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Antimicrobial Metabolites Profile and Inhibitory Activity of Streptomyces xinghaiensis-OY62 Isolated from Soil against Indicator Strains

Aims: The aims of this study were to screen for potential broad-spectrum antimicrobial-producing actinomycetes from the tropical rain forest of Oyo State, Nigeria, to assess the effects of cultural conditions on antimicrobial metabolites, characterize the metabolites and determine its antimicrobial activity against indicator strains. Place and Duration of the Study: Department of Microbiology, University of Ibadan, Ibadan, between April 2014 and August 2016. Methodology: Ten soil samples were purposively collected between April and June 2014 for the isolation of Actinomycetes. The isolated strain was identified culturally and molecularly using 16S rDNA. The effect of cultural parameters on antimicrobial activity was done by a standard method. The antimicrobial metabolites were produced by submerged fermentation. Partial purification was carried out by column chromatography. Chemical characteristics of the metabolites were determined Original Research Article Adeyemo and Onilude; SAJRM, 1(3): 1-15, 2018; Article no.SAJRM.41569 2 by Fourier transformed infra-red spectrometer (FTIR) and gas chromatography coupled with a mass spectrometer. The antimicrobial activity was done by agar well diffusion and macro broth dilution. Results: Isolate OY62 had broad-spectrum antimicrobial activity and it was identified using its 16S rDNA gene as Streptomyces xinghaiensis-OY62 (KU934248). The highest antimicrobial activity against indicator strains was recorded between pH 7 and pH 8, 0.8% (w/v) sodium chloride, at elevated temperature 55C, casein+KNO3 as a nitrogen source, starch or absence of carbon source and incubation period of fifteen days. Aliphatic alkene, hydroxyl, carboxylic acids, amides and carbonyls were functional groups detected while thirteen antimicrobial metabolites were characterized. The MIC against indicator strains was between 6.25 mg/L to 12.5 mg/L. Streptomyces xinghaiensis-OY62 exhibited broad-spectrum activity against indicator strains. Conclusion: The observed results showed that potential broad-spectrum antimicrobial-producing strains of Streptomyces could be isolated from the soil of southwestern Nigeria, which could be useful in the production of antimicrobials that can inhibit the growth of resistant pathogens, reduce microbial infections and death.


INTRODUCTION
Streptomyces are Gram-positive filamentous bacteria found predominantly in the soil. They have a morphology cycle that is very complex [1]. They are known to produce a wide range of secondary metabolites including antimicrobial compounds [2]. Most of these secondary metabolites are bioactive in nature. They have unique structures and their formations are regulated by fermentation medium, growth rate, feedback control, enzyme inactivation and induction [3]. They are not useful in the growth or reproduction of the producing microorganism. They are usually synthesized in a consortium of similar metabolites that are chemically different [4]. Examples of secondary metabolites are phenol 2, 4-bis (1, 1-dimethyl ethyl and dibutyl phthalate that had been reported to possess antimicrobial property [5,6]. In addition, aliphatic compounds such as nonadecene and tetradecene have been identified in the solvent extract of different strains of Streptomyces [7]. Furthermore, Silber et al. [8] showed that bis (2-ethylhexyl) phthalate possessed inhibitory property against Micrococcus luteus, Vibrio harveyi and Pseudoaltermones piscida. Many of these secondary metabolites are antibacterial, antifungal, anti-tumour in nature and they have found applications in the treatment of human infections [9].
Bioactive compounds have a tremendous impact on human health during the second half of the century [10]. They have been reported to aid mineral absorption, oxidative stress protection, glucose metabolism, lowering of cholesterol, antithrombic, hypotensive, antimicrobial as well as immunomodulatory agents [11].
The relevance of bacteria as natural sources of bioactive compounds has been a subject of interest to pharmaceutical industries [11].
Therefore, due to continuous emergence and reemergence by pathogenic microorganisms to commercially available antimicrobial drugs, there is a need to search and screen for more antimicrobial metabolites from actinomycetes. It is estimated that these group of bacteria produce more than seven thousand metabolites [12]. Hence, the general objective of the study was to screen for antimicrobial-producing actinomycetes from the uncultivated tropical rain forest of Oyo State, southwestern Nigeria, to profile antimicrobial metabolites produced by Streptomyces xinghaiensis-OY62 and to assess its inhibitory activity against some indicator strains.

Soil Samples Collection
Ten soil samples were purposively collected at a depth of 10- 15

Isolation of Actinomycetes
Ten-fold serial dilution was carried out on each of the soil samples using the description of Harrigan and McCance [13]. The isolation was carried out using Pour Plate technique with media that included glycerol asparagine agar, tyrosine agar and starch casein agar [14,15]. The plates were incubated at room temperature (28°C-30°C) for seven days.

Screening for Antimicrobial Activity of the Actinomycete Isolates
The antibacterial activity of the actinomycete isolates against test strains was done by crossstreaking technique following the procedure of Oskay [16].

Cultural and Morphological Studies of the Selected Actinomycetes Isolate
The cultural and morphological characteristics of the selected actinomycetes were carried out following the procedures described in International Streptomyces Programme ISP by Shirling and Gottlieb [15].

Molecular Identification Using the 16S rRNA Gene Sequence
Chromosomal deoxyribonucleic acid DNA was extracted using the versatile quick-prep method for genomic DNA [17]. The 16S rRNA analysis of the isolated strains was carried out using the method described by Pospiech and Newmann [17].   (4,28,35,45,55 and 60), carbon source (w/v) (glucose, galactose, maltose, sucrose, glycerol, starch and control), nitrogen source (ammonium sulphate, peptone, yeast extract, malt extract, casein, potassium nitrate, casein+ potassium nitrate and control) as well as production period in days (3, 5, 7, 10 and 15) on the inhibitory activity of Streptomyces xinghainesis-OY62 was assessed by inoculating 500 mL of basal medium with 5.0% (v/v) 24 h old culture of Streptomyces xinghainesis-OY62 followed by incubation at 30°C. However, the best-supporting fermentation condition of a previously determined parameter was kept constant in subsequent parameter under study.
The inoculated flasks were placed on an Orbital Shaker (Platform Shaker MSZ-100A) at 150 rpm and incubated for seven days, after which the fermentation was terminated. The fermented broth was filtered using Whatman No. 1 and treated with 50% (w/v) ammonium sulphate, so as to remove interfering proteinous materials from the antimicrobial extract. The treated filtrate was centrifuged at 5000 rpm for 20 min (Wincom 80-2). Additionally, an equal volume (1:1) of the supernatant and ethyl acetate (Loba Chemicals, India) were strongly shaken together for 30 min in separating funnel to effect the extraction of the antimicrobial metabolites by the solvent. The separating funnel with the mixture was further allowed to stand for another 30 min. The supernatant, solvent contained extract was collected and concentrated at 60°C using Rotary Evaporator (Rotatory Evaporator, RE-52A) [9]. The crude antimicrobial extract was used for the assessment of the cultural conditions using agar well diffusion assay.

Agar Well Diffusion
The effect of cultural conditions on the antimicrobial activity of the Streptomyces xinghaiensis-OY62 against the following indicator strains: Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Campylobacter jejuni ATCC 33291 and Pseudomonas aeruginosa ATCC 9077 was carried out. A well of 6.0 mm diameter was made at the centre of Petri dishes containing Mueller Hinton agar, which had been previously seeded with a 24 h culture of the indicator strains, which had their turbidity adjusted to 0.5 McFarland standards (1.5x10 8 CFU/mL). Each well was filled with crude extract (50.0 μL) and the plates were incubated at 37°C (Memmert 854, Schlabach, Germany). The zones of inhibition were read in triplicate using a ruler. The 6.0 mm of the well was subtracted from the values obtained.

Partial
Purification and Characterization of the Antimicrobial Compounds Produced by

Streptomyces xinghaiensis-OY62
Ten litres of sterile starch casein broth was inoculated with 5.0% (v/v) 24 h seed inoculum of Streptomyces xinghaiensis-OY62 and incubated at 55°C. This was followed by separation of the mycelial debris from the fermented broth by filtration using Whatman No.1 filter paper. The filtrate was mixed with ammonium sulphate, 50% (w/v). The ammonium sulphate treated filtrate was centrifuged at 5000 rpm for 20 min, after which the supernatant was mixed with an equal volume (1:1) of ethyl acetate (Loba Chemicals, India) and shaken in a separating funnel for 30 min for the extraction of the antimicrobial metabolites. The separating funnel with the mixture was allowed to stand for another 30 min. The supernatant containing the antimicrobial metabolites was pooled together and concentrated at 60°C using a Rotary evaporator (Rotary evaporator, RE-52A) [9]. The crude antimicrobial extract was transferred to a water bath at 50°C to dry off the°remaining ethyl acetate.
Furthermore, one gram of the crude antimicrobial extract was mixed with 3.0 mL of ethyl acetate. The dissolved crude antimicrobial extract was loaded into a silica gel (100-200 mesh) column chromatography, column dimension 2.0 cm inner diameter x 25 cm length. The column was eluted with n-hexane and ethyl acetate (1:4 v/v) and thirty fractions, 3.0 mL each was collected. All fractions collected were observed for the presence of antimicrobial activity and fractions that exhibited inhibitory activity against indicator strains were pooled together and concentrated at 60°C using Rotary evaporator (Rotary evaporator, RE-52A). The partially purified antimicrobial extract was used for the chemical characterization as well as the minimum inhibitory concentration (MIC) assay against indicator strains.

Chemical characterization of the antimicrobial compounds produced by Streptomyces xinghaiensis-OY62
The functional groups present in the partially purified antimicrobial compounds produced by Streptomyces xinghaiensis-OY62 was determined using Fourier transformed infrared (FTIR) spectrometer. The partially purified extract was mixed with potassium bromide (KBr). The spectrum obtained from the partially purified antimicrobial compound was recorded on Shimadzu AUX220 spectrophotometer that was in the range of 4000 cm

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration of the partially purified antimicrobial compounds
The Minimum Inhibitory Concentrations (MICs) of the partially purified antimicrobial extract of Streptomyces xinghaiensis-OY62 was carried out using macro broth dilution method as described by Andrews [22]. The tubes without turbidity after MIC was recorded were streaked on nutrient agar for indicator strains. The streaked plates were incubated at 37°C and were observed for growth after 24 h. The petri dish without growth that was inoculated with the lowest concentrations of the antimicrobial extract was recorded as the minimum bactericidal concentrations (MBCs) of the partially purified antimicrobial extract.

Statistical analysis
Data were collected in triplicates and were subjected to Duncan Multiple Range Test for Mean ± SD.

Isolation, Identification and Antimicrobial
Screening against Screening Microorganisms Seventy-five actinomycetes were isolated from ten soil samples collected purposively across Oyo State, Nigeria. Ten of the actinomycetes exhibited antimicrobial activity, out of which isolate OY62 was observed to have broadspectrum antimicrobial activity. Table 1 showed that isolate OY62 grew abundantly on all the four media used, aerial and reverse side colours were between brown to golden brown. It produced no diffusible pigment and it was Gram-positive. The 16S rRNA nucleotide sequence of the isolate showed that it has 99% similarity with Streptomyces xinghaiensis at NCBI Genbank. The 16S rRNA gene sequence has been deposited at NCBI GenBank and assigned accession number KU934248. Table 2 showed that antimicrobial activity was low at extreme pH 4 and 9, with lowest activity against Pseudomonas aeruginosa ATCC 9077 (15.3 mm) and Bacillus subtilis ATCC (10.7 mm) respectively but reached its best antimicrobial activity between pH 7 and 8. The highest antimicrobial activity was 35.7 mm against Enterococcus faecalis ATCC 29212.

Effect of Sodium Chloride on Antimicrobial Activity
The influence of sodium chloride concentration as presented in Table 3 revealed that a concentration of 0.8% (w/v) gave the highest antimicrobial activity against indicator strains, with Campylobacter jejuni ATCC 33291 most sensitive with a zone of inhibition of 28.3 mm and B. subtilis ATCC 6633 least sensitive with zone of inhibition of 20.0 mm.

Effect of Temperature on Antimicrobial Activity
The effect of temperature on inhibitory activity on indicator strains showed that a temperature of 55 o C was most suitable for the antimicrobial production and activity by Streptomyces xinghaiensis-OY62 against indicator strains (Table 4). A zone of inhibition of 32.7 mm was recorded against Campylobacter jejuni ATCC 33292. Low antimicrobial activity was recorded at extremes temperatures of 4°C and 60°C.

Effect of Carbon Source on Antimicrobial Activity
The result presented in Table 5 revealed that all the carbon sources used in the course of this study gave good antimicrobial activity by S. xinghaiensis-OY62 against test strains. However, the production medium without any carbon source also supported the production of antimicrobial compounds and activity with zones of inhibition that ranged between 26.3 mm and 31.7 mm were recorded (Table 5).

Effect of Nitrogen Source on Antimicrobial Activity
It was observed that the combination of potassium nitrate and casein gave the best antimicrobial activity against indicator strains ( Table 6). The antimicrobial activity against indicator strains measured in the zone of inhibition with casein+KNO 3 was between 28.3 mm and 37.7 mm. The activity was very poor in the absence of nitrogen source.

Effect of Incubation Period on Antimicrobial Activity
The recorded incubation period showed that Streptomyces xinghaiensis-OY62 had the highest antimicrobial activities against test strains by the fifteen days of incubation (Table 7). The zones of inhibition against the indicator strains were between 30.0 mm and 37.7 mm by the fifteenth day of fermentation.

Chemical Characterization of the Partially Purified Antimicrobial Compounds
The IR spectrum of the antimicrobial compounds synthesized by Streptomyces xinghaiensis-OY62 had peaked at wave numbers 609.89 cm and amide at wave numbers 3493.20 cm -1 and 1728.28 cm -1 (Fig. 2).

Minimum Inhibitory and Bactericidal Concentrations
The MIC and MBC of the partially purified antimicrobial extract against Bacillus cereus ATCC 10876 and Staphylococcus aureus ATCC 700699 indicator strains was 6.25 mg/L and 12.5 mg/L respectively. However, E. coli ATCC 35218, Salmonella typhimurium ATCC 13311, Klebsiella pneumoniae ATCC 8309 and Pseudomonas aeruginosa ATCC 9077 had MIC 12.5 mg/L and MBC 25.0 mg/L respectively (Table 9).

DISCUSSION
The morphological and cultural characteristics of Streptomyces xinghaiensis-OY62 are similar to the report of Oskay [16] ( Table 1). The partial sequence of the 16S rRNA gene of isolate OY62 showed that the isolate had 99% similar nucleotide sequence to other strain of Streptomyces xinghaiensis (Fig  1). The genetic and physiological factors of the strains could be responsible for the similarity observed. Sodium chloride has been reported to exert osmotic pressure on the production strain that results to increase in the number of metabolites excreted into the production medium. The observed result is in agreement with the report of Reddy et al. [26]. The similarity could be attributed to the high osmotic pressure which could aid the excretion of antimicrobial compounds from the cells.
The observed elevated temperature was higher than previous reports of Rakesh et al. [27] and Rehman and Mahesh [28] who had previously reported a temperature of 45°C. The difference could be due to different strains used for production, the thermo-stability of the metabolizing enzymes as well as the stability of the antimicrobial metabolites produced at elevated temperature. At such an elevated temperature, the tendency for mesophilic contaminants to survive in the production vessel is reduced and the rate of biochemical reaction is increased thereby reducing the production time. Streptomyces misakiensis strain IFO 12891

Fig. 3. Separation by GC-MS analysis of antimicrobial compounds produced by Streptomyces xinghaiensis-OY62
Furthermore, starch and absence of carbon source supported good antimicrobial activity against indicator strains (Table 5). Starch has been reported to support good antimicrobial production and activity against test microorganisms [29]. However, the production of antimicrobial metabolites in the absence of a carbon source could be due to the possibility of  The suitable incubation period for the synthesize of antimicrobial compounds by Streptomyces xinghaiensis-OY62 was observed at day fifteen of fermentation as shown in Table 7. This, however, was higher than previous submissions of Bundale et al.
[23] and Khattab et al. [31] who had earlier reported between five and ten days respectively. The difference obtained could be as a result of differences in the strains used and the nature of the nitrogen and carbon sources used for fermentation.
The IR spectrum analysis of the partially purified antimicrobial compounds synthesized by Streptomyces xinghaiensis-OY62 had peaked at various wave numbers indicating the presence of functional groups such as unsaturated alkenes, aromatic rings, amide, hydroxyl as well as carbonyl groups (Fig. 2). These are in agreement with earlier reports of Ayari et al., Coates [32,33]. However, the differences observed in wave numbers even, though indicating the same functional groups could be attributed to the spatial arrangement of the molecules and their stretch during vibration [33].
The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of partially purified antimicrobial compounds produced by S. xinghaiensis-OY62 against the test strains ranged between 6.25 mg/L and 12.5 mg/L respectively ( Table  4). The results are higher than the previous report of Al-Bari et al [34], Arasu et al. [35]. The differences observed could be due to different strains of indicator organisms and level of purity of the antimicrobial compounds.

CONCLUSION
The outcome of this research study showed that Streptomyces xinghaiensis-OY62 (KU934248) was isolated from soil of tropical rain forest of Oyo State, Nigeria. The strain exhibited broadspectrum antimicrobial activity against indicator strains. Hence, it may be useful in the production of antimicrobial agents which may find application in the in the treatment of microbial infections.