isolates of S. aureus strain. Based on those antibacterial assessments, it was found that most of the isolate, namely APM-21 was very active against MRSA as gram positive bacteria. This was majorly attributed to the morphological differences which presence of lipopolysaccharide in gram negative bacteria as hydrophobic layer resulted impermeable condition to lipophilic compounds. As for gram positive bacteria reported have only an outer peptidoglycan layer which is not sufficient as permeability barrier, thus will be more susceptible to the metabolites [11].

We further report here the antibiofilm activity from those three potential actinomyces extracts. Biofilms are reported as the dominant microbial lifestyle, and are present in numerous conditions including industrial, clinical, and food treatment environments. The biofilm structure is crucial in antibiotic resistance mechanism because it protects microbial cells from the host immunity and avoids the penetration of antibiotics [27]. In this study, for the first time, the antibiofilm activity of extract from actinomycetes strains which isolated from Indonesia regions on MDR bacterial strains, were studied. The results showed that APM-11 extract (2× MIC) has the best antibiofilm ability against biofilms formed by E. coli, P. aeruginosa, K. pneumoniae, and B. subtilis with the inhibition value range from 61.18 to 72.27% (Fig. 3B). As for MRSA biofilm formation was inhibited at the highest inhibition value of 72.4% by APM-21 extract (2× MIC) (Fig. 3C). In present study were also investigated the eradication of cells biofilm which resulted in slightly similar with antibiofilm activity as APM-11 and APM-21 showed the highest percentage of eradication cells biofilm (Fig. 3D-F). It should be noted that several antibiofilm agents were previously reported from numerous actinomycetes strains, including Streptomyces sp. against S. aureus (MRSA) [28], Streptomyces sp. SBT343 against S. epidermidis [29], and S. lienomycini BOGE18 against S. aureus WS12 MDR strain [24] with diverse antibiofilm capacity. However, as compared to these previous studies, i.e., compared to S. lienomycini BOGE18 extract against S. aureus MDR strain, our extract has a lower inhibition antibiofilm value. This is presumably due to the sources of actinomycetes isolates not being the same, thus the active compounds have different characters and bioactivities.

Furthermore, three selected isolates were characterized by using nucleotide sequencing and identified as S. cyaneus (APM-7), S. coerulescens (APM-11), and S. panayensis (APM-21) (Table 6). Based upon the identity acquired by morphological, and phylogenetic analysis in the selected strains, we could assume the possibility of involvement from diverse active compounds which responsible for their broad-spectrum antibacterial activity. Interestingly, some previous studies have reported that those three identified Streptomyces spp. exhibited antibacterial activity against both gram-positive and gram-negative bacteria on non-MDR strains. Several compounds responsible for the antibacterial activity of the three Streptomyces spp. isolates have been identified i.e., cirramycin-B, di-(2-ethylhexyl) phthalate and cephamycin which isolated from S. cyaneus, S. coerulescens G60, and S. panayensis, respectively [30–32].

In our continuing effort to investigate the potency of three selected actinomycetes isolates, we have prompted further analysis to identify the chemical constituents present in those three actinomycetes extract through LC-MS/MS analysis. The results obtained indicated that our three extracts contained two putative compounds, namely rancinamycin III and enteromycin (Fig. 6A-C). These two compounds were previously reported to have been identified from several actinomycetes strains with highly antibacterial activity (Table 7) [33–34]. Therefore, the strong antibacterial activity of our three potential extracts is thought to be due to the presence of these two compounds. In addition, as for APM-7 extract also contained other putative compounds, namely 7-deoxyechinosporin, bromoisorumbrin, and maremycin which were reported to have antibacterial, cytotoxicity, and antifungal activities, respectively [35–38]. Furthermore, as for APM-11 extract, other putative compounds were also identified, namely ashimide and thiazohalostatin which were reported as having cytotoxic and cytoprotective activities, respectively [39–40]. Interestingly, the corresponding extract which had the strongest antibacterial activity against MRSA, namely APM-21 extract, was also identified other two putative compounds known as antibiotic from actinomycetes group, namely Paramagnetoquinone C and Caerulomycin I. For the record, these two compounds are known to have strong antibacterial activity [41–42]. Therefore, our results confirm that the antibacterial along with antibiofilm properties of these three potential extracts might be due to the presence of these promising compounds.

In conclusion, three soil Streptomyces spp. encode APM-7, APM-11, and APM-21 exhibited potential antibacterial, and antibiofilm activities against MDR clinical isolates. The MIC value of the antibacterial compounds against the test pathogens indicated the potent activity which highlights its prospective and could be a promising candidate in the new antibacterial agent towards MDR strains especially of MRSA, since the isolate of APM-21 showed the strongest activity. Taken together, our findings demonstrated that naturally occurring Streptomyces spp. have a remarkable potential to produce active constituents against bacteria and disruption of its biofilm structure which enabling discovery of novel antibiotics. Further ongoing characterization and structure elucidation of the active compounds are required to provide deep insight into the development of antibiotic candidate derived from Indonesian Streptomyces spp. isolates.

Funding sources

This work was partly funded by the Research Health Organization of National Research and Innovation Agency (BRIN), Indonesia through “Prototype of directed therapy drug materials program 2024” and supported by “National Collaborative Research 2023 (RiNa)” from the Directorate of Research and Innovation, IPB University, Indonesia.

Acknowledgement

Authors thank Dr. Tjandrawati Mozef from The Research Center for Pharmaceutical Ingredients and Traditional Medicine, BRIN, Indonesia, and Apriza Yuswan for their generous assistance and discussion during this research.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

MEP and JAP planned the work, and contributed to the article’s conception, writing and editing, and made critical revisions to the manuscript. MEP, JAP, SS, LOAFH, RK, ES, and VP conducted a study of the antibacterial, antibiofilm activities, and molecular identification of selected actinomycetes strain. GP, and RTD performed compounds profiling and interpreting LC-MS/MS data. All authors read and approved the final version of the manuscript.

Data Availability

Data underlying the reported findings are deposited in the NCBI dataset at: GenBank https://www.ncbi.nlm. nih.gov/genbank/ as follows: APM-7 (OR066163), APM-11 (OR066164), APM-21 (OR066165).

Consent to Participate

The authors have agreed to participate in the publication of the paper.

Consent for Publication

All authors have agreed to publish the paper.

Competing Interest

The authors declare no competing interests exist in this study.

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Antibacterial and antibiofilm activities from soil Streptomyces spp. isolated from Muna Island, Indonesia against multidrug-resistant clinical isolates.

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Abstract

Figures

Introduction

Materials and Methods

Isolation of soil actinomycetes

Screening for antibacterial activity by double layer method

Fermentation and extraction of active metabolites

Disc diffusion assay

MIC and MBC values calculation

Antibiofilm assessment by crystal violet

Eradication of cells biofilm

Molecular identification of potential actinomycetes

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)

Statistical analysis

Results

Screening for antibacterial activity

MIC and MBC determinations

Antibiofilm ability

Eradication of cells biofilm

Conventional and molecular identification

LC-MS/MS analysis of 3 potential actinomycetes extracts

Discussion

Conclusion

Declarations

References

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