Effect of production parameters and inhibitory activity of antimicrobial compounds produced by co-cultured strains of Streptomyces xinghaiensis-OY62 and S. rimosus-OG95

2.1 Actinomycetes strains used for the present study

Two strains of Streptomyces used for the study, Streptomyces xinghaiensis-OY62 (KU934248) and Streptomyces rimosus-OG95 (KU934251) were obtained from the stock cultures at Microbial Physiology and Biochemistry laboratory, Department of Microbiology, University of Ibadan.

2.2 Indicator strains

Four indicator strains: Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Campylobacter jejuni ATCC 33291 and Pseudomonas aeruginosa ATCC 9077 were used for agar well diffusion assay while seven indicator strains that included Bacillus cereus ATCC 10876, Escherichia coli ATCC 35218, Klebsiella pneumoniae ATCC 8309, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 11311, Pseudomonas aeruginosa ATCC 9077 and Candida albicans ATCC 10231 were used for the determination of Minimum Inhibitory and Minimum Bactericidal Concentrations.

2.3 Effect of production parameters on the antimicrobial activity of co-cultured strains of Streptomyces xinghaiensis-OY62 and Streptomyces rimosus-OG95 on indicator strains

The basal medium used for the assessment of the effect of production parameters of co-cultured strains of Streptomyces on the indicator strains was starch casein broth (g/L): Soluble starch 15.0, potassium phosphate dibasic 2.0, Potassium nitrate 2.0, Sodium chloride 2.0, Casein 0.30, Magnesium sulphate heptahydrate 0.05, Calcium carbonate 0.02, Iron II sulphate heptahydrate 0.01 at pH 7.2 (Sharon et al., 2014).

The effect of pH (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0), sodium chloride concentration (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0), temperature (4 °C, 28 °C, 35 °C, 45 °C, 55 °C and 60 °C), carbon source (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) and incubation period in days (3, 5, 7, 10 and 15) on antimicrobial activity of co-cultured strains of Streptomyces xinghainesis-OY62 and S. rimosus-OG95 was studied by inoculating 500 mL of the basal medium with 2.5% (v/v) each of 24 h old culture of Streptomyces xinghainesis-OY62 and S. rimosus-OG95. However, the best result of a previously determined parameter was kept constant in the next parameter under study.

The inoculated flasks were incubated at 28–30 °C on an Orbital Shaker (Platform Shaker MSZ-100A) at 150 rpm for seven days, after which the incubation was terminated. The fermented broth was filtered using Whatman No. 1 and treated with 50% (w/v) ammonium sulphate, so as to remove interfering metabolites from the antimicrobial extract. The mixture was centrifuged at 5000 rpm for 20 min (Wincom 80-2). Furthermore, equal volume (1:1) of the supernatant and ethyl acetate were vigorously shaken together for 30 min in separating funnel to effect the extraction of the antimicrobial metabolites. The separating funnel with the mixture was further allowed to stand for another 30 min. The supernatant was collected and concentrated at 60 °C using Rotary Evaporator (Rotatory Evaporator, RE-52A). The crude antimicrobial extract was used for agar well diffusion assay.

2.4 Agar well diffusion assay

Agar well diffusion method was used to determine the antimicrobial activity of the co-cultured strains of Streptomyces used in this study against indicator strains (Kumari et al., 2013). A well of 6.0 mm was made at the center of petri dishes containing Mueller Hinton agar. These plates had been earlier swabbed with a 24 h culture of Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Campylobacter jejuni ATCC 33291 and Pseudomonas aeruginosa ATCC 9077 that have their turbidity adjusted to 0.5 McFarland standards (1.5 × 10⁸ CFU/mL). Each well was filled with crude extract (50.0 μL) and the plates were incubated at 37 °C (Memmert 854, Schnabach, Germany). The zones of inhibition were measured in triplicate using ruler. The 6.0 mm of the well was subtracted from the readings.

2.5 Partial purification and characterization of the antimicrobial compounds produced by co-cultured Streptomyces xinghaiensis-OY62 and Streptomyces rimosus-OG95

Eight liters of starch casein broth was inoculated with 2.5% (v/v) 24 h seed inoculum of each Streptomyces xinghaiensis-OY62 and S. rimosus-OY95 and incubated at 45 °C. After the incubation period, the fermented broth was separated from the mycelial debris by filtration using Whatman No. 1 filter paper. The filtrate was treated with 50% (w/v) ammonium sulphate (Kandula and Terli, 2013). The treated filtrate was centrifuged at 5000 rpm for 20 min. The supernatant was mixed with equal volume (1:1) of ethyl acetate and shaken vigorously in a separating funnel for 30 min for extraction of the antimicrobial metabolites, after which it was allowed to stand for 30 min. The supernatant containing the antimicrobial metabolites was collected and concentrated at 60 °C using a Rotary evaporator (Rotary evaporator, RE-52A) (El-Naggar et al., 2001). The crude concentrate was then transferred to a water bath at 50 °C to dry off the remaining solvent.

One gram of the crude antimicrobial extract was dissolved in 3.0 mL ethyl acetate. The dissolved crude antimicrobial compound was loaded into a silica gel (100–200 mesh) column chromatography, column dimension 2.0 cm inner diameter × 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 were screened for the presence of antimicrobial compounds and fractions within the range showing antimicrobial activity were pooled together and subjected to the second round of purification using column chromatography. The partially purified antimicrobial compounds was loaded into the silica gel column of 2.0 cm inner diameter and 25.0 cm column length and eluted with hexane:ethyl acetate (1:4 v/v). Fractions were collected and spotted on silica gel coated plates. Fractions that possessed activity and similar retention factor were pooled together concentrated at 60 °C using Rotary evaporator (Rotary evaporator, RE-52A). Spots complex were identified when plate were exposed to iodine crystals. The partially purified antimicrobial extract was used for Minimum Inhibitory Concentration (MIC) assay against indicator strains and chemical characterization.

2.6 Chemical characterization of the antimicrobial compounds produced by co-cultured strains of Streptomyces xinghaiensis OY62 and S. rimosus OY95

The chemical characterization of the partially purified antimicrobial compounds produced by Streptomyces xinghaiensis OY62 and Streptomyces rimosus OG95 was carried using Fourier transformed infrared (FTIR) spectrophotometer. The sample was homogenized with potassium bromide (KBr). The spectrum of the partially purified antimicrobial compound was recorded on Shimadzu AUX220 spectrophotometer that was in the range of 4000 cm⁻¹–400 cm⁻¹ (Sanghvi et al., 2014). The chemical structures, formulae and identification were determined using gas chromatography–mass spectrophotometer (GC–MS) Shimadzu QP 2010 (Anupriya et al., 2016).

2.7 Determination of Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations of the partially purified antimicrobial compounds

The Minimum Inhibitory Concentrations (MICs) of the partially purified antimicrobial extract was determined using macro broth dilution method as described by Andrews (2001). The tubes without turbidity (no growth) after MIC were read and streaked on nutrient agar for bacterial indicator strains and the test tube of Candida albicans was streaked on potato dextrose agar. The streaked plates were incubated at 37 °C. The plates were observed for growth after 24 h and the plates without growth inoculated with the lowest concentrations of the antimicrobial extract was recorded as the minimum bactericidal concentrations (MBCs).

3.1 Partial purification and characterization of the antimicrobial compounds

The retention factor (Rf) of the partially purified antimicrobial extract produced by co-cultured S. xinghaiensis-OY62 and S. rimosus-OG95 was 0.97. The FTIR spectrum showed the presence of functional groups such as aromatic rings at wave numbers 484.15 cm⁻¹ and 651.96 cm⁻¹, carbonyls and amide at 1728.28 cm⁻¹ and 3729.81 cm⁻¹ (Fig. 1).

Close

Fig. 1

FTIR spectrum of antimicrobial compounds produced by co-cultured Streptomyces xinghaiensis-OY62 and Streptomyces rimosus-OG95.

Export to PPT

The GC–MS spectrum (Fig. 2) of the partially purified antimicrobial compounds synthesized by co-cultured S. xinghaiensis-OY62 and S. rimosus-OG95 showed that ten antimicrobial metabolites were identified and characterized (Table 7, Fig. 3a–h). Dibutyl phthalate was mostly produced (41.4%) followed by Bis (2ethylhexyl) phthalate having 37.9%. The least produced antimicrobial metabolite was Phenol, 2,4-bis (1,1-dimethylethyl)- that had 1.65% (Table 7).

Close

Fig. 2

GC–MS spectrum of antimicrobial compounds produced by co-cultured strains of Streptomyces xinghaiensis-OY62 and Streptomyces rimosus-OG95.

Export to PPT

Close

Fig. 3

(3a–3h) Chemical structures of antimicrobial compounds produced by co-cultured strains of S. xinghaiensis-OY62 and S. rimosus-OG95 using GC–MS spectral analysis: (3a). Phenol, 2, 4-bis (1,1-dimethyl, (3b) 1,2-Benzene dicarboxylic acid bis (2-methyl propyl ester, (3c) Phthalic acid isobutyl 2-pentyl ester, (3d) 1,2-Benzene dicarboxylic acid, butyl octyl ester (3e) 9-Octadecenoic acid, methyl ester, (E), (3f) 9-Octadecenamide, (3g) Dibutyl phthalate and (3h) Bis (2-ethylhexyl) phthalate.

Export to PPT

3.2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of partially purified antimicrobial extract produced co-cultured strains of S. xinghaiensis-OY62 and S. rimosus-OG95 ranged between 3.12 mg/L and 12.5 mg/L and 12.5 mg/L to 25.0 mg/L respectively against the indicator strains. However, gentamicin (control) had MIC that ranged between 0.78 mg/L to 3.12 mg/L and MBC of 1.56 mg/L to 6.25 mg/L against indicator strains (Table 8).