The sensitivity of Bacteria to Antibiotic and other Chemotherapeutic agents

Most microbiologists distinguish two groups of antimicrobial agents used in the treatment of infectious disease: antibiotics, which are natural substances produced by certain groups of microorganisms, and chemotherapeutic agents, which are chemically synthesized
Antibiotic sensitivity is the susceptibility of bacteria to antibiotics.
Purpose:
Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo.
Scientific principle of antibiotic susceptibility testing (AST) depend on Minimum Inhibition Concentration (MIC) which represent the minimum concentration of the antibiotic that will inhibit this particular isolate. This can be recognized either by serial dilution method which can give MIC and or MLC (minimum lethal concentration) or by measuring zone of inhibition using disk diffusion method.
Component of Antibiotic Sensitivity Testing
1- The identification of relevant pathogens in exudate and body fluids collected from patients.
2- Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs.
3- Assay of the concentration of an administered drug in the blood or body fluid of patient required controlling the schedule of dosage.
Examples of Antibiotic Sensitivity Testing Methods 1. DILUTION METHODS
The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment. Micro dilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format. Macro dilution testing uses broth volumes at about 1.0 ml in standard test tubes.
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The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment. Each antimicrobial agent is tested using a range of concentrations commonly expressed as (?g) of active drug/ ml of broth (i.e., ?g/ ml). Typically, the range of the concentrations tested for each antibiotic are series of doubling dilution (e.g., 16, 8, 4, 2, 1, 0.5 ?g/ ml).
For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC. The MIC is thus the minumum concentration of the antibiotic that will inhibit this particular isolate. The test is only valid if the positive control shows growth and the negative control shows no growth. A procedure similar to broth dilution is agar dilution. Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited.
Antimicrobial susceptibility testing using Macro dilution method
Antimicrobial susceptibility testing using micro-broth dilutions
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2. DISK DIFFUSION METHOD
Culture medium is evenly inoculated with the organism to be tested and plotting paper discs containing the antibiotics are put on the surface. During incubation, antibiotic diffuses radially from the disc in to the medium. If the organism is sensitive to the drug in the concentrations achieved, its growth is prevented in a circular zone around the disc.

Factors Affecting Size of Zone of Inhibition
Inoculum density
Timing of disk application Temperature of incubation Incubation time
Size of the plate
Depth of the agar medium(4mm) Proper spacing of the discs (2.5 cm) Potency of antibiotic disk Composition of medium
Acidic pH of medium
Reading of zone

The Kirby – Bauer method:

1" Preparation of plates:

Only Mueller – Hinton medium can be used. The medium is prepared and
sterilized as directed by the makers. difibrinated blood(blood that has been treated to denature fibrinogen without causing cell lysis), may be necessary for tests on fastidious organisms in which case the medium should be allowed to cool to50°C before 5 per cent of blood is added. The medium should be poured into Petri dishes on a flat horizontal surface to a depth of 4 mm. (25ml in an 8.5 cm circular dish). Poured plates are stored at 4 °C and used for one week of preparation. PH should be checked at the time of preparation and should be (7.2 O 7.4) Mueller-Hinton agar MH agar is considered the best medium to use for routine susceptibility testing of nonfastidious bacteria for the following reasons: It shows acceptable batch-to-batch reproducibility for susceptibility testing · It is low in sulfonamide, trimethoprim, and tetracycline inhibitors· It supports satisfactory growth of most nonfastidious pathogen. A large body of data and experience has
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been collected concerning susceptibility tests performed with this medium.

2" Preparation of inoculums:

Preparation of inoculum
1. Using a sterile inoculating loop or needle, touch four or five isolated colonies of the organism to be tested.
2. Suspend the organism in 2 ml of sterile saline.
3. Vortex the saline tube to create a smooth suspension.
4. Adjust the turbidity of this suspension to a 0.5 McFarland standard by adding more organism if the suspension is too light or diluting with sterile saline if the suspension is too heavy.
5. Use this suspension within 15 minutes of preparation.

3" Inoculation:
Plates are inoculated with in 15 min of preparation of the suspension so that the density of the inoculum dose not change. A sterile cotton – wool swab is dipped in to the suspension and surplus removed by rotation of the swab against the side of the tube above the fluid level. The medium is inoculated by even streaking of swab over the entire surface of the plate in three direction.
A.
B.
FIG.. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton agar
plate. (A) Inoculate the plate with the test organism by streaking the swab in a back-and-forth motion very close together as you move across and down the plate. Rotate the plate 60° and repeat this
action. Rotate the plate once more and repeat the streaking action. This ensures an even distribution of inoculum that will result in a confluent lawn of growth. (B) Diagram illustrating the pattern the swab should follow as it is drawn across the plate.

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4" Antibiotic disc:

After the inoculum has dried, single discs are applied with forceps, or a sharp needle and pressed gently to ensure even contact with the medium. Not more than six discs can be accommodated on an 8.5cm circular plate.
Discs should be stored at 4 °C in sealed containers, and should be allowed to come up to room temperature before the containers are opened. Discs should be used before the expiry date on the label.

5"Incubation:

Plates are incubated inverted for (16O18) hr at (35 – 37) °C.

6" Reading of zones of inhibition:
The diameters of the zones are measured to the nearest (mm) with calipers
or millimeter rule.
Interpretation:
* Sensitive: Infection treatable with normal dosage. * Intermediate: Infection that may respond to therapy with high dosage . * Resistant: Not treatable with this agent.

Fig. Kirby-Bauer disk diffusion susceptibility test protocol, measuring zone sizes. Using a ruler or caliper measure each zone with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light. The size of the zone for this organism-antibiotic combination is 30 mm.
Dr. Zainab Almahdi M.Sc Luma Witwit
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