DEFINITION

SENSITIVITY TESTING It is the degree of activity of the

selected antimicrobial agent against the infecting bacterial strains.

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.

INTRODUCTION

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-Bawer (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 needsCareful standardizationBroth and agar mediumAntimicrobial solutionIncubation 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 ie by alteration in the

structure of their own DNA.• genetic exchange ie 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 resistanceii.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