MICROBIAL ASSAY AND ANITMICROBIAL SPECTRUM

Antimicrobial Drugs:
Antibiotic: Substance produced by a microorganism that in small amounts inhibits the growth of another microbe. Antibiotic producing microbes include: o Gram-Positive Rods: o Fungi Penicillium notatum: Penicillin Cephalosporium spp.: Cephalothin Bacillus subtilis: Bacitracin Bacillus polymyxa: Polymyxin

o Actinomycetes:

Streptomyces venezuelae: Chloramphenicol Streptomyces griseus: Streptomycin Streptomyces nodosus: Amphotericin B Micromonospora purpurea: Gentamycin

Sources of Antibacterial Agents Natural - mainly fungal sources Semi-synthetic - chemically-altered natural compound Synthetic - chemically designed in the lab

Minimal Inhibitory Concentration = MIC (At which drug concentration is the bacterial population inhibited)

The original antibiotics were derived from fungal sources. These can be referred to as natural antibiotics Organisms develop resistance faster to the natural antimicrobials because they have been preexposed to these compounds in nature. Natural antibiotics are often more toxic than synthetic antibiotics. Benzylpenicillin and Gentamicin are natural antibiotics. Semi-synthetic drugs were developed to decrease toxicity and increase effectiveness Ampicillin and Amikacin are semi-synthetic antibiotics Synthetic drugs have an advantage that the bacteria are not exposed to the compounds until they are released. They are also designed to have even greater effectiveness and less toxicity. Moxifloxacin and Norfloxacin are synthetic antibiotics There is an inverse relationship between toxicity and effectiveness as you move from natural to synthetic antibiotics

Classification of antibiotics
Class by coverage Gram negative Vs. Gram Positive Anaerobic vs aerobic Atypical Class by mechanism of action Inhibit cell wall synthesis Inhibit protein synthesis Inhibit nucleic acid synthesis Class by group Beta lactams penicillins Cephalosporins Carbapenems

 Glycopeptides Aminoglycosides Macrolides Quinilones Sulphonamides Tetracyclines

Classification:
Cell wall synthesis inhibitors Beta-lactams (penicillins, cephalosporins, aztreonam, imipenem) Poly-peptides (bacitracin, vancomycin) Protein synthesis inhibitors Aminoglycosides Tetracyclins Macrolides Chloramphenicol Clindamycin Folate antagonists Sulfonamides Trimethoprim Quinolones

Spectrum of Antimicrobial Activity Broad Spectrum: Effective against many different types of bacteria (e.g.: both gram positive and negative). Examples: Tetracycline

Narrow Spectrum Antibiotics: Effective against a subset of bacteria (either gram positive and negative). Examples: Penicillin, Isoniazid (Mycobacteria only}

ANITMICROBIAL SPECTRUM
The term broad-spectrum antibiotic refers to an antibiotic that acts against a wide range of disease-causing bacteria. A broad-spectrum antibiotic acts against both Gram-positive and Gram-negative bacteria, in contrast to a narrow-spectrum antibiotic, which is effective against specific families of bacteria. An example of a commonly used broad-spectrum antibiotic is ampicillin. The antibiotics are designed in such a way that they bind active sites of different pathogens.

Microbial assay mean determination of ingredients of antibiotics and their role on inhibition of growth of microbes. For this purpose standard preparations of antibiotics with a known activity are used for standardization. There are 3 most critical situations for assaying antibiotics. A) control. B) Pharmacokinetics i.e. Quantitative relationship of the rates of drug adsorption, distribution, metabolism and elimination (ADME) process. The data used to estiblish doses forms and frequency for desired pharmacological responses on human beings and on animals. C) Antimicrobial chemotherapy i.e. specific drugor chemical is invariable employed to treat an infectious disease. Idealy the chemical must destroy the pathogen completing without harming the host. Production Large scale production of antibiotics and microbes for existing potency and strict quality

Antimicrobial susceptibility test It is the determination of an antimicrobial agent that will inhibit the growth of microorganism in vitro using a tube dilution method, agar cup method or disc diffusion method. Antibiotics are not equally effective against all different microorganism. Susceptibility testing focuses primarily on the interaction of antimicrobial agents, the organisms and their resistance mechanisms. Why Do Susceptibility Testing To provide a guide for therapy

 Allows selection of the most appropriate agent Least expensive Narrowest spectrum Most effective To monitor the evolution of resistance Antibiotic resistance has an impact on individual health and public health. Types of resistance seen and frequency Which drugs you can expect to be successful Emerging and new resistance seen in the community Their effect is of three types 1. Cylinder plate method 2. Filter paper Method 3. Turbidimetric method

Cylinder Plate method

This method depends upon diffusion of the antibiotics from a vertical cylinder via a solidified agar layer in a petridish or plate to an extent such that the observed growth of the incorporated microorganosm is prevented totally in a zone just around the cylinder coating a solution of antibiotics.

Filter Paper Method

These tests are commonly done by placing small filter paper disc coating antibiotics on the surface on an agar plate heavily seeded with the test organism. The plate is incubated. A zone of inhibition surrounding the filter paper disc is observed if the test organism is sensitive to the antibiotics.

Turbidimetric Method
It depends on inhibition of growth of a microbial culture in a particular uniform solution of antibiotics in fluid medium in comparison to a medium without antibiotics.

Standard Preparation & Unit of activity

The assay & standardization are done in similar fashion. An agar plate with standard test organism and measured volume of antibiotics are placed in glass or metal cylinder on the surface of incubated agar. The diameter of the zone of inhibition is measure in accordance with antibiotics concentration in taken cylinder. The authentic samples of appropriate antibiotics for which the potency has been previously determined with reference to the appropriate international standard. The standard preparations ay be expressed either in international units (IU) or in microgram or mg-1 with respect to pure antibiotics. Different media prepared for the test organism containing peptones, pancreatic juices, casein, beef extract, dextrose, yeast extract, glycerin, NaCl, KH2PO4 & agar.

Method of Preparation of Test Organisms

Maintain a test organism on slant of medium using 3 ml saline solution for one week. Centrifuge and discard the supernatant liquid. Resuspend the sediments with 50- 70 ml of supernatant of saline solution and keepthe suspension for 30 min. Store the suspension under refrigeration. Maintain the test organism on 10 ml agar slant of another growth medium. Incubate at 32- 350C for 24 hours.

Selective Inhibition/Toxicity
Due to the differences in structure and metabolic pathways Harm microorganisms, not the host.

Four major sites:

Cell wall Ribosomes DNA Cell membrane

Antimicrobial Mechanisms of Action

u Inhibition of Cell Wall Synthesis:

Interfere with peptidoglycan synthesis. u u u Result in cell lysis. Low toxicity. E.g.: Penicillin and vancomycin.

Inhibition of Protein Synthesis: u u Interfere with prokaryotic (70S) ribosomes, also found in mitochondria. Anti-ribosomal antibiotics impair ribosomes by binding to either 50S or 30S ribosomal subunits. Ribosomes are essential for translation of mRNA into proteins No translation L No protein synthesis No protein synthesis L No growth u u u Most have broad spectrum of activity Tetracycline, chloramphenicol, erythromycin, and streptomycin.

u u u

Injury to the Plasma Membrane: Cause changes in membrane permeability. u u u Result in loss of metabolites and/or cell lysis. Many polypeptide antibiotics. E.g.: Polymyxin B (antibacterial) or miconazole (antifungal).

Inhibition of Nucleic Acid (DNA/RNA) Synthesis: Interfere with DNA replication and transcription. u u May be toxic to human cells. E.g.: Rifampin and quinolones..

Inhibition of Synthesis of Essential Metabolites: Involve competitive inhibition of key enzymes. u u Closely resemble substrate of enzyme. E.g.: Sulfa drugs inhibit the synthesis of folic acid.