Kirby-Bauer -Antibiotic Sensitivity Test - Discussion

 v  To determine the susceptibility of various bacterial species to various antibiotics and synthetic agents.

Antibiotics – biochemical substances produced by microorganisms that inhibit the growth of, or kill, other microorganisms.

Many drugs are now completely synthetic or the natural drug is manipulated to change its structure called semisynthetics.

There is a large number of antimicrobial agents available for treating diseases caused by microorganisms. Such drugs are now an essential part of modern medical practice.

The antimicrobial agents used in medical practice are aimed at eliminating the infecting microorganisms or at preventing the establishment of an infection.

To be of therapeutic use, an antimicrobial agent must exhibit selective toxicity; it must exhibit greater toxicity to the infecting pathogens than to the host organism. A drug that kills the patient is of no use in treating infectious diseases, whether or not it also kills the pathogens.

As a rule, antimicrobial agents are of most use in medicine when the mode of action of the antimicrobial chemicals involves biochemical features of the invading pathogens not possessed by normal host cells.

Characteristics of an antibiotic

• It should be toxic to the infecting organism while harmless to the host cells and the microbiota of the host.

• It should stay in toxic form for a sufficient amount of time to affect the infecting microorganism. If it changes to another form or is broken down in the body, it may not be useful.

• Sufficient amounts of it should reach the site of infection to kill the infecting agent.

• The infecting agent should be sensitive to it.

Bacteria respond in different ways to antibiotics and chemosynthetic drugs, even within the same species. For example, Staphylococcus aureus is a common normal flora bacterium found in the body. If one isolated this bacterium from 5 different people, the 5 isolates would likely be different strains, that is, slight genetically different.

It is likely that if antibiotic sensitivity tests were run on these isolates, the results would vary against the different antibiotics used.

The determination of antibiotic susceptibility of a pathogen is important in selecting the most appropriate one for treating a disease. There are several different procedures used by clinical microbiologists to determine the sensitivity of microorganisms to antibiotics.

Two such procedures are commonly used. The first one (the Kirby-Bauer Disc Method) is used to determine which antibiotic is the most effective against a certain pathogen. The second (MIC) is used to determine the lowest concentration that is needed to kill the pathogen at the site of infection.

The Kirby-Bauer test for antibiotic susceptibility (also called the disc diffusion test) is a standard that has been used for years.

First developed in the 1950s, it was refined and by W. Kirby and A. Bauer, then standardized by the World Health Organization in 1961. Now clinical labs use automated tests. However, the K-B is still used in some labs, or used with certain bacteria that automation does not work well with.

This test is used to determine the resistance or sensitivity of aerobes or facultative anaerobes to specific chemicals, which can then be used by the clinician for treatment of patients with bacterial infections.

The presence or absence of an inhibitory area around the disc identifies the bacterial sensitivity to the drug

Kirby-Bauer Test to Measure Antibiotic Sensitivity.

In Kirby-Bauer testing, bacteria are placed on a plate of solid growth medium and wafers of antibiotics (white disks) are added to the plate. After allowing the bacteria to grow overnight, areas of clear media surrounding the disks indicate that the antibiotic inhibits bacterial growth. The concentration of antibiotic that diffuses into the media decreases with increasing distance from the source.

Therefore, the more sensitive the bacteria are to a given antibiotic, the larger the clear bacteria-free zone that forms around the disk containing that antibiotic

The basics

The bacterium is swabbed on the agar and the antibiotic discs are placed on top.

The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc.

If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc- the zone of inhibition 

The zone sizes are looked up on a standardized chart to give a result of sensitive, resistant, or intermediate.

1. Dip a sterile swab into the broth and express any excess moisture by pressing the swab against the side of the tube.
2. Swab the surface of the agar completely (do not leave any unswabbed agar areas at all).

3. Pictures below show what happens when the plate is not swabbed correctly with uniformn coverage of the bacterium over the entire agar.
   

• After completely swabbing the plate, turn it 90 degrees and repeat the swabbing process. (No need of re-moistening the swab.)
• Run the swab around the circumference of the plate before discarding it in the discard bag.
• Allow the surface to dry for about 5 minutes before placing antibiotic disks on the agar.
• Use a pair of forceps to take an antibiotic disc from the dispenser- the forceps have to be sterile. Place the forceps in alcohol, flame the forceps until they catch on fire, let the flame go out----sterile forceps.
• Lightly touch each disc with your sterile inoculating loop to make sure that it is in good contact with the agar surface.
• Incubate agar side of the plate down at 37^o C.

Interpretation

1. Place the metric ruler across the zone of inhibition, at the widest diameter, and measure from one edge of the zone to the other edge. Holding the plate up to the light is better to measure the zone.
2. Zone diameter measured in millimeter units. If there is no zone at all, report it as 0---even though the disc itself is around 7 mm.
3. Zone diameter is reported in millimeters, looked up on the chart, and result reported as sensitive, resistant, or intermediate.
   
4. Record the results.

Antibiotic (Antimicrobial Agent)	Resistant (< or = mm)	Intermediate (mm)	Susceptible (= or > mm)
Amoxicillin (other)	<13	14-17	>18
Amoxicillin (Staph)	19		20
Ampicillin (other)	11	12-13	14
Ampicillin (Staph)	28		29
Carbenicillin (other)	17	18-22	23
Carbenicillin (Pseudomonas)	13	14-16	17
Cefoxitin	14	15-17	18
Chloramphenicol	12	13-17	18
Ciprofloxacin	15	16-20	21
Clindamycin	14	15-20	21
Erythromycin	13	14-22	23
Gentamycin	12	13-14	15
Kanamycin	13	14-17	18
Methicillin (Staph)	9	10-13	14
Oxacillin (Staph)	10	11-12	13
Streptomycin	14	15-20	21
Sulfamethoxazole-trimethoprim	10	11-15	16
Tetracycline	14	15-18	19
Tobramycin	12	13-14	15
Vancomycin	9	10-11	12

Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug.

The MIC is currently the standard test run for antibiotic sensitivity testing because it produces more pertinent information on minimal dosages.