exploring the antimicrobial properties of actinobacteria isolated from soil samples

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ABSTRACT: Actinobacteria are one of the leading microbes known for producing excellent secondary metabolites. These metabolites are known to possess antibacterial, antifungal, neuritogenic, anticancer, antialgal, antimalarial and anti-inflammatory activities. The purpose of this study was to explore the antimicrobial properties of actinobacteria isolated from soil samples collected from the nursery of VIT University, Tamil Nadu India. Actinomycetes were isolated by serial dilution and pour plate technique. The isolates obtained were purified and screened for their antimicrobial activities Muller Hinton Agar was used for screening the antimicrobial activity of Actinomycetes using agar well diffusion technique. Primary screening was done using cross streak method. The bioactive compound was extracted from efficient actinobacteria using solvent extraction method. Using Kirby- Bauer method the antimicrobial activity of crude and solvent extract was performed. MIC for ethyl acetate extract was determined by agar wall diffusion method. Out of 30 colonies, 4 actinobacteria were selected (DMPVIT-1 to DMPVIT-4) and screened for antimicrobial property against Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus. Among the 30 isolates, DMPVIT-3 was found to be effective against Pseudomonas aeruginosa, Salmonella typhis in primary screening (cross streak method). The DMPVIT-3 potential isolate was inoculated in production media for secondary screening.Based on these results we concluded that DMPVIT-3 possesses high antimicrobial activity.

Key Words: Actinobacteria, Cross streak method, Antimicrobial activity, well diffusion method, MIC and Kirby Bauer method.

INTRODUCTION

The emergence of multi drug resistance among common bacterial pathogenshas been increasingly important to fight pathogens with the help of new molecules. The improper usage of antibiotics contributes a major role for drug resistance in pathogenic microbes. Microorganisms acquire resistance towards common antibiotics by altering their metabolism and genetic material [1, 2]. Due to development of dug resistance, treatment of bacterial infection has become a serious problem [3].However, due to certain defects and escalation of drug resistance it emphasizes the requirement of advance antimicrobial agents having potential activity towards Gram positive bacteria [4].Microorganisms are an important aspect of the environment and human life. They are present everywhere in the nature even in the soil sediments. Natural products are boundless source for important novel compounds having antagonistic activity against pathogenic organisms. Actinobacteria from terrestrial origin produce hundreds of antibiotics which are widely used at present. Some differences could be expected among organisms existing in marine and terrestrial environments due to variation in the physical, chemical and biological factors [5]. It is apparent that the terrestrial environment is a potent source for finding new actinobacteria and new antibiotics or biologically active substances [6,7].

Actinobacteria are gram positive filamentous bacteria which are supreme secondary metabolite producers [8].Actinobacteria hold a prominent position and are virtually unlimited source for novel compound having many therapeutic applications. About 70% of bioactive compounds have been isolated from actinobacteria [9]. Actinobacteria are the most economically and biotechnologically valuable prokaryotes able to produce wide range of bioactive secondary metabolites, such as antibiotics, antitumor agents, immunosuppressive agents and enzymes. These metabolites are known to possess antibacterial, antifungal, .neuritogenic, anticancer, antialgal, antimalarial and anti-inflammatory activities [10]. Actinobacteria has the capacity to synthesize many different biologically active secondary metabolites such as cosmetics, vitamins, nutritional materials, herbicides, antibiotics, pesticides, anti-parasitic and enzymes like cellulose and xylanase used in waste treatment. Due to pharmacological limitations and prevalence of antibiotic resistant pathogens the search of new antimicrobial drug from actinobacteria are raised.

There are about 23000 bioactive secondary metabolites which are produced by microorganisms. Over 10000 of the reported compounds are produced by Actinomycetes. Thus actinobacteria represents 45% of all bioactive microbial metabolites discovered. Also in Actinomycetes, there are about 7600 compounds which are produced by Streptomyces sp.Streptomyces sp. are one of the most prolific species and can produce a great many antibiotics (around 80% of the total antibiotic production) and active secondary metabolites. Streptomyces has the most secondary metabolites and are potent antibiotics, which has made them the primary antibiotic-producing organisms exploited by the pharmaceutical industry. The present work was undertaken to isolate potent actinobacteria form soil sample to elucidate their antimicrobial activity against clinical isolates.

MATERIALS AND METHODS

Sample Collection

Soil samples were collected from nursery VIT University, Tamil Nadu, (13.0900° N, 80.2700° E) India during August 2014.Soil samples were collected at a depth of 10-25cm.Samples were collected in sterilized container and transferred to the laboratory and stored in refrigerator at 4áµ’C until further processing.

Isolation of Actinobacteria

Isolation and enumeration of actinobacteria were performed on selective media such as actinomycetes isolation agar (AIA) and starch casein agar. The soil samples were serially diluted up to 10-7 and 100µL of the serially diluted samples were inoculated into the media. All these media were supplemented with nalidixic acid to avoid bacterial contamination and cyclohexamide (100 µg/mL) to avoid fungal contamination.Inoculated plates were incubated at 28ºC for 7 days [11].

Antimicrobial activity of Actinobacterial isolates

Test organism

The clinical isolates was collected from Narayani Hospital, Ariyur, Vellore District, Tamil Nadu, India..Bacterial isolates includes Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Staphylococcus aureus. The Test organism are maintained in glycerol stock andstored at -20°C.

Antibiogram

The four clinical isolates were screened for their sensitivity towards standard antibiotics included, Ampicillin (10mcg/disc), Methicillin (10mcg/disc), Vancomycin(30mcg/disc), Penicillin(10U/disc),Chloramphenicol (30mcg/disc), Polymycin-B(10µg/disc).

The Drug sensitivity test was performed by disc diffusion method on Mueller Hinton agar (MHA) plates. Bacterial test pathogens were prepared by inoculating into nutrient broth overnight. Bacteria were inoculated on Mueller Hinton agar plates by lawn culture method using sterile cotton swabs. The standard antibiotics discs were placed on the agar surface using a sterile forceps. Plates were incubated at37°C for 24 hours and were observed for zone of inhibition [12].

Primary Screening of actinobacteria for antibacterial activity - cross streak method

Primary screening of 4 actinobacteria isolates were performed by cross streak method on nutrient agar plates (NA) [13]. The actinobacterial isolates were inoculated in straight line on NA plates and incubated for 7 days. The clinical isolates were cross streaked on the same plate in perpendicular manner. The plates were incubated at37°C for 24 hours. The plates were examined for the zone of inhibition.

Fermentation Process

The potent actinobacterial isolate was inoculated into production broth (SS media) containing soluble starch-25g, glucose-10g, yeast extract-2g, CaCo3-3g, trace elements-1ml, distilled water-1000ml. Flask was kept on the rotary shaker incubator at a speed of 120 rpm at room temperature for 7 days. After fermentation, the medium was harvested and centrifuged to remove cell debris. Filtrate was collected and lyophilized and stored at 4 ÌŠC for further use [14].

Extraction of bioactive compounds

The bioactive metabolites were recovered from the harvested medium by solvent extraction method. The filtrate was mixed with ethyl acetate, chloroform, butanol (1:1 v/v) and shaken vigorously for 1 hour in a solvent extraction funnel. Solvent and filtrate mixture were stabilized for 24-48 hrs. After 48 hrs the solvent phase are separated from aqueous phase. The solvent extracts were concentrated and used for antibacterial activity [16, 17].

Secondary screening (Agar well diffusion method)

Secondary antimicrobial screening of actinobacteria was detected by agar well diffusion method on Muller Hinton agar [18].Different clinical isolates such as P.aeruginosa, S.typhi, E.coli, and S.aureus was inoculated on MHA plates using sterilized cotton swabs. In each of these plates, wells were cut out using a sterilized gel borer. The crude and solvent extract were used against test pathogen, 100µl of extracts were loaded into each well. Plates were incubated at 37ºC for 24 hours. After incubation all plates were examined for the presence of inhibition zone around the wells [19].

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) for ethyl acetate extract was determined by agar well diffusion method [20]. Test organism was lawn cultured on the Muller Hinton Agar plates. Agar surface was bored by using a gel borer. The extract was dissolved in ethyl acetate to obtain a concentration of 50µg, 100µg, 150µg, 200µg. A 100 µl of extract was loaded into different wells.All test plates were incubated at 37º C for 24 hours.

Taxonomic exploration

The efficacious actinobacteria were characterized by morphological and biochemical method and the results were compared with Nonomura key 1974, Shirling and Gottlieb 1966 and with Bergey’s manual of Determinative Bacteriology [21]

Morphological characteristics

Actinobacteria isolate were inoculated in seven different international streptomyces project (ISP) mediums (ISP 1 to ISP 7) and incubated for 7 days at room temperature. The colonies were observed under a microscope and colony morphology was noted with respect to aerial mycelium color, nature of colony and reverse side color.

Assimilation of carbon source

The ability of different actinobacteria species in utilizing various carbon sources is analyzed. viz., arabinose, xylose, inositol, mannitol, fructose, rhamnose, sucrose and as sources of energy were studied based on the method recommended by ISP. These carbon molecules were sterilized by ether sterilization [22].

RESULTS

Isolation of actinobacteria

Soil sample was collected from the nursery of VIT University, Tamil Nadu, India. A total of 30 actinobacteria colonies were isolated based on colony morphology and microscopic appearance (Table 1).The actinobacteria was cultured in AIA and starch casein agar. The starch casein agar enhanced more actinobacteria colonies, when compared to other media.

Table 1

Isolation of Actinomycetes using different media

Medium

Number of plates inoculated

Total number of actinomycetes isolated

Number of actinomycetes recovered

Actinomycetes isolation agar

15

30

20

Starch casein agar

15

15

10

IMAG0482.jpg

C:\Users\user\Desktop\IMAG0484.jpg

Figure 1: Colony Morphology of Actinobacteria isolates

Table 2

Comparison of morphological characteristics of DMPVIT 3

Characteristics

DMPVIT-3

Colour of aerial mycelium

Blackish grey

Melanoid pigment

-

Reverse side pigment

-

Soluble pigment

-

Spore chain morphology

Spiral

Table 3

Utilization of carbon source

Utilization of sole carbon sources

DMPVIT-3

Arabinose

+

Xylose

+

Inositol

+

Mannitol

+

Fructose

+

Rhamnose

+

Sucrose

+

Antibiogram

The clinical isolates were screened for antibiogram by disc diffusion method on MH agar plates. The result exhibits that tested drugs did not show any zone of inhibition the test samples.

Antimicrobial activity of isolated actinobacteria

Actinobacteria isolates were screened for antimicrobial activity against the clinical isolates. Among 30 actinobacteria isolates, only 4 isolates (DMPVIT-1, DMPVIT-2, DMPVIT-3, and DMPVIT-4) showed activity towards test organism in cross streak method. The zone of inhibition was (18.6±0.3) mm, (14.96±0.3) mm (13.42±0.2) mm,(15.4±0.3)mm respectively. Out of these 4 isolates, DMPVIT-3 showed good antimicrobial activity in agar well diffusion method. The potential isolate DMPVIT-3 was inoculated into production media (SS media). The bioactive compound was extracted in different polarity solvents and the extracts were screened for antimicrobial activity against the clinical isolates. The crude extract (13.33±1.05) mm, and ethyl acetate extract (22.24±0.4) mm exhibited potent activity against test organism, other solvent extract did not show any activity against the bacteria.MIC test was performed with the extract against the clinical isolates and found to be 200µg/ml. After fermentation the media was centrifuged and the supernatant was screened for antimicrobial activity. Out of 4 plates, 2 plates showed inhibition zone.

Table 4

Secondary screening (DMPVIT-3) – Agar well diffusion

Test organism

Concentration

Zone of inhibition

S.typhi

  • 50µg/ml
  • 100µg/ml
  • 150 µg/ml
  • 200 µg/ml
  • 10mm
  • 14mm
  • 16mm
  • 19mm

P.aeruginosa

  • 50µg/ml
  • 100µg/ml
  • 150 µg/ml
  • 200 µg/ml
  • 12mm
  • 15mm
  • 16mm
  • 23mm

Table 5

Minimum inhibitory concentration (DMPVIT-3)

Microorganism

MIC (µg/ml)

S.typhi

200

P.aeruginosa

200

C:\Users\user\Desktop\20141015_123149.jpg

C:\Users\user\Desktop\20141015_123212.jpg

Figure 2: Zone of inhibition of DMPVIT-3

DISCUSSION

Natural compounds obtained from soil source plays important key to discover various new drugs[23]. Actinobacteria are the most potent industrially important organism that are capable of synthesizing bioactive compounds like enzymes , hormones ,vitamins and other secondary metabolites. These bioactive compounds are highly difficult to synthesize artificially. Hence these microbial compounds are most prominenet sources for discover and production for new drugs[24].

Actinobacteria are one of the major groups oforganisms present in both terrestrial and marineenvironment. Primary and secondary metabolitesproduced by these organisms are biologically activeandserves as a dominant source to discover new drug molecule(Imada, 2005). Actinobacteria are the largest antibiotic producing group in the microbial worlddiscovered so far (Saadoun and Gharaibeh, 2003). Avariety of bioactive compounds from actinobacteriaarecommercially available to treat various dreadfuldiseases.In order to discover new and novel compounds for this purpose The actinobacteria exist in various habitats in nature even in the marine environment. Actinobactereia dwelling the terrestrial environment are more unique and diverse with the ability to produce unique chemical entities5. The terrestrial ones from the soil have been extensively used for the production of secondary metabolites useful to human. This interest is undoubtedly linked to reports that soil actinobacteria are proving to be a productive resource for the discovery of new medicines.

Most of the 70% commercial antibiotics are obtained from soil actinobacteria.[25].multi drug resistance pathogens serve as a hospital borne pathogen and plays a dominant role in many clinical problems globally[26]. In 2011, Karthik et al reported marine sediments were good sources for isolation of actinobacteria and M2 good for isolation marine action bacteria [27]. Similarly Bhaskaran et al reported that starch casein agar, was found to be good for the soil actinobacteria population. However, in the present study the maximum number of colonies was isolated starch casein agar. Hence the soil physiochemical properties may play a major role in the selection of isolation media. Out of 30 isolates which are isolated from soil was subjected to primary screening, among them only 4 isolates showed antimicrobial activity. As the isolate DMPVIT-3 showed maximum inhibition zone, it was selected for secondary screening.

The bioactive compounds are extracted from natural sources through several techniques solvent extraction is usually employed for the extraction of secondary metabolites for the culture filtrates. Different polarities of organic solvent have been utilized for the extraction of bioactive compounds from actinobacteria.[28] The extracts from ethyl acetate showed maximum antimicrobial activity against the bacteria. The ethyl acetate extract of DMPVIT-3 showed potent activity against clinical isolate. The MIC of the extract from DMPVIT-3 was 200µg/ml.

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