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The Impact of Secondary Metabolites Produced by Streptomyces on Uro-pathogens

Suhndh Ahmed Mohmmed Musa

Department of Botany, Faculty of Science, University of Khartoum

Abstract: Soil streptomyces are known as a rich source to produce secondary metabolites. This study was conducted to examine the effect of the Streptomyces sp. extracts against the uro-pathogen isolates from urinary tract infections in pregnancy from Kassala state. Cultural, morphological and physiological characterization of 15 microorganisms isolates from four soil samples indicated that all isolates belonged to the Streptomyces genus. All Streptomyces isolates produced chitinase enzyme except isolate SU11. After 72 hours all of the isolates were produced L-asparaginase enzyme. Ethyl acetate extracts of Antimicrobial were used for secondary screening against uro-pathogen. The high activity has been observed against Gram-positive bacteria with a percentage of 73.3% when compared to gram-negative bacteria. Whilst 26.7% has a high activity against Gram-negative comparing with Gram-positive bacteria, and different activity was shown against candida spp.

Keywords: Streptomyces, Chitinase, L-asparaginase, Antimicrobial, Uro-pathogen

Received July 10, 2020; Revised August 12, 2020; Accepted August 21, 2020

Citation: Suhndh Ahmed Mohmmed Musa, The Impact of Secondary Metabolites Produced by Streptomyces on Uro-pathogens. American Journal of Microbiological Research, 8 (3). 103-109. January 2020. doi:10.12691/ajmr-8-3-4

1. Introduction

Antimicrobial resistance is a worldwide health problem [1]. In the past new drug discovery come from natural sources [2]. Actinomycetes is big group of microorganisms that are widely distributed in insect microbiomes, freshwater, marine and terrestrial environments [3,4]. These are not only an antibiotic producer but also an important source of bioactive compounds [5,6]. Streptomyces, an Actinomycetes member, produces secondary metabolites and enzymes commercially important in medical and agricultural applications combating problems caused by pathogenic bacteria and/or fungi [7,8]. Some Streptomyces spp. produces antibiotics during sporulation [9]. Their ability to produce different antibiotics attracted researchers [10]. L-asparaginase produced by microorganisms has been widely used as an effective therapeutic agent against acute lymphoblastic leukemia, lymphosarcoma as well as in the food industry [11,12]. Some Streptomyces spp are known to produce L-asparaginase [13]. Microorganisms like Serratia and Actinomycetes (principally Streptomyces) are good producers of chitinases [8,14,15]. It inhibits fungal growth and controls its pathogenicity in plants [16], but it is not fully effective in all cases [17].

According to Hamza et al soil microbiology was not given due attention in Sudan, despite the high variability in soil types and the environment [18].

The aims of this study isolation and identification of Streptomyces bacteria from different localities in Kassala state, detection of their ability to produce secondary metabolites(Antimicrobial and Enzyme), and application of these products to investigate their ability to inhibit the uro-pathogens isolated from pregnant women urine in Kassala state.

2. Material and Methods

2.1. Soil Samples Collection and Pre-treatment

Soil samples were collected from different locations in Kassala state. The samples were taken from a depth of 5-10 cm after removing approximately 2-3cm of the earth's surface and were then mixed with 1% CaCO3 and left to dry for in open air 3-5 days.

2.2. Isolation of Streptomyces spp.

Isolation of Streptomyces spp was performed by the soil dilution plate technique [19]. In this method usually (10⁻³-10⁻⁵) 0.1 ml aliquot was pipetted from each dilution and spread onto Starch Casein Agar (SCA). The inoculated Petri-dishes were incubated at 27°C for 3-5 days.

2.3. Characterization of Streptomyces spp Isolates

Cultural, morphological and physiological features of the presumptive Streptomyces spp isolates were determined as described by International Streptomyces Project (ISP) documented by Shirling and Gottlieb [20]; Locci [21] and Bergey’s Manual for Determinative Bacteriology [22].

Colony characteristics followed by [23,24].

2.4. Morphological Characterization of Streptomyces spp

A- Aerial mass and reverse color

Aerial mass and reverse colors were detected according to Shirling and Gottlieb by using ISP2 [20].

B- Diffusible soluble pigments

Streptomyces cultures were detected by the method of Shirling and Gottlieb on the ISP7 medium [20].

2.5. Microscopic Characteristics

Gram staining method followed by Collins et al. [25]. Spore chain morphology required culturing Streptomyces isolates on ISP-2 agar medium using cover slip culture technique as described by [26].

2.6. Physiological Characterization

A- Melanoid Pigments

The production of melanoid pigments by Streptomyces cultures was detected by the method of Shirling and Gottlieb on the ISP7 medium [20].

B- Carbohydrate Utilization

Carbohydrate utilization test was performed by using 10 % (w/v) of the carbon sources. The carbon sources were: L- arabinose, D- xylose, meso- inositol, D- mannitol, D- fructose, rhamnose, raffinose, xylose and sucrose. [20].

C- NaCl Tolerance

Sodium chloride tolerance was detected according to [27]. Streptomyces isolates were cultured in yeast extract agar medium. After sterilization, gradient concentrations of NaCl (0, 2.5, 5.7,5 and 10%) were aseptically added.

2.7. Screening of Streptomyces Isolates for Enzyme Production

A- Chitinase production

The isolates were cultured in the chitin agar (CCA) medium [28]. After incubation, 0.1% Congo red solution was fed over the plates, the appearance of clearance zone around the colonies after seven days of incubation was noted and measured.

B- L-Asprginase production

The isolated cultures were re-cultured in the ADS medium with phenol red. The cultures were incubated at 37°C at pH 6.5, change of the medium colour to pink indicates the production of L-Asprginase enzyme [29].

2.8. Antimicrobial Activities

A- Preliminary screening of the Streptomyces isolates for antibiotic production

Antibiotic activities of the Streptomyces isolates were tested in vitro against uro-pathogene (bacteria and fungi). Antibacterial activities of pure Streptomyces isolates were performed by the cross- streak method [30].

B- Screening for secondary metabolites

- Culture and extraction of bioactive compounds

Screening for the secondary metabolites was done by using Streptomyces Antibiotic Activity broth medium. Method described by [31]. Ethyl - acetate extraction method was used [32]. The solvent layers were collected in tubes and evaporated to dryness at 40oC [33]. The dried extracts were used for antibiotic screening.

- Biological activities of the extracts

Inhibitory activities of the Streptomyces extracts were examined against uro-pathogenies (E. coli, Vibrio cholera, Neisseria, Listeria, Staphylococcus and Candida sp.).

3. Result

3.1. Isolation and Cultural Characterization of Streptomyces spp

Fifteen strains were isolated from 4 soil samples on Starch Casein KNO3 agar medium, colony characteristics (color, Shape, Size, margin, surface, Elevation, texture and opacity) were study (Table 1) and (Figure 1). In all strain the surface was powdery, with raised elevation, the texture was dry and the opacity was opaque consequently these were not included in the table. The strains were purified, given codes prefixed with SU1 to SU 15.

Table 1. Soil type and Cultural characteristics of Streptomyces isolate

Figure 1. Cultural characteristics of Streptomyces isolates

3.2. Morphological Characterization of streptomyces spp

3.2.1. Aerial Mass and Reverse Color

Different colure was recorded by different isolate White, Pink, Cream and White, Pink and White, Gray and Gray and White in (Aerial mycelium) while Yellow, Cream, Brown and Gray in (Reverse color) (Table 2, Figure 2).

3.2.2. Diffusible Soluble Pigments

The ability to produce soluble pigments other than Melanoids was noted as producers (+) and not producers (-), 46.7% produced and 53.3 % was not produced. (Table 2, Figure 3).

3.3. Microscopic Characteristics

All the isolates were Gram-positive bacteria with extensive branching (Table 2, Figure 4).

3.4. Physiological characterization

3.4.1. Melanoid Pigments

The ability to produce Melanoids other than soluble pigments was noted as producers (+) and not producers (-), 46.7% were positive and 53.3 % were negative (Table 2, Figure 3).

Table 2. Morphological, some of Physiological and Microscopic characteristics of Sstreptomyces isolates.

Figure 2. Aerial mass colour (forward side) and reverse colour (rearward side) of Streptomyces isolate SU2

Figure 3. Melanoid and soluble pigments of Streptomyces isolate SU6

Figure 4. Different shapes of Streptomyces hyphae

Table 3. Streptomyces isolates growth in different carbohydrate source

3.4.2. Carbohydrate Utilization

Table 3 shows that all tested isolates were able to grow on media supplemented with (Mannitol, Sucrose and Raffinose) when used as a carbohydrate source.

In the table cells with (-) indicated growth not better than negative control; the (+) mean growth is better just like negative control but not like positive control and in cells with (++) the growth better like a positive control.

3.4.3. NaCl Tolerance for Streptomyces Isolates

The NaCl tolerance test for the 15 Streptomyces isolates revealed that all concentrations 2.5, 5, 7.5 and 10% as well as the control, supported either low, medium or high growth with no constant pattern isolates wise or NaCl concentration wise.

3.5. Chitinase Enzyme Production

All Streptomyces the isolates produced chitinase except isolate SU11. High production is shown by isolate SU1 (Table 4 and Figure 5).

3.6. L-asparaginase Enzyme Production

The L-Asparaginase production was recorded over a period of 72 hrs. After 24h only isolated SU1 and SU2 produced the enzyme. After 48h more isolates produce the enzymes and after 72h all isolates were able to produce the enzyme (Table 4 and Figure 5).

3.7 Screening of Antimicrobial

All of the Streptomyces isolates were subjected to primary screening for antimicrobial production by using cross a streak method (Figure 6) against uro-pathogens. These isolates which demonstrated antimicrobial activities against uro-pathogens were again subjected to secondary screening disk method. High inhibitión zone was shown against Gram- negative bacteria by Streptomyces isolates SU (1, 2, 4, 5, 7, 9, 10, 12, 13, 14, 15) and against Gram- positive bacteria by Streptomyces isolates SU (3, 6, 8, 11). Various in inhibition zones results showed against all candida sp. (Figure 6).

Table 4. Chitinase and L Asparagine Production by Streptomyces isolates

Figure 5. Enzymes produce by Streptomyces

Figure 6. Inhibición zone by streptomyces aginst uro-pathogen

4. Discussion

Soil is an ecological niche of many organisms living together, Williams et al. [34] reported that the soil is a natural habitat for Streptomyces sp., and wide species of Streptomyces was isolated from it [35,36]. In this study, morphological and physiological characterization indicated that 15 isolates from soil type from Kassala belonged to the Streptomyces genus. These isolates were similar in surface, elevation, texture and opacity and differ markedly in colour, shape, size and margin. All isolates which had pale brown color had the same Cultural characteristic except for isolate SU5 which was observed. Different aerial mass and reverse colors of Streptomyces isolates were recorded According to the Bergey’s manual of systematic bacteriology [21].

The present study detected 46.7% positive and 53.3% negative for melanoid and soluble pigments. White series producing melanoid pigments were found during this study but not by Taddei et al. [37] who recorded melanoid pigments in other series. Microscopically the current Streptomyces isolates showed branched hyphae and were Gram-positive confirming Claessen et al. findings [38]. Utilization of carbon sources plays an important role in species differentiation by Streptomyces sp. [39]. During this study, all the isolates were able to grow in L-arabinose, D-xylose, meso-inositol, D-glucose, D-mannitol, D-fructose, rhamnose, raffinose and sucrose. Similar results obtained in Streptomyces sp. strain 3B which failed to assimilate meso-inositol and D-mannitol as a carbon source [40]. NaCl tolerance test was proved that the optimum growth at zero concentration, while another study reported that the optimum of NaCl, for most Streptomyces strains, was (5-10 % NaCl) [41,42]. In this study Streptomyces were produced enzyme and anti-microbial, other study verification it was able to produce secondary metabolites, like antibacterial, antifungal, anti-cancer, and anti-HIV [43,44,45].

In this work, L-aspreginase and chitinase were produced by the isolates of Streptomyces. This confirmed earlier findings. Production of L-asparaginase by Streptomyces karnatakensis and Streptomyces venezuelae was reported by [46]. On the other hand, Chitinase production was detected by Kolla et al. from Streptomyces sp. ANU 6277 and Meriem et al. from Streptomyces griseorubens C9 isolates [47,48]. L-asparagine was increased through time, [49] reported that the production began after 24 h and gets to the top rung after 72 h of incubation and Amena et al. demonstrated that the production of L-Asparaginase increase through time at constant pH and temperature [50]. Different zones of chitin hydrolysis were formed by all Streptomycetes isolates except for one isolate (SU11), high concentration produced by most strains [51].

Preliminary screening of antibacterial activity indicated that all the isolates they have antibacterial activity against one or more isolates of uro-pathogen. After that, all of it were subjected to secondary screening. Ethyl acetate extracted were used, the extraction by ethyl acetate of secondary metabolites showed clear activity against pathogenic bacteria than other solvents [52]. Secondary screening showed different activities from that of primary screening, similar results reported by [53]. Variability in activities obtained in primary and secondary screening test may be attributed to the use of different media (solid, liquid media) or improved techniques which may lead to production of different secondary metabolites, also some compounds may be lost during the organic solvent [54].

Some Streptomyces extract activity against Gram- positive bacteria were high compare with Gram negative bacteria, while some Streptomyces extract activity against Gram-negative bacteria were high compare with Gram-positive bacteria. [55,56,57,58] reported that the inhibition zone of Streptomyces was relatively larger against Gram-positive bacteria compared with Gram-negative bacteria. Some Streptomyces extracts have an inhibition zone against candida sp., [59,60,61] reported that different strains of Streptomyces showed various degrees of antifungal activity against A. niger and C. albicans. Streptomyces isolate SU 13 and SU 15 high produce Chitinase and L Asparagine enzyme but it differed in antimicrobial activity. When isolate su11 doesn’t produce Chitinase put have antimicrobial activity against fungi, Inbar and Chet explain that chitinases do in fungal cell walls [62].

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