Studies On The Antibacterial Activity of The Actinomycetes Isolated From The Khumbu Region of Nepal

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Studies on the antibacterial activity of the Actinomycetes isolated from the

Khumbu Region of Nepal.

Bhagabati Pandey 1, Prakash Ghimire1 and Vishwanath Prasad Agrawal 2


1
Tribhuvan University, Kirtipur Nepal, E.Mail: bati@wlink .com.np; 2 Academician of Royal Nepal
Academy of Science and Technology, E.Mail: vpa@wlink.com.np

Abstract

Antibacterial activity of actinomycetes isolated from Lobuche area (5000-5300 meter in height) and
Lukla area (2660 meter in height) in Khumbu region has been studied. A total of 106 actinomycetes
were subjected to primary screening by perpendicular streak method against Gram-positive (Bacillus
subtilis and Staphylococcus aureus) and Gram-negative (Enterobacter aerogens, Escherichia coli,
Klebsiella species, Proteus species, Pseudomonas species, Salmonell typhi and Shigella species) test
bacteria. It was observed that 2 isolates were active against only Gram-negative bacteria, 8 against
Gram-positive and 26 against both Gram-positive and Gram-negative bacteria.
Altogether 36 putative isolates were subjected to secondary screening by agar well method to further
test the capabilities of primarily screened organisms. Selected isolates (20) from the secondary
screening belonged to the genera Streptomyces (10), Streptoverticillium (4), Saccharopolyspora (3),
Micromonospora (2) and Actinosynema (1).
Finally 2 isolates (Streptomyces species and Saccharopolyspora species) were selected for further study
on the basis of (a) broad spectrum activity and (b) larger zone of inhibition in comparison to others. The
antibacterial substances were extracted with ethyl acetate from isolate-inoculated starch-casein broth
fermented for 7 days at 28°C by solvent extraction method. Minimum bactericidal concentration (MBC)
of ethyl acetate extract against Staphylococcus aureus were 1.25 mg/ml for Saccharopolyspora species
and 5mg/ml for Streptomyces species. Thin layer chromatography (TLC) of the ethyl acetate extracts
were carried out in duplicate using Chloroform: methanol (4:1) as solvent system and Tetracycline as
reference antibiotic. Under UV light they gave greenish yellow spots with Rf value 0.88 for the
antimicrobial from Streptomyces species and 0.90 for that from Saccharopolyspora species. In
bioautography (using Staphylococcus aureus as test organism) inhibition zones were obtained and they
were associated with the yellowish green spots of the chromatogram as detected under UV light. This
may indicate the same compounds were responsible for the antibacterial activity of those actinomycetes
isolates.

Introduction

The actinomycetes are Gram positive bacteria having high G+C (>55%) content in their DNA. The
name ‘Actinomycetes’ was derived from Greek ‘aktis’ (a ray) and ‘mykes’ (fungus) and given to these
organisms form initial observation of their morphology. Actinomycetes were originally considered to be
an intermediate group between bacteria and fungi but now are recognized as prokaryotic organisms.
The majority of actinomycetes are free living, saprophytic bacteria found widely distributed in
soil, water and colonizing plants. Actinomycetes population has been identified as one of the major
group of soil population (Kuster 1968), which may vary with the soil type.
The actinomycetes are noteworthy as antibiotic producers, making three quarters of all known
products; the Streptomyces are especially prolific and can produce a great many antibiotics and other
class of biologically active secondary metabolites. They cover around 80% of total antibiotic product,
with other genera trailing numerically; Micromospora is the runner up with less than one-tenth as many
as Streptomyces. If we include secondary metabolites with biological activities other than antimicrobial,
actinomycetes are still out in front, over 60%; Streptomyces spp. accounting for 80% of these
(Hopwood, et al., 2000).
Due to large geographic variation, there is large variation in soil type and their contents in Nepal
and hence it is quite likely that the distribution of antibiotic producing actinomycetes is also variable.
This study is carried out to screen the antibiotic producing actinomycetes from higher altitude, Khumbu
region, of Nepal which is situated at the lower part of Mount Everest base camp.

Materials and methods

The specimens (actinomycetes) used in this study were isolated from the soils of Khumbu region. Soils
from different places of Khumbu region were brought to the laboratory in aseptic condition.
Actinomycetes from the soil had been isolated by pour plate technique on Starch-casein agar and
Glycerol-arginine agar after serial dilution in distilled water. Dry colonies of actinomycetes were
selected and isolated. Thus isolated colonies had been preserved in Glycerol based media and stored at -
20°C. The actinomycetes of which antimicrobial activity should be determined were revived by
streaking on Starch-Casein agar and incubated at 28°C for 7 days.

Screening of actinomycetes for antimicrobial activity: The screening method consists of two steps;
Primary screening and secondary screening.
In primary screening the antimicrobial activity of pure isolates were determined by perpendicular streak
method (Egorov, 1985) on Nutrient agar (NA). The test organisms used were; Bacillus subtilis,
Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species,
Pseudomonas species, Salmonella typhi and Shigella species.
Secondary screening was performed by agar well method against the standard test organisms
Escherichia coli, Staphylococcus aureus, Salmonella typhi, Bacillus subtilis, and Proteus spp.

Characterization of actinomycetes: The potent actinomycetes selected from secondary screening were
characterized by morphological and biochemical methods. Morphological methods consist of
macroscopic and microscopic methods. The microscopic characterization was done by cover slip culture
method (Kawato and Sinobu, 1979).The mycelium structure, color and arrangement of conidiospore and
arthrospore on the mycelium was observed through the oil immersion (1000X). The observed structure
was compared with Bergey’s manual of Determinative Bacteriology, Ninth edition (2000) and the
organism was identified. Various biochemical tests performed for the identification of the potent isolates
are as follows: Casein hydrolysis, Starch hydrolysis, Tween 20 hydrolysis, Urea hydrolysis, Esculin
hydrolysis, Acid production from sugar, NaCl resistance, Temperature tolerance.

Fermentation process: Fermentation was carried out in a 1L Erlenmeyer flask following the procedure
as described by Liu et.al (1992).

Isolation of antibacterial metabolites: Antibacterial compound was recovered from the filtrate by
solvent extraction method following the process described by Westley et.al, 1979.and Liu et.al, 1986.
Ethyl acetate was added to the filtrate in the ratio of 1:1(v/v) and shaken vigorously for 1 hour for
complete extraction. The ethyl acetate phase that contains antibiotic was separated from the aqueous
phase. It was evaporated to dryness in water bath at 80°-90°C and the residue obtained was weighed.
Thus obtained compound was used to determine antimicrobial activity, minimum inhibitory
concentration and to perform bioautography.

Determination of the antimicrobial activity: The antimicrobial activity was determined by agar well
method (Sen. et al., 1995). The partially purified extract obtained by the evaporation of the ethyl acetate
extract was dissolved in 1 ml 0.2M phosphate buffer (pH 7.0). Then 100µl of it was loaded into well
bored and test organism (0.5 McFarland turbidity standard) swabbed Muller Hinton agar plates. The
plates were incubated at 37°C for 18-24 hrs and examined. The diameter of the zones of complete
inhibition was measured to the nearest whole millimeter.

Determination of minimum inhibitory concentration: It was determined by the serial dilution of the
antimicrobial in nutrient broth, two fold dilution at each time, against Staphylococcus aureus.
Thin layer chromatography and Bioautography: Silica gel plates, 10X20 cm, 1mm thick, were
prepared. They were activated at 150°C for half an hour. Ten microliters of the ethyl acetate fractions
and reference antibiotics were applied on the plates and the chromatogram was developed using
chloroform: methanol (4:1) as solvent system. The plates were run in duplicate; one set was used as the
reference chromatogram and the other was used for bioautography. The spots in the chromatogram were
visualized in the iodine vapour chamber and UV chamber.
Muller Hinton agar inoculated with Staphylococcus aureus was poured over the chromatogram and the
plate was incubated overnight at 37°C in sterile condition. The next day the inhibition zones were noted
and the Rf values of the antimicrobials were determined.

Results

Out of 106 actinomycetes subjected for primary screening process, only 36 isolates showed the activity
against test organisms. Of the 36 isolates, 2 were active against only gram negative organism, 8 against
gram positive organisms and 26 against both gram positive and gram negative organisms. Among them,
31 of the isolates were active against Bacillus subtilis, 27 against Staphylococcus aureus, 17 against
Escherichia coli, 15 against Salmonella typhi and 14 against Proteus species.
Out of the 36 isolates that were subjected for the secondary screening, 23 isolates were active against
Bacillus subtilis, 23 against Staph. aureus, 17 against E. coli, 10 against Proteus species and 6 against
Salmonella typhi.
The results can be represented in the bar diagram as follows:
Bacillus subtilis
E.coli
Proteus vulgarius
Salmonella typhi
Bacillus subtilis
Nos. of active isolates

40 Staphylococcus
31 Citrobacter spp. aureus

Nos. of active isolates


27 Enterobacter aerogens
30 Escherichia coli
Klebsiella spp 25 23 23
17
20 14 15 14 Proteus vulgarius 20 17
13 Salmonella typhi
Shigella spp 15 10
10 6 7
Staphylococcus aureus
10 6
0 5
Test bacteria 0
Test bacteria
Fig 1: Activity shown by actinomycetes Fig2: Activity shown to test bacteria in
in primary screening. secondary screening.

Identification: The identification of the potent antibiotic producing strains reveals that most of the
specimens belong to the genus Streptomyces (10) followed by Streptoverticillium(4),
Saccharopolyspora(3), Micromonospora (2) and Actinosynema (1).
Two potent isolates were selected for fermentation on the basis of their broad spectrum of activity and
largest zone of inhibition. They were found belonging to the genera Streptomyces and
Saccharopolyspora.

Minimum inhibitory concentration: The minimum inhibitory concentration for the extract from
Streptomyces spp was 5mg/ml and that from Saccharopolyspora spp was 1.25mg/ml.

Thin layer chromatography and Bioautography: The spot given by the extract of Streptomyces spp
was a circular with Rf value 0.88 and that of Saccharopolyspora spp was an extended spot with Rf value
0.90. The fluorescence colours of the spots were greenish yellow. The reference antibiotic, Tetracycline,
didn’t move with the solvent system.
In bioautography the Streptomyces spp gave an inhibition zone of 20mm diameter and the
Saccharopolyspora spp gave inhibition zone of size 10x80mm. The reference antibiotic Tetracycline
gave the inhibition zone of 25mm diameter at the origin.
Discussion

The putative isolates of primary screening when subjected to secondary screening, showed different
activity from that of primary screening; some of the active isolates didn’t show the activity in the
secondary screening while some showed little activity and some showed improved activity. According
to Bushell (1993), during the screening of the novel secondary metabolite, actinomycetes isolates are
often encountered which show antibiotic activity on agar but not in liquid culture.
The result of primary and secondary screening revels that most of the active isolates were active
against gram positive bacteria (Bacillus subtilis and Staph. aureus) than gram negative bacteria. The
reason for different sensitivity between gram positive and gram negative bacteria could be ascribed to
the morphological differences between these microorganisms, gram negative bacteria having an outer
polysaccharide membrane carrying the structural lipopolysaccharide components. This makes the cell
wall impermeable to lipophilic solutes, The gram positive should more susceptible having only an outer
peptidoglycan layer which is not an effective permeability barrier (Scherrer &Gerhardt, 1971).
Although various biochemical tests were performed, it was unable to identify the actinomycetes up to
species level due to the lack of other tests. According to Kutzner (1972) for proper identification of
genera and species of actinomycetes, besides morphological and physiological properties, various other
biochemical properties such as cell wall chemo type, whole-cell sugar pattern, peptidoglycan type,
phospholipids type and G+C% of DNA should be determined.
The minimum inhibitory concentration (MIC) for the antimicrobial extracted from Streptomyces
spp was 5mg/ml and that from Saccharopolysporaso spp was 1.25mg/ml. This show that the
antimicrobial from Saccharopolyspora spp was more active than from Streptomyces spp but there are
various factors affecting the activity. The Streptomyces spp can be a poor fermenter than the later one or
the solvent used for extraction may not be suitable for it or the compound may not be properly extracted
by the solvent.
The MIC is not a constant for a given agent, because it is affected by the nature of the test
organism used, the inoculum size, and the composition of the culture medium, the incubation time, and
aeration.
For complete characterization of an antibiotic it should be isolated in pure form as a single component
but this is impractical in a screening programme like this. However, a little effort was made in this
approach. According to the TLC separation, the two extracts yielded components with Rf values similar
to the antibacterial compounds as visible on bioautogram. In addition, the inhibition zones were
associated with yellowish green spots which had been detected under UV radiation. This may mean that
the same compounds are responsible for antibacterial activity of those isolates.
Although the antimicrobial agents obtained in this study can’t be declared as new antibiotics, there is the
probability of finding new antibiotics in Nepal because of its wide biodiversity. For proper identification
of the antimicrobial extracts it is necessary to obtain in pure form, which requires a series of purification
process and different chemical analysis such as HPLC, Spectroscopy and other sophisticated techniques.
As we know the land of Nepal is virgin in this field, so lots of works should be done to explore the new
antibiotics because any new antibiotics and its producing organism have been a great demand.

References:

Agrawal, V.P., 2002. Biodiversity of Khumbu region: population study of actinomycetes, Submitted to Royal Nepal
Academy for Science and Technology.
Becker, B.M. Lechevalier, R.E.P. Gordon, Lechavier, H.A., 1964. Rapid differentiation between Nocardia and
Streptomyces by paper chromatography of whole cell hydrolysates. Applied Microbiology. 12, 421-423.
Berdy, J., 1967. Recent developments of Antibiotic Research and Classification of Antibiotics According to Chemical
Structure, Advances in Applied Microbiology, 309.
Bergey’s manual of determinative bacteriology, 2000. Actinomycetales. 9th edition.
Hugo, W.B., Russel, A. D., 1983. Pharmaceutical Microbiology, 3rd edition, Blackwell Scientific publications.
Kawato, M., Shinobu, R., 1959. A simple technique for the microscopical observation, memoirs of the Osaka
University Liberal Arts and Education, 114.
Waksman, S.A., 1968. Actinomycetes; Nature, Formation and Activities, Applied Microbiology. 5, 235-293.
Williams, S. T., Cross, T., 1971. Actinomycetes. Applied microbiology. 4, Academic Press.

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