Extraction, Separation and Characterization of Antibiotic Compound produced by Streptomyces rimosus (ACW9) Active against MDRs Uropathogens

Abstract

Soil is a well-studied ecological niche for the discovery of beneficial biologically active chemicals like therapeutically relevant antibiotics. Actinobacteria have been recognised as one of the largest microbial groups in soil that are capable of producing a wide range of beneficial secondary metabolites and compounds with varying characteristics. Therefore, the current study aimed to assess antimicrobial and antioxidant activities of Streptomyces rimosus ACW9 isolated from wheat farm soil. Molecular identification (16S rRNA ribotyping) was used to identify strain ACW9. Ethyl acetate crude extract displayed promising antibacterial activity against clinically isolated uropathogens, Enterobacter sp. (20 mm) and Bacillus subtilis (14 mm). In addition, the extract showed 62.3% DPPH scavenging with IC50= 1.829 mg/ml. The characterization of the purified metabolites from strain ACW9 was done by GCMS, FTIR, and HPLC techniques. The profound metabolites of ethyl acetate extract were identified by GCMS were cyclohexane, (-2-Nitro-2-Propyl), hexadecane, and pentanoic acid. It may be concluded that actinobacteria isolated from wheat farm soil is a promising source for medicinally important bioactive compounds.

Introduction

I. INTRODUCTION

Urinary tract infections (UTIs) are common bacterial infections that can occur in any part of the urinary system, including the kidneys, ureters, bladder, and urethra [1], [2]. The bladder and urethra are the most infected parts of the lower urinary tract [3]. Each year, around 150 million people globally have UTI, resulting in huge socioeconomic costs. UTIs are more frequent in women than men due to many risk factors, including coitus, female anatomy, vaginal infection, diabetes, and an individual history of UTI. Nearly 50-60 % of women experienced this infection at least once in their lives [4], [5]. Gram-negative bacteria such as Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Citrobacter spp. Among Gram-positive bacteria, Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae are the most common and predictable types of bacteria that can cause UTI infections. Antibiotics are routinely used to treat UTIs, which results in long-term changes in the susceptibility and resistance patterns of different bacterial strains implicated in the pathogenesis [6], [7]. The increased frequency of multi-drug resistance (MDR) pathogens as well as cross resistances with existing antibiotics pose a serious threat to public health [8].

The search for a novel bioactive compound is desperately needed to combat this drug resistance issue. Therefore, the classic strategy of isolating and cultivating novel microbes from natural environments such as wheat field soil is still beneficial and has resulted in the identification, production, and commercialization of antibiotics. Soil microorganisms are important for the development of novel pharmaceuticals, nutriment materials, cosmetics, enzyme inhibitors, immune modifiers, and vitamins [9], [10]. Among the soil microorganisms, the actinomycetes constitute a major group with 80 species [11], practically all of which are from terrestrial soils, where they live mostly as saprophytes and endophytes with significant chemical and morphological variation but originating from a distinct evolutionary route [12]. Actinobacteria are gram-positive bacteria with high G+C content in their DNA and a characteristic filamentous appearance [13]. They are recognised as the most ubiquitous source of secondary metabolites, particularly antibiotics as well as anticancer, antiparasitic, antiviral, and antifungal compounds [14], [15]. More than 70–80% of all known antibiotics have been isolated from actinomycetes and are used in medicine and agriculture [16]. The genus Streptomyces is the biggest producer of antibiotics. Several microbial secondary metabolites are reported to be rich sources of therapeutic drugs [17].

The present study aimed to evaluate some actinomycetes from soil samples collected from wheat farm fields for harnessing their antimicrobial activity against certain pathogens that are known to cause urinary tract infections.

II. MATERIALS AND METHODS

A. Isolation of Actinobacteria from soil sample

For the isolation of strain ACW9, soil sample was collected in a Nasco sampling bag (Himedia) from wheat farm field in Haridwar, India. Actinobacterial strains were isolated on actinomycetes isolation agar (AIA) using the 10-fold serial dilution technique. All the plates were incubated at 28°C for 6-7 days. The isolated colonies were maintained on glycerol yeast agar medium plates at 4°C until further analysis.

B. Test Human Uropathogens

In our study, the following uropathogens, Enterobacter tabaci (MW785203), Pseudomonas aeruginosa (MW970251), Bacillus subtilis (MW785179), Staphylococcus aureus (MW952818) were used to assess the antimicrobial activity. All these uropathogens were clinical isolates. All test strains were maintained in nutrient agar slant at 4°C.

C. Primary Screening By Cross Streak Method

The antagonistic activity of isolated strain ACW9 was determined using the cross-streak method. Strain ACW9 was streaked in the centre of the plate and incubated at 28°C for 5-6 days. After that, freshly grown test microbial strains were streaked perpendicular to the edge of the initial streak and incubated at temperature 35°C for another 24-48 hours. The distance of inhibition between strain ACW9 and the test uropathogens was used to calculate inhibitory activities [18].

D. Characterization Of Active Actinobacteria ACW9

Morphological characteristics were studied by culturing ACW9 on AIA and broth medium, as described in Bergey's Mannual of Determinative Bacteriology.

Scanning electron microscopy was used to examine the morphological spore pattern [19]. For physiological characterization, different carbon sources were accompanied in basal medium at a concentration of 1% and incubated at different temperatures (20°C, 25°C, 30°C, 35°C, 40°C), pH (5-9), and NaCl (1-5 % w/v) ranges. Biochemical attributes like production of some specific enzymes were studied using catalase, oxidase, urease, and gelatin hydrolysis. All the results were demonstrated as either positive or negative.

E. Molecular identification -16S rRNA ribotyping of ACW9

16S rRNA gene sequencing method was employed for molecular characterization. The genomic DNA of ACW9 was isolated using method described by [20].

Actino specific forward primer 16S (5'-GGATGAGCCCGGCCTA-3') and 16S reverse primer (5'-CGGTGTGTACAAGGCCCGG-3') were used to amplify the 16S ribosomal sequence from genomic DNA in a thermal cycler. The Blast tool was used to compare the similarity of the obtained sequence.

The phylogenetic tree was built using neighbour joining method using MEGA X. The 16S rRNA sequence was submitted to NCBI GenBank using Banklt protocol.

F. 16S rRNA Bioinformatics

Mfold webserver (http://unfold.rna.albany.edu/) was used to predict the secondary structure stability of 16S rRNA in terms of Gibbs free energy. The sequence of Streptomyces rimosus was submitted to the Mfold server and run on the default setting. Furthermore, restriction sites were also assessed using the NEB cutter (nc2.neb.com/nebcutter2/).

G. Bioactive production and fermentation of ACW9

Seed culture of ACW9 was inoculated in 500 mL of ISP-2 medium and incubated at 180 rpm, 30°C for 7 days. After incubation period, culture broth was centrifuged at 5000 rpm for 20 min and supernatant was collected separately. 

Bacterial metabolites from supernatant were extracted in a separating funnel thrice with an equal volume of ethyl acetate (1:1). Finally, organic ethyl acetate layer containing metabolite was collected and concentrated using the vacuum rotatory evaporator. The crude extract thus obtained was recuperated in Dimethyl sulfoxide (DMSO) and bio-assayed against test organisms.

H. Antagonistic action against test uropathogens

Disc diffusion method was used to assess the antimicrobial activity of crude extract against test uropathogens. Test organisms were subcultured into broth medium and incubated at 34°C for 24 hours. Using sterile nutrient broth, the growth density was adjusted until the desired turbidity at the 0.5 McFarland standard was obtained and swabbed on Muller Hinton agar plates. Paper discs were loaded with 100 µl of crude extract, and DMSO and levofloxacin were used as negative and positive controls, respectively. The loaded discs were placed on test uropathogen cultured plates and incubated for 24-48 hours at 34°C. Following incubation, the zone of inhibition around the discs was measured. Each experiment was carried out in triplicate.

I. DPPH radical scavenging activity of active isolate ACW9

DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical scavenging assay of crude extract was measured using the method described by [21]. In brief, different concentrations of methanolic extract of ACW9 ranging from 0.5-2.5 mg/mL were added with DPPH solution (0.005% in methanol). After 30 min of incubation at room temperature, the absorbance (OD517 nm) of the sample was recorded using methanol as a base line correction. The average absorbance value was calculated after recording all three readings in triplicate. Radical scavenging activity was expressed as the inhibition percentage of free radicals by the sample and calculated by using the formula:

% DPPH radical scavenging activity = [(A control − A sample)/A control] × 100.

The (IC50) value was also calculated. The IC50 (The half -inhibitory concentration) value is the dose required to cause 50% inhibition.

J. Partial identification of the extracted secondary metabolites

1. FT-IR Analysis

The IR spectra of ACW9 ethyl acetate extract were recorded in FTIR, Perkin Elmer, USA Spectrum RX1. For analysis, KBr pellets were prepared by adding 1mg of dried extract to 300 mg of KBr (IR grade), dried under vacuum at 100°C and compressed to disc.  The pellet thus obtained was scanned in a frequency range of 400-4000cm-1 with a resolution of 4cm-1. The spectrum plot of intensity vs wavenumber thus obtained was interpreted. 

2. Gas Chromatography-Mass Spectrometry (GCMS)

The crude extract of Streptomyces rimosus strain ACW9 was analysed using Gas Chromatography-Mass Spectrometry (GC-MS) on a Perkin Elmer-SQ8T-680. The apparatus includes a DB 35-MS capillary standard nonpolar column with dimensions of (30mm × 0.25mm × 0.25um) film. Helium gas (1ml/min) was used as the carrier gas to inject 0.2um-filtered, with temperature ranging from 350°C to 60°C for 15 minutes to 30 minutes. The compounds were identified using the NIST and Willey library attached to GCMS instrument.

3. HP-LC analysis

HPLC is a chromatographic technique for separating a mixture of compounds in order to identify, measure, and purify the individual components of the mixture. In this study, we used the Shimadzu LC-2010AHT to analyse an unknown compound. Column (C18) was used for sample analysis, and different mobile phases were prepared based on the samples. Before injection, the sample was mixed 50:50 with the solvent and filtered through a Millipore filter. Approximately 10 µL of the sample filtrate was injected into column C18 (250mm × 4.6mm × 5mm).

The sample was run for 30 min and the retention time was noted. The UV-VIS detector was set at 245nm and the flow rate was 0.50ml/min. The ACW9 chromatogram's peaks were identified by comparing the elution time to the standard, and the compound was determined.

III. RESULTS AND DISCUSSION

A. Primary screening of strain ACW9

As a part of our research for more potent antibiotic, one promising isolate, ACW9 was found to produce a broad-spectrum activity with strong antibacterial activity against several pathogenic multi drug resistant uropathogens.

The primary screening clearly shows that ACW9 possessed good antimicrobial potential against all the tested uropathogens. This strain was, therefore selected for further isolation of bioactive metabolites, purification, characterization.

B. Identification of ACW9

The bacterial strain ACW9 was found to be Gram-positive, chain forming bacterium with rough surface (Fig.1). The colony showed diffusible brown pigment along the entire colony margin. The morphological, physiological, and biochemical traits of strain ACW9 are mention in detail in (Table.1).

The 16S rRNA gene coding sequence of the strain was analyzed for sequence homology by BLAST tool in NCBI platform. The sequence with highest similarity to closely related strains was retrieved from the database in FASTA format and aligned by CLUSTALW program. Neighbor joining method was used to study the phylogenic relationships among different strains (Fig.2). The strain got maximum homology with strain Streptomyces rimosus NBRC12907 accession number NR_112332.1 (99.49%). Therefore, on the basis of 16S rRNA gene sequence, the isolate ACW9 was designated as Streptomyces rimosus ACW9. The 16S rRNA sequence of ACW9 was deposited in GenBank, NCBI with accession number (MW 767045).

C. Prediction of the secondary structure of 16S rRNA sequence and analysis of restriction sites

The folding of 16S rRNA was predicted to fully understand the thermodynamic stability of the gene sequence (Fig.3a). The Gibbs free energy of 16S rRNA sequence in folded state was found to be -480.10Kcal/mol. This study suggests that minimum free energy (MFE) of the 16S rRNA sequence, indicating high nucleotide stability in strain ACW9 with optimal configuration. GC-AT content from 16S rRNA coding sequence comes out to be 59% and 41% respectively (Fig.3b). The concept of studying free energy associated with the folding of 16S rRNA gene sequence might provide preliminary information to make a concurrent prediction on the stability of the genes. In our study, Streptomyces rimosus ACW9 showed −480.10 kcal/mol free energy of secondary structure, which is more or less in accordance with the previous studies [22], [23].

D. Antimicrobial activity of ACW9 crude extract against MDRS of UTI infection

The strain ACW9 was grown in ISP-2 medium for production of biometabolites. ISP-2 medium was extracted using ethyl acetate solvent. Ethyl acetate was extensively used as a specific solvent for biometabolite extraction. The antimicrobial activity of ethyl acetate crude extracts was assessed by disc diffusion method. The ethyl acetate extract of strain ACW9 was found to be effective against all the tested human uropathogens. The strain ACW9 showed a broad range of antimicrobial activity against both Gram-negative and Gram-positive pathogens. The results of antimicrobial activity of strain ACW9 are given in (Table. 2). The researcher [24] observed a marked difference in the crude extracts in comparison to pure drug that was already in clinical use. In another study, [25] reported extracellular ethyl acetate extract of Methylobacterium sp. with zone of inhibition of 9 mm against B. subtilis.  In several reports, ethyl acetate was mostly used as an extraction solvent to isolate the crude extracts from actinomycetes [26], [27]. Earlier, several studies reported that most of the antimicrobial secondary metabolites were from extracellular actinomycetes [28], [29], [30].

Table 2. Antibacterial activity of ethyl acetate extract of S. rimosus (ACW9) against selected Uropathogens

± (Stander error), ND (non detected)

E. Antioxidant property of crude extract

Several diseases are implicated in high levels of reactive oxygen species (ROS) in the body, including diabetes [31], cardiovascular disease, inflammation and arthritis, cancer, and neurodegeneration [32], [33], [34]. Furthermore, there seems to be growing interest in the use of naturally derived antioxidants rather than synthetic antioxidants by the food industry [35]. DPPH is a stable free radical with maximum absorbance at 517 nm. The decolorization of violet colour of DPPH into yellow colour indicated the reduced form of DPPH demonstrated the extract scavenging ability. The extract of ACW9 showed substantial scavenging activity in range between 18.5±0.033 to 62.3±0.033 with significant dose dependent inhibition of DPPH activity with an IC50 value of 1.829 mg/ml. The results of inhibition are clearly presented in (Fig. 4a-b).

G. GC-MS analysis

The ethyl acetate extract was subjected to GC-MS analysis, and the fractional profiles revealed the presence of various chemical compounds, each with their specific retention time (Fig.6) which depicted that the ethyl acetate extract generated a total of 13 peaks after 30 minutes of run time. The NIST database was used to locate the probable compound as well as its molecular weight for all the compounds present at respective peaks (Table. 3). The major identified compounds were (1). Pentanoic acid, (2). Dodecane, 1- fluoro, (3). Hexadecane, (4). Cyclohexane, (2-Nitro-2- Propyl), (5). 1-Docosene, (6). 5-Ecosane-E, (7). 1-Heptacosanol, (8). N-Tetracosanol-1, (9). Octadecanoic acid, (10). Heptadecane, (11). Pentadecanoic acid, (12). Tetradecanoic acid (13). 9-Octadecanoic 1,2,3 Propanetriyl ester [EEE] is present in the sample ACW9. However, when compared to other chemical components in the extract, Cyclohexane, (-2-Nitro-2-Propyl), and Hexadecane had the highest peak area 15.2% and 16.8%, respectively, and are thus recognised as main contributors. The present results showed the presence of different kinds of fatty acids which are biosurfactants similar results were obtained for glycolipid biosurfactant properties displayed by S. puniceus RHPR9 can be understood [43]. GC-MS analysis revealed the following compounds, cyclohexane, (2-Nitro 2-Phenyl) and hexadecane, which have both been previously reported as antimicrobial [44], [45], [46]. Hexadecane has been reported as an antimicrobial and antifungal compound [47]. Hexadecane plays a major role in many biological activities as a preservative in food as well as in drugs [48]. The bioactive compounds reported in the present study, dodecane, hexadecanoic acid, 1-Heptacosanol, N-tetracosanol, were previously reported as antifungal and antibacterial [49]. Hexadecanoic acid is the most effective antibacterial fatty acid in mice, and it is also the most effective antibacterial fatty acid in humans [50], [51], [52]. Other studies have found octadecanoic acid and tetradecanoic acid to be antioxidants, antimicrobials, and immunomodulators [53]. Despite the fact that it is known to have antibacterial and antifungal properties, there have been few reports on tetradecanoic acid [54], [55]. Octadecanoic acid has antifungal, antitumor, and antibacterial properties [56]. Even though it is known to have antibacterial and antifungal properties, there have been few reports on tetradecanoic acid [57], [58].

H. HPLC analysis

The antibacterial compounds produced by ACW9 were detected using ethyl acetate extract via HPLC analysis. The sample was prepared as mentioned in USP. The obtained chromatogram of ACW9 was compared with the standard antibiotic based on retention time. HPLC chromatogram of ACW9 revealed a major peak with a retention time of 3.457 minutes, which resembles that of standard oxytetracycline. The study suggests, that the strain ACW9 produces oxytetracycline along with other compounds. Corn-cob was used as a substrate in the production of oxytetracycline by Streptomyces rimosus TM-55 in a solid-state fermentation [65]. In the HPLC report of ACW9, major compound may be similar to oxytetracycline, like in the previous report [66]. At retention time 3.4, the extract of isolate ACW9 showed a major peak (Fig.7). When the peak was compared to standards, it was observed that it resembled the oxytetracycline standard peak, intimating that oxytetracycline is the second of the broad-spectrum tetracycline group of antibiotics. Tetracycline is an antibiotic that is used to treat a variety of Gram-positive and Gram-negative pathogens, but it is most effective against Enterobacter sp. and Pseudomonas sp. in the present research. Tetracycline is an antibiotic that is effective against multidrug resistant pathogens. Streptomyces strain ACW9 is a potent antimicrobial producer that can be further studied for other bioactive against different resistant uropathogens due to the presence of all of the important antimicrobial metabolite producers. The current study's findings suggested that the potential activity of the extract against uropathogens could be attributed to cyclohexane and hexadecane, which were also identified by [58, 46].

IV. AUTHORS' CONTRIBUTIONS

Archana Singh conceptualized the study, developed the methodology, wrote the original draft, performed the experiment, and contributed to the validation process. Padma Singh provided supervision throughout the study. Both authors have reviewed and approved the manuscript for submission to your esteemed journal.

V. ACKNOWLEDGEMENT

The authors are thankful to the Department of Microbiology, Girl’s campus, Gurukul Kangri Deemed to be University, Haridwar providing all the necessary facilities required to carry out the research work. The authors are also thankful to Biokart India Pvt Ltd, Bangalore providing molecular facilities.

Conclusion

In conclusion, the extracellular crude extract of Streptomyces rimosus ACW9 showed good activity against all the tested uropathogenic bacteria. The present results suggest that the isolated actinomycete S. rimosus (ACW9) could be used as an antioxidant and antibacterial agent against the tested microbial pathogens.

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