ANTIMICROBIAL SENSITIVITY

TESTING
 DEFINITION
SENSITIVITY TESTING
It is the degree of activity of the selected
antimicrobial agent against the infecting
bacterial strains.
 INTRODUCTION
 Usually almost all the bacteria in infectious
disease are drug resistant.
 Hence sensitivity test is performed to select
the correct antimicrobial drug of choice.
 It may also help to identify the pathogen.
 LIMITATIONS
o It helps us to measure only the antimicrobial
activity against a bacteria under laboratory
conditions and not in the patients.
o The patients clinical condition, type and site
of infection, drug hypersensitivity, ADME,
characters of the patients are not taken in to
consideration in sensitivity testing techniques.
 Sensitivity testing can be performed by
 Diffusion technique and
 Dilution technique
1. Agar diffusion sensitivity test
 A disc of blotting paper is impregnated with a
known volume and appropriate concentration
of antimicrobial placed on a plate of sensitivity
agar inoculated with test organism.
 The antimicrobial diffuses from the disc in to
the medium. After 24 hours, the culture is
examined for areas of growth around the disc.
 Growth for sensitive strains are inhibited for a
distance while for resistant strains it grows up
to the edge of the disc.
 The zone of inhibition caused by the
antimicrobial is compared with the control.
 The volume, moisture content, PH,
constituent of agar medium, concentration,
storage and application of dose influence the
diffusion technique.
 Agar diffusion sensitivity tests are carried out
either by Kirby-Bauer (KB) method, ICS
method or by Stocks method.
 Modified KB method is recommended by the
National Committee for clinical Laboratory
Standards (NCCLS) and the WHO.
2. DILUTION SENSITIVITY TESTS.
 Dilution sensitivity tests usually measures the
minimum inhibitory concentration (MIC) or
minimum bactericidal concentration (MBC)
required to kill the bacteria.
 Here dilutions of antimicrobials are added to
the broth or agar.
 A standardised inoculum of test organism is
added.
 After overnight the lowest antimicrobial required
to prevent visible growth is taken in to
consideration.
 Dilution technique needs
Careful standardization
Broth and agar medium
Antimicrobial solution
Incubation time and
Dilution time
General requirements for sensitivity testing.
1.Sensitivity testing agar.
Suitable media include Mueller Hinton
agar, Iso sensitest agar and Gibco sensitivity
testing agar no.2.
Mueller Hinton agar(MHA)
Composition
 Meat infusion 2.0 g/l
 Casein hydrolysate 17.5 g/l
 Starch 1.5 g/l
 Agar-agar 13.0 g/l
2.ANTIMICROBIAL DISC
 This disc should be refrigerated at a
temperature instructed by the manufacturer.
 This should not be used after expiry date.
 The working stock disc should be warmed to
room temperature, avoid keeping in direct
sunlight.
 ANTIMICROBIAL RESISTANCE

 Antimicrobial resistance can arise in bacteria

in several ways.
 Microbes acquire resistance after a change in
their DNA.
 Such changes may occur by
 genetic mutation i.e. by alteration in the structure
of their own DNA.
• genetic exchange i.e. by acquisition of extra-
chromosomal DNA from other bacteria.
 Genetic exchange is the most common
cause of serious clinical drug resistance
because it can produce resistance to
multiple drugs.
 In genetic exchange , the resistance genes
are transferred from one bacterial species to
another by means of discrete, movable,
extra chromosomal DNA elements called
TRANSPOSONS.
 Transfer of transposons between bacteria can
occur by
• Conjugation ie direct physical mating between
bacteria.
• Transduction ie through the agency of
bacteriophages.
• Transposition ie by means of plasmids which are
transferable, extra chromosomal DNA molecule.
 DRUG RESISTANCE

It refers to unresponsiveness of a micro-

organism to an antimicrobial agent.
They are of 3 types:
i. Natural resistance
ii.Acquired resistance
iii.Cross resistance.
 NATURAL RESISTANCE

 Some microbes have always been resistant to

certain AMA.
 They lack the metabolic process or the target
site which is affected by the particular drug.
 eg. gram negative bacilli are normally
unaffected by Pencillin G
 M.tuberculosis is insensitive to tetracyclines
 ACQUIRED RESISTANCE

• It is the development of resistance by an

organism (which was sensitive before) due to
the use of an AMA over a period of time.
 CROSS RESISTANCE

• Cross-resistance is the tolerance to a usually

toxic substance as a result of exposure to a
similarly acting substance.
• It is a phenomenon affecting e.g. pesticides
and antibiotics as an example