1. INTRODUCTION:
Antibiotics are compounds produced by the
microorganisms that are able to inhibit the growth of other microorganisms1.
They have more economic value in health as it cures many infectious diseases.
The soil is a host of several microorganisms like bacteria, actinomycetes,
viruses etc. The soil serves as a versatile host of several microorganisms and
these microorganisms produces both primary and secondary metabolites which
controls the growth and development of other biota including flora and fauna2-3.
The antibiotics are the commercially most important exploited secondary
metabolites produced through microbial mediated fermentation. There are diverse
bacteria which are reported to produce different antibiotics with specific
functions4. Many drug resistant organisms have been arising and
hence there is a need to identify a compound which possesses the antimicrobial
activity against diverse pathogenic species.
The present research focuses on the
isolation and identification of the microorganism from the soil which secretes
the secondary metabolite with the antimicrobial action. The growth of the
isolated organism is optimized for the production of the secondary metabolite.
The antimicrobial activity of the secondary metabolite was confirmed by the
antibiotic sensitivity test.
2. MATERIALS AND
METHODS:
2.1. Chemicals:
Agar, peptone, Simmons citrate agar,
Christinens urea agar, Mueller Hinton agar and Whatmann filter paper No.3 were
obtained from Himedia, India. Sodium chloride, glycerol, ferrous sulphate,
diammonium hydrogen phosphate and calcium carbonate were purchased from Sigma Aldrich,
India. The reagents were prepared using sterilized double distilled water. All
the chemicals, media and the glass wares used in the investigation were
sterilized before use.
2.2. Collection
and preparation of soil sample:
The soil samples for the isolation of
microorganisms were collected from Madha Engineering College, Kundrathur,
Chennai. 1.0 g of soil was weighed and was suspended in 100 mL of sterilized
water and was serially diluted from 10-1 to 10-5 fold.
2.3. Isolation of
microorganism:
The media for the isolation of
microorganism from the soil was formulated with agar, 28 g/L; peptone, 10 g/L
and NaCl, 15 g/L substituted with glycerol, 15 mL/L; ferrous sulphate, 0.5 g/L;
diammonium hydrogen phosphate, 0.5 g/L; agar, 30 g/L and calcium carbonate, 2.0
g/L. The media was sterilized in autoclave at 121˚C for 15 min and 15 lbs
pressure. The media was then poured in the sterilized glass petriplates and
allowed to get solidified. The soil sample from the last dilution was
inoculated into the petriplate by simple streaking method and incubated at 37°C
for 24 h. The organism were isolated and stored in glycerol stock for future
investigations.
2.4. Screening of
microorganisms for the production of the antibiotic:
The
crowded plate technique was performed to isolate the antibiotic producing
colonies from the isolated colonies. The colonies grown in the incubated plated
were then sub-cultured and observed for the clear zone of inhibition. The
colony which is showing the clear zone were then sub-cultured and maintained in
agar slants at 37°C for 24 h for further investigation.
2.5. Biochemical characterization of the
isolated bacteria
2.5.1. Gram’s staining:
A
smear of bacterial culture was made on a grease free slide and then heat fixed.
The smear was then flooded with the crystal violet stain and washed with
distilled water. Then the slide was flooded with Gram’s iodine and removed by
gentle wash. The smear was then decolorized with distilled water and the
counter stain saffranin was added and viewed under the microscope.
2.5.2. Citrate utilization test:
Simmons
citrate agar slants were prepared by adding 3 mL of sterilized media into the
test tubes. The isolated bacterial strains were inoculated into the slants and
were incubated at 37°C for 24 h. The surface of the inoculated slants was
examined for the evidence of growth and color changes.
2.5.3. Catalase test:
The
catalase test was performed by the introduction of small amount of the
bacterial culture with the help of capillary tube. The release of the air
bubbles was observed and compared with the control.
2.5.4. Urease test:
Christinen’s
urea agar slant was prepared, inoculated with the isolated microorganisms and
incubated at 37°C for 24 h.
2.5.5. Triple sugar iron test:
Triple
sugar iron test was done for the confirmation of carbohydrate utilization. The
TSI slants were prepared and the isolated microorganisms were inoculated and
incubated at 37°C for 24 h and observed.
2.6. Extraction of the metabolite produced after
fermentation:
The
fermentation broth was centrifuged at 10000 rpm for 10 min to
yield the metabolites devoid of cell debris and this mixture was used for the
extraction of metabolites using equal volume of ethyl acetate, dried over anhydrous sodium
sulfite and evaporated to dryness in rotary vacuum evaporator5-8.
2.7.Assessment of antimicrobial activity of
the metabolite:
Kirby-Bauer
disc diffusion method was performed to analyze the efficiency of the
antibiotic. The Mueller Hinton agar was prepared, sterilized and poured into
the sterilized petriplates and allowed to solidify. The metabolites were tested
on the pathogenic bacteria and fungal species. Staphylococcus aureus, Salmonella
typhi, Pseudomonas aeruginosa, Aspergillus fumigatus, Aspergillus niger and Penicillium chrysogenum were used for
the analysis of the antibiotic. The metabolites from the isolated bacterial
strain were coated on the Whatmann filter paper No. 3 and air dried and made
into desired shape of discs. The discs were then placed in the plates along
with the control and incubated at 37°C for 24 h and observed for the zone of
inhibition against the pathogenic organisms.
3. RESULTS AND DISCUSSION:
3.1. Isolation and screening of the
secondary metabolite
The
microorganisms present in the soil sample were isolated by serial dilution
technique. The antibiotic producing microbes were then screened by the crowded
plate technique. The colonies that were observed in the plate were counted by
using a colony counter and the morphology of the colonies was also observed.
The clear zone producing colonies were identified and selected for further
investigation. The selected colony is then sub-cultured on the fresh nutrient
agar supplemented media. The streak plate method was performed for the
isolation of pure culture.
3.2. Biochemical characterization of
bacterial strain
The
morphological feature of the isolated strain of bacteria was clearly visible
with Gram’ staining and the motility test. The isolated culture was identified
as rod shaped and Gram negative bacteria. The hanging drop technique was
performed to check for their motility. The bacteria were swamming near the edge
of the slide, indication that they are motile and possessed flagella.
The
biochemical tests were performed on the isolated culture and the results were
positive for citrate utilization test. Simmons citrate agar slants showed a
color change from green to blue upon incubation and hence it is positive for
citrate utilization. The triple sugar iron slants showed a red slant at the
bottom of the tube. The isolated culture does not showed groin the MacConkey agar
indicating it as a non lactose fermentor.
3.3. Assessment of antimicrobial property
of the metabolite
The
antibiotic production was confirmed by the antibiotic sensitivity test. The
isolated bacteria was tested against the fungal (Aspergillus fumigatus, Aspergillus niger and Penicillium chrysogenum)
and bacterial (Staphylococcus aureus,
Salmonella typhi and Pseudomonas aeruginosa) pathogens. The
maximum zone of inhibition was observed against the pathogens as shown in the
Table 1. The maximum zone of inhibition observed in Salmonella typhi and Penicillium
chrysogenum was 14 and 20 mm.
Table 1 Zone of inhibition exerted by the
bacterial metabolite against the different microbial strains
|
S.No.
|
Organism tested
|
Zone of inhibition (mm)
|
|
1
|
Staphylococcus aureus
|
12
|
|
2
|
Salmonella typhi
|
14
|
|
3
|
Pseudomonas aeruginosa
|
10
|
|
4
|
Aspergillus fumigatus
|
14
|
|
5
|
Aspergillus niger
|
24
|
|
6
|
Penicillium chrysogenum
|
20
|
4. CONCLUSIONS:
The
extraction of antibiotics from the potential microorganisms paves the way to
resist the multi drug resistant pathogenic organisms. Many species have been
isolated from various sources to produce the secondary metabolite, antibiotics.
The soil harbors diverse microorganisms and hence serves as the potential
source for the isolation and screening of bacteria to produce the antibiotics.
The isolates were collected from the soil and screened for the antimicrobial
activity on the pathogenic organisms. The antimicrobial activity was confirmed
with the antibiotic susceptibility test with the zone of clearance. From this
study, we conclude that this secondary microbial metabolite can be used for the
treatment of microbial related diseases. The mechanism of action needs to be
carried out further in order to understand the application and usage of this
antibiotic as a therapeutic drug.
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