Antimicrobial Control Agents

Mr. Shadi ALashi

Antimicrobial control agents Usually, microbial controls are used to avoid contamination of pure

cultures, prevent infection, or treat existing diseases.

A microbiocidal effect kills microorganisms.

A microbiostatic effect prevents the reproduction of microorganisms.

Antiseptics are chemicals used on living tissues to inhibit the growth of microorganisms.

Disinfectants are chemicals used on nonliving surfaces to inhibit the growth of microorganisms.

Chemotherapeutic agents are chemicals used to destroy or inhibit the growth of microorganisms in living tissues.

Antimicrobial Chemotherapy : is the use of chemicals to inhibit or kill microorganisms in the host.

Selective Toxicity: This means that the agent used must inhibit or kill the microorganism without seriously harming the host.

Based on their origin, there are 2 general classes of antimicrobial chemotherapeutic agents:

1. Antibiotics: substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms.

2. Antimicrobial Chemotherapeutic Chemicals: chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

ANTIMICROBIAL CHEMOTHERAPEUTIC AGENTS

Antimicrobial agents are :

Cidal in action: they kill microorganisms.OR Static in action : they inhibit microbial growth long enough for the body's own

defenses to remove the organisms.Antimicrobial agents also vary in their spectrum: Broad spectrum : Drugs which are effective against a variety of both gram-

positive and gram-negative bacteria.

Narrow spectrum : Drugs which are effective against just gram-positive bacteria, just gram negative bacteria, or only a few species are termed.

If a choice is available, a narrow spectrum is preferable since it will cause less destruction to the body's normal flora. In fact, indiscriminate use of broad spectrum antibiotics can lead to super-infection by opportunistic microorganisms.

UV causes damage to nucleic acids by inducing covalent bonds between adjacent thymine bases, resulting in thymine dimerization. The thymine dimers change the structure of DNA, preventing DNA replication and RNA transcription.

HAW TO CHOOSE ANTIMICROBIAL DRUGS?

Narrow spectrum OR broad spectrum.

Old age OR child.

Male OR female.

Pregnant OR lactating women.

In patient OR out patient.

Type of microorganisms.

Site of infection.

The Action of Antimicrobial Drugs

Several tests may be used to tell a physician which antimicrobial agent is most likely to combat a specific pathogen:

1. Tube dilution tests:• A series of culture tubes are prepared, each containing a liquid medium and a

different concentration of a chemotherapeutic agent. The tubes are then inoculated with the test organism and incubated for 18-24 hours at 37 oC . After incubation, the tubes are examined for turbidity (growth).

Minimum Inhibitory Concentration (MIC):Is the lowest concentration of chemotherapeutic agent capable of preventing growth of the test organism.

Minimum Bactericidal Concentration (MBC):Is the lowest concentration of the chemotherapeutic agent that allows less than 0.1% of the inoculum to survive , also called “ Minimum Lethal Concentration (MLC)”

2. The agar diffusion test (Bauer-Kirby test):• In this test, the in vitro response of bacteria to a standardized antibiotic-

containing disc has been correlated with the clinical response of patients given that drug.

In the development of this method, a single high-potency disc of each chosen chemotherapeutic agent was used. • Zones of growth inhibition surrounding each type of disc were correlatedwith the minimum inhibitory concentrations of each antimicrobial agent (as determined by the tube dilution test). • The MIC for each agent was then compared to the usually-attained blood level inthe patient with adequate dosage. Categories of "Resistant,“ "Intermediate," and "Sensitive" were then established.

• It’s a new technique for direct detection of MIC, a graduated increasing concentration of the antibiotic is fixed along a rectangular plastic test strip which is applied to the surface of an inoculated agar plate, after over night incubation a tear drop shaped inhibition zone is seen.

• The zone edge intersect the graded test strip at the MIC of the antimicrobial.

3. Epsilometer test:

Disk-Diffusion Test

E Test

E Test

Additive (indifferent) effect: the activity of two drugs in combination is equal to the sum (or a partial sum) of their independent activity when studied separately.

Synergistic effect: the activity of two drugs in combination is greater to the sum of their independent activity when studied separately.

Antagonistic effect: the activity of two drugs in combination is less to the sum (or a partial sum) of their independent activity when studied separately.

Effects of Combinations of Drugs

Effects of Combinations of DrugsAdditive (indifferent) effect

Effects of Combinations of DrugsSynergistic effect

Effects of Combinations of DrugsAntagonistic effect

Antimicrobial susceptibility testing by the disk diffusion method (Kirby-Bauer) & Antibiotic profil

Purpose of ProcedureTo test isolated bacteria for its susceptibility to antimicrobial agents

Specimen RequirementsIn general a pure growth of the isolate.

Equipment• Sterile cotton swabs.

Reagents Required• Mueller Hinton Agar plates.• Appropriate antibiotic discs or dispenser.• McFarland Standard (0.5).• Pure culture of the test organism.

The basic steps for the Bauer-Kirby method of antimicrobial susceptibility testing are:

a. Prepare a standard turbidity inoculum ((0.5) McFarland Standard ) of the test bacterium so that a certain density of bacteria will be put on the plate.

b. Inoculate a (90 mm diameter, 4 mm agar depth of Mueller-Hinton agar plate) with the standardized inoculum so as to cover the entire agar surface with bacteria.

c. Place standardized antibiotic-containing discs on the plate.

d. Incubate the plate at 37 oC for 24 hours.

e. Measure the diameter of any resulting zones of inhibition in millimeters (mm).

f. Determine if the bacterium is susceptible, intermediate, or resistant to each antimicrobial agent using a *standardized table.

* Standarized table according to manufactory pamphlet.

Mueller Hinton AgarINGREDIENTS• Beef Extract.• Digest of Casein ( peptone ).• Starch.• Agar ....... 1.7 %.• Final pH is ~7.3 at 25°C.

Pour cooled Mueller Hinton agar into sterile Petri dishes

on a level, horizontal surface to give a uniform depth of

about 4 mm and cool to room temperature.

Mueller Hinton Agar

• McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range.

• Were mixing specified amounts of barium chloride and sulfuric acid together.

• Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in the solution.

• A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4).

• Turbidity of 0.5 McFarland standard is approximately equal 1.5X10^8 CFU/mL of bacterial suspension.

0.5 McFarland standard

McFarland standards

Procedure:

1. Adjust the concentration of the inoculum by comparing the turbidity of Normal Saline tube to that of a 0.5 McFarland standard.

Note: If the turbidity of the inoculum is greater than that of 0.5 McFarlandstandard, dilute with sterile normal saline. If the turbidity of the standard isgreater than the inoculum, add more of the test organism.

2. Dip a sterile cotton swab into the adjusted inoculum tube and drain excessfluid by pressing the swab against the walls of the test tube.

3. Hold Muller-Hinton plate half or partially open and streak the plate using thewet cotton swab covering all the area even at the sides.

4. Place the plates aside for about 10 – 15 minutes at room temerature. Allow the inoculum to dry.

5. Using a sterile forceps or needle, apply a set of suitable antibiotic disks. Five to six disks for each plate, or 8 disks if you use the automatic dispenser.

6. Let the plates stand for 10 - 15 minutes at 4oC, then incubate in inverted position at 37 oC for 18-24 hours.

Procedure: continue ……

7. Using a ruler or caliper, measure the zone of inhibition around eachantimicrobial disk and record it.

8. Consult the special chart provided by the manufacturer of the antimicrobialdisks and interpret results as Sensitive, Resistant, or Intermediate.

Procedure: continue ……