Antibacterial test methods for c.

abyssinica
against common urinary tract infection pathogen

Sisay .f

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 Outline
• Objective
• Introduction
• Hypothesis
• research questions
• Antibacterial test methods

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 Objective
– To asses the antibacterial activity of C.
abyssinica .

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 Introduction
• UTI is one of the most common disease both
developing and developed world with high
incidence rate in female.
• The causative agent are E.coli (75 to 95 %) ,
E.faecalis , k. pneumonia, and p.argenosea.
• Currently the treatment failures associated with
multidrug-resistant strain and high recurrence rate
is a global concern to public health .
• So new compounds from the natural product are
needed to combat this issue

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 Antibacterial test model for clutia abyssinica
• Hypothesis: “C. abyssinica has activity against urinary
tract infection “
• Research questions :
– Does it has an activity against common pathogens for urinary
tract infection
– What type of antibacterial activity it has? ,
• bactericidal or bacteriostatic
– Does it has secondary metabolites that have antibacterial
activity?
– What is Bacterial response to the extract
• sensitive or Resistance ,
– What is the minimum inhibitory concentration and minimum
bactericidal concentrationsisay,( fikru
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 Preparation of the plant for antimicrobial test
• Identification of Plant ,
– with the help of botanical expert
• Collection of the Plant.
– wash with water
– drying under shade to avoid/protect surrounding contamination and
dust
• Choose solvent for extraction
– Acetone, methanol, ethanol and water common solvents for
preliminary investigations of antimicrobial activity in plants
– Solvents being used should be tested as controls in order to ascertain
that the antibacterial activity is not due to the solvent.
• Choose appropriate standard protocol
– USA Clinical and Laboratory Standards Institute (CLSI) for bacteria and
yeasts testing
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sisay, fikru
 Determination of bacterial sensitivity
Agar well disk diffusion
• Apply standard amount of bacterial suspension to the
agar medium uniformly
• Then, a hole with a diameter of 6 mm is punched
aseptically with a sterile cork borer
• extract solution at desired concentration is introduced
into the well.
• Then, agar plates are incubated under suitable
conditions depending upon the test bacteria .
• The plant extract diffuses in to the agar medium and it
inhibits the growth of bacteria .
 Cont..
the diameter of inhibition zone (in mm) is measured and
interpreted as
> 9mm : Sensitive,
< 9mm : Resistant
 Cont..
• Advantage
– Simple to perform and cheap
– ability to test multiple type of bacteria on the same agar
plate except for swimming bacteria
– Easy to interpret results provided
• Limitation
– Selective diffusion
– poor performance for slow-growing and fastidious bacteria
– cannot distinguish bactericidal and bacteriostatic effects
– It does not MIC and MBC
– Manual measurement of zones of inhibition is tedious
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 Determination of MIC
Agar dilution
– Different predetermined concentration ( two-
fold dilution range ) of our plant extract applied
on solid agar plates which contain bacteria of
interest
– follow the growth of bacteria and compared with
a control culture which does not contain our
extract
– the highest dilution at which the sample
prevents the growth of the microorganism (MIC)
is determined.
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 Cont…
• Advantage of agar dilution
– The ability to test multiple bacteria, except bacteria
that swarm, on the same set of agar plates at the same
time
– the visibility of Bacterial growth is not affected by the
color of our plant extract like broth dilution
– No need of reagents
• Limitation of agar dilution
– Manual dilution is so tedious

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 Determination of MBC
Broth macro dilution
– bacterium suspension is tested against varying
concentrations of plant extract (usually serial two fold
dilutions) in a liquid medium .
– after appropriate incubation period , the tubes
examined for visible bacterial growth as evidenced by
turbidity
 Cont..
• To conform the growth or death of bacteria specific
volume from each tube is taken and incubated
again on the agar medium .
• The highest dilution on the agar medium with no
bacterial growth i.e. minimum bactericidal
concentration (MBC) is determined.
• Advantage of broth macrodilution
• the generation of a quantitative result (MIC and MBC)
• Limitation of broth macrdilution
– manual task of preparing the extract solutions for
each test tube is tedious .
– the relatively large amount of reagents and space
required for each test .
 secondary metabolite Identification

TLC -Bioautography
 It involves localizing the antibacterial activity on a
chromatogram.
 The antibacterial agent is transferred from the
TLC plate or paper chromatogram to an
inoculated agar plate by diffusion and the zones of
inhibition is visualized

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 Cont..
 Bioautographic assays can be divided in three groups
Direct bioautography, the developed TLC plate is dipped
into or sprayed with a bacterial suspension then
incubate in suitable condition and follow the growth

Direct bioautography

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 Cont…
Contact bioautography, where the antibacterial
compounds are diffuse from the TLC plate to an
inoculated agar plate through direct contact.
After some minutes or hours to allow, the chromatogram is
removed and the agar plate is incubated
Agar overlay or immersion bioautography, where a
seeded agar medium is applied onto the TLC plate.
Zone of inhibition identify
antimicrobial lead

Agar overlay bioautography

Contact bioautography
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 Cont…
• Advantage of bioautography
– It allows rapid localization of activity even in a complex
matrix
– Both non-polar and polar compound are examined for
activity
– Avoid the time-consuming isolation of inactive compounds
– It used for all bacteria type and also spore and it is also
cheap
• Limitation of bioautography
– adsorbent and solvent inference
– the differential diffusion of components
– Can’t used to determine MIC and MBC
– Need further investigation sisay,
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other technique 18
 References
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Evaluating Antimicrobial Activity : A Review $.” Journal of Pharmaceutical Analysis 6 (2): 71–79.
2. Choma, Irena M, and Edyta M Grzelak. 2014. “Bioautography Detection in Thin-Layer
Chromatography Bioautography Detection in Thin-Layer Chromatography.” Journal of
Chromatography A 1218 (19): 2684–91.
3. Coudron, Philip E, and Charles W Stratton. 1995. “Use of Time-Kill Methodology To Assess
Antimicrobial Combinations against Metronidazole-Susceptible and Metronidazole-Resistant
Strains of Helicobacter Pylori” 39 (12): 2641–44.
4. Das, K, R K S and Tiwari, and D K Shrivastava. 2010. “Techniques for Evaluation of Medicinal
Plant Products as Antimicrobial Agent : Current Methods and Future Trends” 4 (2): 104–11.
5. Eloff, Jacobus Nicolaas. 2019. “Avoiding Pitfalls in Determining Antimicrobial Activity of Plant
Extracts and Publishing the Results.” BMC Complemntry and Alterative Medicne 3 (19): 1–8.
6. Grzelak, Edyta, Wioleta Jesionek, and Irena Choma. 2013. “Applications of Novel Direct
Bioautography Tests for Analysis of Antimicrobials: A Review,” no. November.
7. Puttaswamy, Sachidevi, Sagar Kishore Gupta, Hariharan Regunath, Leo Patrick Smith, and
Shramik Sengupta. 2018. “IMedPub Journals A Comprehensive Review of the Present and
Future Antibiotic Susceptibility Testing ( AST ) Systems Keywords :,” 1–9.

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 Thank you

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