Screening for new antibiotics

Bacteriostatic and bactericidal antibiotics Antibiotics are categorized as bactericidal if they kill the susceptible bacteria or bacteriostatic if they reversibly inhibit the growth of bacteria. In general the use of bactericidal antibiotics is preferred but many factors may dictate the use of a bacteriostatic antibiotic. When a bacteriostatic antibiotic is used the duration of therapy must be sufficient to allow cellular and humoral defense mechanisms to eradicate the bacteria. If possible, bactericidal antibiotics should be used to treat aggressive infections.

Methods for antibiotic detection Diffusion methods: -These methods based on the diffusion of antibiotics in agar inoculated with sensitive microorganisms. - The effects of antibiotics are revealed by the production of zones of inhibition.

Three categories of diffusion are employed: 1- One-dimentional (linear) diffusion in test tubes or capillaries. 2-Two- dimentional diffusion e.g., radial diffusion from cups cut in agar layers. 3- Three dimentional diffusion from cylinders or disc located on the surface of the agar layer.

Sensitivity of microorganisms to antibiotics Measurement of the antibiotic sensitivity of an organism in the laboratory is designed to predict whether an infection will respond to treatment with that antibiotic or not.

Role of laboratory •MIC/ MBC measurement Sensitivity Testing: •Disc diffusion •MIC/ MBC measurement

The MIC is the lowest concentration of the antibiotic that results in inhibition of visible growth (i.e. colonies on a plate or turbidity in broth culture) under standard conditions. The MBC is the lowest concentration of the antibiotic that kills 99.9% of the original inoculum in a given time. OR The lowest concentration of antibiotic that allows less than 0.1% of the original inoculum to survive.

Measurement of Sensitivity of microorganisms to antibiotics In the laboratory, sensitivity is most often measured using a disk diffusion test. Antibiotic solutions of particular concentrations are dried onto filter paper disks. These are then applied to a lawn of the microbe under examination which has previously been inoculated onto an appropriate solid medium.

The effectiveness of the antibiotic is relative to the inhibition zones of the bacterial growth, the more the diameter, the more potent the tested antibiotic.

Stokes controlled sensitivity test a control sensitive bacterium is inoculated on part of a plate and the tested bacterium is plated on the remainder. Disks of antibiotics are placed at the interface and the zones of inhibition are compared. The use of a sensitive control shows that the antibiotic is active, so that if the test organism grows up to the disk it may safely be assumed that the test organism is resistant to that drug.

The bacterium in the diagram is susceptible to drug "x" but resistant to drug "y". The disc containing drug "y" contains active antibiotic as shown by the zone of inhibition it causes in the control bacterium.  

Determination of the minimum inhibitory concentration of antibiotics using broth dilution method: A series of broths are mixed with 2 fold serially diluted antibiotic solutions and a standard inoculum is applied. After incubation, the minimum inhibitory concentration MIC is the first broth in which growth of the organism has been inhibited. The more resistant an organism is, then the higher will be the MIC.

The MIC test of a bacteriostatic drug. If the bacteria removed from the drug can grow on drug free medium at highest concentrations, the drug is known to have bacteristatic action.

If the bacteria removed from the drug can not grow on drug free medium at most concentrations, the drug is known to have bactericidal action. One tube difference is allowed in this test

Disadvantages Solutions?? Only one antibiotic & one organism can be tested each time Time-consuming Solutions?? Disc diffusion method Microbroth dilution method

Microbroth Dilution Method Microdilution plates: “Microdilution/ Microbroth dilutions” 96 wells/ plate: simultaneously performed with many tests organisms/ specimens, less reagent required