ANTIMICROBIAL ACTIVITY OF ACTINOMYCETES.pptx

STUDY OF
ANTIMICROBIAL
ACTIVITIES OF
ACTINOMYCETES
Presented by
Kiran Sanjyal
Kritisha guragain
Laxmi Ojha Nawang sherpa

OBJECTIVES
•i) To isolate Actinomycetes spp. From soil sample
•ii) To evaluate and characterize antibiotic activity
of Actinomycetes

INTRODUCTION
•Actinomycetes are unicellular
organisms that are found in a
variety of habitats as
discussed in the earlier class.

DISCOVERY OF Actinomycetes
•initially described by Ferdinand Cohn
in 1875 observed a filamentous organism
in a concretion from a human lacrimal
duct and named it Streptothrix foersteri.
•Later, C.O. Harz in 1878 named an organism
that he isolated from a case of
bovine lumpy jaw, as Actinomyces bovis.

•The term actinomycete means “ray fungus” in
Greek and actinomycetes are still referred to as
“ray fungi”.

CLASSIFICATION
•Kingdom: Bacteria
•Phylum: Actinobacteria
•Subclass: Actinobacteridae
•Order: Actinomycetales

•Actinomycetes belong to one of the
largest group of Microorganisms
Actinobacteria
•Volume 4 of Bergey’s Manual
typically consisting of High G C
content 50-55%

• Facultatively anaerobic, or Actinomyces meyeri and Actinomyces israelii obligate
anaerobic
Morphology and Chemotaxonomy.
Morphological
• They resemble Fungi morphogically and also, live in solid places like some fungi.
Mycelium formation
• A special type of vegetative reproduction; formation of asexual spores
Fragmenting hyphal forms (Nocardia spp.)
• Permanent and highly differentiated branched mycelia (e.g., Streptomyces spp., Frankia)
• Formation of branched substrate hyphae that break up into flagellated motile elements
(Oerskovia)

Colony form substrate and aerial
mycellium.
Aerial mycellium :
thicker than substrate..
Formation of exospore
An important criteria for
identifying
Streptomyces
( cottony, powdery).

Substrate mycellium –
growing in the solid substratum
(soil or agar) as a dense mat.
varies in shape, size, colour, thickness.
Example: Micromonospora, Actinoplanes

• Chemotaxonomy
use of the distribution of chemical components to group
organisms
according to the similarities of their cellular chemistries
• Classification and identification of actinomycetes
-Structure and composition of their peptidoglycans
-presence or absence of nonproteinogenic amino acid 2,6-diaminopimelic acid
-Cellular fatty acid
-Distribution of different types of phospholipids
-Sugar in cell wall composition
• Group A cell walls contain arabinose and galactose;
• group B cell walls contain madurose (
• group C consists of those with no diagnostic sugars;
• group D cell walls contain arabinose and xylose
• group E cell walls contain galactose and rhamnose. In addition, the presence of 3′-
O-methyl-rhamnose in Catellatospora

Importance of Actinomycetes
•These organisms are one of the most
important sources of structurally diverse,
clinically used antibiotics and other
valuable bioactive products, as well as
biotechnologically relevant enzymes.
•In soil, they are involved in the
decomposition and mineralization cycle
with the production of an extracellular
enzyme such as cellulase, chitinase and
lignin peroxidase, etc. They play an
important role in soil biodegradation for

As in Antimicrobial property,
Antimicrobial property refers to the production of such metabolic
product that in very small amount is detrimental or inhibitory to
other microorganisms.
Produced by fermentation technology,
3/4th of all known antibiotics are synthesized with the use of
actinomycetes.

WHY DO YOU THINK
ACTINOMYCETES
PRODUCE SO MANY
ANTIMICROBIALS?

•free-living bacteria
•many actinomycetes have evolved to live in symbiosis
with among others plants, fungi, insects and sponges.
•As a common theme, these organisms profit from the
natural products and enzymes produced by the
actinomycetes.
•For example, for protection against pathogenic
microbes, for growth promotion or for the
degradation of complex natural polymers such as
lignocellulose. At the same time, the actinomycetes
benefit from the resources of the hosts they interact
with.

Discovery of first antibiotic from
Actinomycetes
• After discovery of Penicillin by Fleming
• Selman Waksman and his students screened by looking for
growth inhibition zones surrounding single colonies of a
series of isolated soil microbes on agar plates growing under a
variety of culture conditions.
• test the inhibition on specifically targeted pathogenic
bacteria.
• painstaking work, as thousands of cultures of different
microbes, were isolated and then tested for antibacterial
activity, but which only a small percentage demonstrated.
• Actinomycin from Streptomyces parvulus
• was active against a broad range of bacteria and even showed

• Antibiotics - Secondary metabolites
include -
• Antitumorals (e.g., doxorubicin Streptomyces peucetius and bleomycin
Streptomyces verticillus),
• Antifungals (e.g., amphotericin B Streptomyces nodosus and nystatin
Streptomyces noursei ),
• Immunosuppressives (e.g., FK-506 Streptomyces tsukubaensis and
rapamycin Streptomyces hygroscopicus)
• Insecticides (e.g., Spinosyn A S. spinosa and Avermectin B S. avermitilis),
• Herbicides (e.g., Phosphinotricin several species of Streptomyces soil
bacteria.) and
• many antibiotics of clinical and commercial importances like
Virginiamycin (by S. virginiae) Daptomycin (by S roseosporus)

•DO YOU THINK STREPTOMYCES IS THE ONLY
GENUS TO PRODUCE ANTIBIOTIC?

•Other Antibiotics
• Rifampicin by Amycolatopsis mediterranei (used in anti Tuberculosis therapy)
• Polymyxins (Gram positive bact.) such as Paenibacillus polymyxa (used in eye infections like
conjuntivities)
• Gentamicin is produced by Micromonospora purpurea ( to treat serious bacterial infections )
• Enterocin produced by Salinispora pacificea against foodborne pathogen
•
Calicheamicins produced by Micromonospora echinospora sp. calichensis.
•
Erythromycin- Saccharopolyspora erythraea
• Fusidic acid - by Fucidium coccinium
• Rifamycin - Amycolatopsis mediterranei

PRINCIPLE
• Actinomycetes are screened by streaking pure culture in Nutrient
Agar or Mueller Hinton Agar(MHA) vertically.
COMPOSITION OF MUELLER HINTON
AGAR

• Nutrient agar is a general media and Mueller hinton agar is neither selective
nor differential meaning to say they supports the growth of almost all the bacteria.
• We can easily identify the antimicrobial activity of Actinomycetes due to following
reasons.
1. Peptone in Nutrient agar acts the source of nitrogen or proteins for the bacteria.
2. Mueller-Hinton Agar as used in Antimicrobial susceptibility test has a few properties
that make it excellent for antibiotic use. It contains starch.
3. Starch is known to absorb toxins released from bacteria, so that they cannot
interfere with the antibiotics.
4 The use of distilled water makes them a loose agar. This allows for better diffusion of
the antibiotics produced than most other plates. A better diffusion leads to a better
zone of inhibition. The distilled water provides a medium to dissolve the nutrients so
that it is easier for the bacteria to absorb them.

• Zone of Inhibition is to measure the ability of an antimicrobial
agent to inhibit the growth of microorganisms. If
Actinomycetes produces antibiotics then, it produces line of
inhibition which indicates the antimicrobial activity

•The formation of Zone of inhibition is essentially seen
due to the production of antimicrobials which targets
bacterial structures or functions, such as cell wall
biosynthesis (e.g., vancomycin), translation (e.g.,
streptomycin), RNA transcription (e.g., rifampicin),
DNA replication and synthesis (e.g., novobiocin and
metronidazole), membrane (polimyxins), and in
general they inhibit bacterial growth

Materials required:
•Pure culture of Actinomycetes
•Nutrient Agar Plates
•Test organisms
•Inoculating Loop

PROCEDURE
1. Streak a vertical line of Actinomycetes on Nutrient Agar plate.
2 Incubate the plates at 30°C for 5 days to get a proper growth.
3 Streak perpendicularly the test organisms keeping 0.5mm-1mm gap
from the growth of Actinomycetes.
4 Incubate the plates at 37°C for 24 hours.
5 Measure the zone of inhibition produced by Actinomycetes if there
are any. Antibiotic produced by Actinomycetes diffuses into agar that
may inhibit the growth of some test organisms.

Streaking Actinomycetes strain
- incubate at 30*C for few days
Leave some gap (0.5 – 1cm) and streak test
microbes
- incubate at 37*C for few days
1st…..30*C for 1 week
2nd ….. 37*C for 5 days

Procedure for a similar experiment
conducted
• 15 soil samples taken from Lagankhel, Katunje, Chovar,
Lubhu, Budanilkantha, Chapagaun, Godawari, Sundarijal,
Sankhamul, Gokarna and Chandragiri
Research conducted at KCGRL and St Xavier’s college lab
-soil sample was serially diluted to 10-2
-Starch Casein Agar (SCA) incorporated with
Amoxicillin 20 mg/l and Ketoconazole 30 mg/l
as to eliminate contaminant gram positive soil dwelling
bacteria and fungi respectively,

incubated at 28°C for 7 days.
-The developed colonies were were further sub-cultured in a
test tube containing SCA slants, incubated at 28°C for 7 days
and then stored in the refrigerator as the stock
Primary Screening of isolates
-inhibitory action of isolates against bacterial species
-Actinomycetes isolates were streaked vertically down the
center of the Nutrient agar plate and then incubated at 28°C
until visible growth of actinomycetes appear as a confluent
vertical line of the colony
-test organisms (Pseudomonas aeruginosa, Escherichia coli,
Klebsiella pneumoniae, Salmonella typhi, Salmonella
paratyphi, Bacillus subtilis, Staphylococcus aureus)
-hospital admitted patients

CONCLUSION
• The Antibiotic activity of various actinomycetes was performed
carefully in the laboratory

PRACTICAL SIGNIFICANCE
1 Actinomycetes can be used in development of new antibiotics for
pharmaceutical or agricultural purpose.
2. Antibiotics such as streptomycin tetracyclin etc and are used for
recycling organic matter.

PRECAUTIONS
• 1. Aseptic conditions should be maintained.
• 2- Zone of inhibition should be observed carefully if present.

REFERENCES
1 Available on
https://www.researchgate.net/publication/327941765_Mining_Actinomycetes
_for_Novel_Antibiotics_in_the_Omics_Era_Are_We_Ready_to_Exploit_This_New_
Paradigm/figures?lo=1&utm_source=google&utm_medium=organic
Accessed on 6th March, 2021
2 Available on
https://scialert.net/fulltext/?doi=ajdd.2011.75.84#t3 Accessed on 6th March,
2021
3Available on
https://patient.info/infections/antibiotics-leaflet Accessed on 5th March,
2021
4 Available on
https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(12)61202-1/
fulltext

REFERENCES
7 Available on
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037154/ Accessed on 6th March, 2021
8 Available on
https://www.jbc.org/article/S0021-9258(20)67861-9/fulltext Accessed on 6th March, 2021
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https://www.intechopen.com/books/actinobacteria-basics-and-biotechnological-applications/production-of
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https://www.slideshare.net/9844003833/actinomycetes-122126860 Accessed on 6th March, 2021

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Manandhar S. and Sharma S (2018). Practical approach to Microbiology. National
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March, 2021

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