Lecture 4

Field of Pharmacy Sciences
Bachelor of Pharmacy- PharmD (Clinical Pharmacy) Program
Pharmaceutical Microbiology (PMB202) Lecture : Seven
Dr : Ahmad M. Abbas Date : 28 / 3 /2023

Antimicrobial resistance
Evaluation of
antibiotics • Antimicrobial Susceptibility Testing
• Evaluation of Bacteriostatic Activity

• Evaluation of Bactericidal Activity
• Microbiological Assay Methods

• Rapid Microbiological Assay Methods
Antimicrobial Susceptibility Testing (antibiotic
sensitivity test)
• Definition: the study of the susceptibility of a M.O to a specific antibiotic

• Used to determine the most suitable antimicrobial agent(s) for the treatment of a
certain microbial infection.
• This is carried out by studying the susceptibility of the isolated infectious agent to
different antimicrobial agents and the selecting the most effective one(s) against
it.
• This is usually carried out by agar diffusion method.
Kirby-Bauer Disc diffusion method
• Uses small discs impregnated with antibiotics (of known volume and
concentration)
• These discs are placed on the surface of the seeded growth medium agar plate,
and incubated at 37oC for 24 hrs.
• After incubation, plates are observed for the zone of inhibition.
• The diameter of zone of inhibition is measured & compared with Kirby-Bauer
chart (standard chart)
Measurement of zone of inhibition
• The size of the inhibition zone is affected by:
1. Diffusion rate and Concentration of the test antibiotic
2. Test M.O (growth rate and metabolic activity)
3. Depth of agar
4. pH of the medium
5. Incubation time
Kirby-Bauer Chart
Antibiotic R MS S
Amikacin 14 mm 15-16 mm 17 mm
Ciprofloxacin 15 mm 16-20 mm 21 mm
Ceftriaxone 13 mm 14-20 mm 21 mm
Doxycycline 12 mm 13-15 mm 16 mm
Gentamicin 12 mm 13-14 mm 15 mm
Interpretation
• SENSITIVE
A pathogen reported as sensitive suggests that the infection it has caused is likely to
respond to treatment if the antibiotic to which it is susceptible is used in normal
recommended doses.
• INTERMEDIATE / MODERATELY SENSITIVE

A pathogen reported as moderately sensitive suggests that the infection it has
caused is likely to respond to treatment if the antibiotic is used in larger than normal
doses or when the antibiotic is concentrated at the site of infection
• RESISTANT
A pathogen reported as resistant suggests that the infection it has caused will not
respond to treatment with that antibiotic irrespective of dose or site of infection
Test to Show the Effect of Drug Combination
• This is carried out by using two strips of filter paper, each is loaded (wet) with one
of the 2 test antibiotics.
• Then, they are placed on the surface of seeded growth medium agar plate, at right
angle to each other.
• After incubation, the inhibition zone at the intersection is compared with that at
the ends to indicate antagonism or synergism.
Used to determine
Rapid Microbiological Assay Methods unknown antibiotic
concentration, via
standard curve
1. Urease production
• When certain bacteria grow in a urea-containing medium, they hydrolyze the urea
into ammonia and consequently raises the pH of the medium. This production is
inhibited by aminoglycosides. The change in pH is proportional to the antibiotic
concentration.
2. Luciferase production
• This assay depends on measurement of the luminescence produced by certain
bacteria. The luminescence is produced due to the reaction between intracellular
ATP and luciferase enzyme. The level of ATP is inhibited by the action of
aminoglycosides. The degree of ATP inhibition and consequently the degree of
luminescence is proportional to the antibiotic concentration.
3. Radiotransferase assay
• This method is used for the assay of chloramphenicol and aminoglycosides. It
depends on the fact that the degree of resistance to these antibiotics is associated
with the level of bactreial acetyl-transferase enzyme (that requires acetyl CoA) or
adenyl-transferase (that requires ATP).
• In this assay, radiolabelled acetyl Co A or ATP together with the corresponding

enzyme are used to radiolabel the test antibiotic. After the reaction, the radiolabeled
antibiotic is separated, then the radioactivity is measured which is proportional to
the antibiotic concentration .
• Advantages:
The method is rapid, precise, and more specific than the other traditional
microbiological methods
4- Immunoassay
• Immunoassays depend on the principle of competition between molecules of
test antibiotic and standard labeled drug molecules for binding to a specific sites
on a limited amount of corresponding antibody.
• Within a specific concentration range, increase in the amount of test antibiotic

molecules cause reduction in the number of labeled drug molecules that bind to
the antibody, which can be measured
MIC & MBC
Minimum concentration of antibiotic required to arrest the
multiplication of the bacteria is known as Minimum
Inhibitory Concentration
Minimum concentration of antibiotic required to kill the

bacteria is known as Minimum Bactericidal Concentration
Evaluation of Bacteriostatic Activity
• It gives an idea about Minimum Inhibitory Concentration (MIC)
• MIC value is affected by:

1. Type and stability of the antimicrobial agent
2. Test M.O (growth rate and metabolic activity)
3. Inoculum size
4. Incubation time and temperature
5. The growth medium
6. Depth of the medium (in case of agar diffusion)
Methods of measuring MIC
1. Serial Dilution
The lowest concentration at which no growth is observed is the minimum
inhibitory concentration.
Initial
conc.
Initial
conc.
2- Agar diffusion method (Cup-plate technique)
• A graph is plotted between log concentrations of the antibiotics and
diameters of the inhibition zones (mm). From the graph, the value of
MIC can be determined.
Most commonly
used growth media
in agar diffusion
techniques are
nutrient agar and
Mueller Hinton agar
3
4
1
2
3- Agar dilution method
• In this method, the antimicrobial agent is mixed with liquefied nutrient agar and
poured into sterile plate, to finally give the required concentration.
• After solidification and drying of the surface, the bacterial suspension is
inoculated onto the agar surface in the form of few drops. The lowest
concentration that prevent microbial growth is the MIC.
• The advantage of this technique is that for each concentration of the tested
substance, several organisms can be tested at the same time.
4. Gradient-plate technique
• In this technique, a double layered agar plate is formed: liquefied nutrient agar is
mixed with the test antimicrobial agent and poured into a sterile plate. The mixed
nutrient agar is allowed to set in the form of a wedge (by raising the plate bottom
from one end)
• A second amount of liquefied nutrient agar is poured onto the wedge and
allowed to set with the plate flat on the bench. The plates are incubated
overnight to allow diffusion of the drug
Highest conc.
several organisms
can be tested at the
same time
Lowest conc.
M.O: a b c d
The test organism must be streaked in a direction running for the
highest to the lowest concentration.
M.O: a b c d
5. Ditch-plate Technique
• The test antibiotic solution is placed in a ditch (tunnel) cut in the surface of
nutrient agar plate. Various test organisms are streaked at right angles to the
ditch. The plate is then incubated. The extent of inhibition is noted.
Evaluation of Bactericidal Activity
• It is usually carried out after determination of MIC by serial dilution technique.

• The tube containing the lowest concentration of the antimicrobial agent that
cause inhibition of growth (MIC) is determined.
• Then, the tubes that contain higher concentrations of the antimicrobial agent
(showed no growth) are selected .
• For each tube, the number of viable cells is determined by viable count
technique.
• The antimicrobial agent concentration that causes reduction of the viable cells by
99.9%, as compared to the original count, is considered as the minimum
bactericidal concentration (MBC).

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Field of Pharmacy Sciences

Bachelor of Pharmacy- PharmD (Clinical Pharmacy) Program

Pharmaceutical Microbiology (PMB202) Lecture : Seven

Dr : Ahmad M. Abbas Date : 28 / 3 /2023

• Evaluation of Bacteriostatic Activity

• Microbiological Assay Methods

• Definition: the study of the susceptibility of a M.O to a specific antibiotic

• INTERMEDIATE / MODERATELY SENSITIVE

• In this assay, radiolabelled acetyl Co A or ATP together with the corresponding

• Within a specific concentration range, increase in the amount of test antibiotic

Minimum concentration of antibiotic required to kill the

• It gives an idea about Minimum Inhibitory Concentration (MIC)

• MIC value is affected by:

• It is usually carried out after determination of MIC by serial dilution technique.

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