ANTIMICROBIALS AND

SENSITIVITY TESTING
Antimicrobials
Modes of action of antimicrobials
Kirby Bauer method
Bacterial resistance

Antimicrobial vs antibiotic

Antimicrobial is the name for a chemical

that either kills or prevents the growth
of microbes ('bugs' or 'germs') such as
bacteria, viruses, fungi or protozoa.
An antibiotic is "a chemical substance
produced by microorganisms which has
the capacity to inhibit the growth of
bacteria and even destroy bacteria and
other microorganisms in dilute
solution."

Contd

Antibiotics that kill the bacteria that

they are exposed to are said to be
bacteriocidal
Some antibiotics/disinfectants/chemicals
just prevent the bacteria from growing,
these are bacteriostatic.

How do antibiotics kill bacteria

Five modes of antimicrobial action ARE:

Inhibition of cell wall synthesis: drugs form
complexes with the peptidoglycan layer
precursors and prevents cell wall maturity
Disruption of cell membrane metabolic
functions: drug peptides break open the
membrane and allows for selective receptivity
Inhibition of protein synthesis: drugs interfere
with elongation of the 70s ribosomes and
messenger RNA cleavege.

Inhibition of protein synthesis

Contd

Inhibition of RNA/DNA synthesis: (i.e.,

inhibition of replication of genetic material or
transcription), drug molecules bind to DNA
gyrase and prevent super coiling of the DNA,
they also bind to DNA-dependent RNA
polymerase
Action as antimetabolites: disrupt metabolism
processes e.g. inhibition of folic acid synthesis

KIRBY BAUER DISC DIFFUSION

SENSITIVITY method
MATERIALS NEEDED
Mueller Hinton Agar Plate
Antibiotic discs paper disks
impregnated with a certain
concentration of antibiotics
0.5 McFarland suspension of organism
this turbidity goes with a certain
number of organisms

KIRBY BAUER TEST

PROCEDURE

Take about 3-5 isolated colonies

Inoculate into Mueller Hinton broth or
Normal saline
The turbidity should match 0.5 McFarland
turbidity standard (about 1.5 x 108 CFU/ML)
Inoculate into agar by dipping sterile swab
into suspension, rotate against inside of tube

Contd

Evenly streak the surface of agar plate

Place disk on agar
Incubate at 35-37 decrees for 18-24
hours
Measure zone of inhibition - diameter

Advantages vs disadvantages
It is inexpensive
It is easy to perform
DISADVANTAGES
- Limited space for antibiotics ( 6)
- Takes long to read ( 24 HRS)
Qualitative not quantitative (no MIC)

Contd

Problems with agar or set up affects

test
Agar too deep small zone size
Agar too shallow large zone size
Over inoculation small zones
Under inoculation large zones
Failure of organism to grow

CONTD

An organism is regarded sensitive to a

certain antibiotic if it shows a zone of
inhibition (no growth) around that
particular antibiotic disc
An organism is regarded resistant to a
certain antibiotic if it shows no zone of
inhibition (growth) around that
particular antibiotic disc

Contd

Contd

Bacterial Drug resistance

Caused by bacterial mutations change in a

base sequence of the DNA/RNA strand. A
mutant an organism whose genotype ( its
DNA/RNA base sequence) differs from that
found in nature
By altered protein binding sites
By altered DNA gyrase, RNA polymerase
By decrease in entry or accumulation, by
bacteria changing permeability to the drug

Contd

By inactivation of drug due to

production of enzymes
Acquired Resistance: this is plasmid- (
an extra chromosomal DNA molecule
capable of replicating independently)
mediated resistance that can be passed
from organism to organism

Contd

By active efflux: This a situation where

the organism develops mechanisms
where they can eliminate the drug very
fast before it causes harm