Screening of O-7 Isolate Actinomycete Producing Antimicrobials in Different Growth Conditions against Selected Pathogens

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INTRODUCTION

Actinomycetes are Gram-positive bacteria found in soil, industrially suitable as sources of a wide variety of secondary metabolites, including several antibiotics of medical and commercial significance [1]. Antibiotic-resistant pathogens such as methicillin and vancomycin-resistant strains of Staphylococcus aureus (S. aureus) and others pose a significant risk to the treatment of severe infections [2, 3]. To prevent this, an urgent replacement of the existing antibiotic is required, and the development of new drugs against drug-resistant pathogens is considerable for today [4].

A significant number of commonly used antibiotics, like erythromycin, streptomycin, rapamycin, and gentamycin, are all substances isolated from soil Actinomycetes. Streptomyces and Micromonospora are the two major classes of soil actinomycetes that act as the primary producers of antibiotics. It was reported that Streptomyces responsible for around 80% of the total antibiotic products, whereas Micromonospora closely follows Streptomyces constituted below than one-tenth of the total antibiotic products [5].

Various strains of actinomycetes normally produce many products that assist to improve the isolation and screening of new strains to discover new products [6]. Several studies are currently underway on antibacterial products produced from actinomycetes that are efficient against several types of antimicrobial-resistant bacteria [7].

The purpose of research is to profit from the availability of different types of soil in Sudan, enabling the growth of several species of antibiotic-producing actinomycetes. The development of antibiotics from isolated microbes locally adds to the cost of importing these medications from abroad and helps in the development and promotion of the local industry and export market. This research goal to isolate, select the promising strain, and improving antibiotic production of actinomycetes isolated from Khartoum state soil.

MATERIALS AND METHODS

This was a prospective analytical analysis of many actinomycetes. Samples were isolated from soils collected from different locations in Khartoum state. The crowded plate method was utilized to separate actinomycetes using a variety of media. The isolates were tested for antimicrobial effectiveness against multiple Gram-positive, Gram-negative, yeast, and fungi bacteria. Numerous fermentation factors like temperature, pH, light, agitation, aeration, and fermentation period have also been enhanced for optimal antimicrobial production.

Soil samples collection

Soil samples were taken from multiple places in Khartoum, using clean, dry, and sterile polythene bags, bands, pens, and other accessories. These samples were air-dried for one week, and used for isolation of Actinomycetes [8].

Isolation of actinomycetes

One gram of soil was dissolved in 9 ml of sterile distilled water. The dilution was completed up to 10-5 dilutions. Aliquots (0.1 ml) of 10-2, 10-3, 10-4, and 10-5 were distributed to starch casein agar (SCA). Based on colonial morphology, the culture of actinomycetes was chosen and purified by the International Streptomyces Project [9].

The actinomycete colonies isolated from the crowded plate were selected for further study (Figure 1), which were named as B (30 strains), D (8 strains), K (15 strains), NS (2 strains), and O (7 strains).

Standard strains

The following reference bacterial cultures have been taken from the Department of Pharmaceutics, College of Pharmacy, University of Khartoum, Sudan: Staphylococcus aureus (ATCC 29213), Bacillus subtilis (ATCC 10400), Escherichia coli (ATCC 13706), Klebsiella pneumoniae (ATCC 10031), Proteus vulgaris (ATCC 6380), Pseudomonas aeruginosa (ATCC 15442), Salmonella typhimurium (ATCC 13311), Aspergillus niger (ATCC 16404), and Candida albicans (ATCC 10231).

Screening for antibiotic production

The antimicrobial activity of soil actinomycete isolates was assessed using the agar streak method [10].

Characterization of the isolates

Biochemical characteristics such as melanin pigment, nitrate reduction, H₂S production, citrate utilization, milk coagulation, and the use of carbon sources have been evaluated using standard methods. Cultural characteristics such as aerial color and mycelium substrate have been studied in different media following the guidelines established by the ISP [11].

Morphological characterization

Cultural characterization

Selected actinomycete isolates were studied by inoculation in different sterile media: carbon utilization agar (ISP-9), Czapek’s sucrose agar, glycerol-arginine medium (ISP-5), glucose yeast extract agar, glucose agar, starch agar, and starch casein medium.

Microscopic characterization

The direct method, slide culture method, and Gram-staining method have been used [12]. The isolates showed the properties of actinomycetes. Morphological characteristics were determined as proposed in Bergey’s manual of determinative bacteriology and biochemical tests were performed [13].

Fermentation process for antibiotic production

It is well established that glucose and other favorable carbon sources suppress morphological and chemical differentiation of Streptomyces, and similar observations have been made concerning nitrogen [14]. The isolates were cultivated in a variety of media. The culture filtrates were harvested, and the antibacterial activity was evaluated by agar well diffusion assay [6].

Medium complexity

Various sources of nitrogen, such as yeast extract, ammonium chloride, and peptone, have been tested for maximum antibiotic productivity [15]. Different concentrations of glucose and lactose as a carbon source have been used in the basal medium.

Suitable parameters for fermentation

The pH values of the medium containing 4% glucose, 1% peptone, 0.3% yeast extract, 0.35% CaCO_3, and 0.5% Na Cl have been adjusted by (NaOH) and (HCl). The optimal temperature for the productivity and growth of the strains was determined by keeping the inoculated fermentation media at 27°C, 28°C, 29°C, 30°C, 31°C, 37°C, and 38°C for 4 days in a shaker incubator [16]. Subsequently, the growth of microorganisms and productivity of an antimicrobial activity was evaluated using the cup-plate method [17, 18]. Two groups of conical flasks containing media inoculated with the selected strain and incubated in a rotary shaker into two ways for studying light effectivity. The first group was covered with black foil to prevent light, and the second group was left uncovered. In the end, antimicrobial activity was determined. Three different sizes of conical flasks were used (250, 500, and 750 ml). Each contained 150 ml of fermentation medium and was inoculated with the selected isolate. The antimicrobial activity was determined after the incubation period. The incubation of the seeded flasks took place in two ways. The first group was incubated in a shaker, and the second group was left at room temperature on the bench. The antimicrobial activity was then determined.

The duration of the fermentation process has been optimized to obtain the maximum antibiotic production time for harvesting [17, 19]. Using the above-mentioned media formulation and fermentation parameter conditions, the media were inoculated with the selected strain and incubated for up to 144 hours. The harvesting time of the maximum antibiotic productivity was determined and the antimicrobial activity was also determined using the cup-plate method [20, 21].

Statistical analysis

The statistical methods used in this study include Descriptive statistics (mean, standard deviation, maximum, minimum, Range, and graphs), a T-test for independent samples, and Two Way (ANOVA) to compare the significant difference between levels. Statistical software (SPSS, version 20) has been used in this study.

RESULTS AND DISCUSSION

Cultural and microscopic characterization of the isolates

The cultural (Figures 1a and 1b), and microscopic (Figure 2) characteristics of the isolates confirmed that the isolates were belonging to the genus Actinomycetes.

Antimicrobial activity of Actinomycetes

Isolated actinomycete strains were tested for anti-microbial activity against seven bacteria, one fungal, and one yeast strain by the agar streak method. Out of sixty-two actinomycetes screened, eleven strains showed significant antimicrobial activity against Gram-positive and/or Gram-negative organisms (Table 1). Out of the eleven strains, six actinomycetes were selected for taxonomic characterization using an antimicrobial activity test. However, the strain (O-7) showed a very broad spectrum with higher scores than all other strains.

Table 1. Activity of isolates against different organisms

(+++) = wide Inhibition, (++) = Medium Inhibition, (+) = Low Inhibition, (-) = No Inhibition

Taxonomic characterization

Biochemical tests were carried out for the six selected isolates (Table 2). The strain (O-7) showed positive results in gelatin liquefaction, nitrate reduction, pigment production, acid production, indole, proteolytic activity, methyl red, and starch hydrolysis tests.

Table 2. Biochemical tests of soil isolates

(+) Positive Reaction, (-) Negative Reaction

Morphological and cultural characterization of (O-7) strain

The cultural and morphological characteristics of the strain (O-7) were studied in different media suitable for actinomycetes. Microscopically, the observations revealed that the strain (O-7) is a filamentous Gram-positive microorganism. By studying morphology, cultural and taxonomic characteristics, it has been confirmed that the strain (O-7) belongs to the genus actinomycete.

Fermentation process for antibiotic production

Medium formulation

Carbon source

Based on the results presented in Figures 3a and 3b, the results showed that monosaccharide, glucose, was the best among the different sugars. It has been observed that 5% glucose and 3% lactose are optimal for the production of antibiotics for the (O-7) strain.

Nitrogen source

By determining the influence of nitrogen sources on the inhibition zone, peptone at a concentration of 1% was found to be a good source of nitrogen for the (O-7) isolate (Table 3).

Table 3. Mean, standard deviation, and Two Way ANOVA for nitrogen source (1% each)

     * P ≤ 0.05   ** P ≤ 0.01 

Fermentation parameters

The potential of Hydrogen (pH)

Optimum pH for maximum growth and productivity of (O-7) isolate was measured. From the obtained results, pH 7.0 was found to be the most suitable for the selected isolate (Table 4).

Table 4. Mean, standard deviation, and Two Way ANOVA for (pH)

     * P ≤ 0.05    ** P ≤ 0.01  

Temperature

The temperature optimization study found that (O-7) showed good growth and maximum antibiotic production at 28°C. (Table 5).

Table 5. Mean, standard deviation, and Two Way ANOVA for Temperature (in ⁰C)

     * P ≤ 0.05   ** P ≤ 0.01   

Light

The results indicated that the culture flasks incubated in the light had a high level of activity against the test organisms. The effect of light on the productivity of (O-7) has been determined (Figure 4).

Aeration and agitation

The effect of aeration and agitation on the productivity of (O-7) was determined in Figures 5a and 5b.

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