ANTIMICROBIALS: INTRODUCTION; MODE OF ACTION OF ANTIBIOTICS A Presentation By Ms R.Venkatajothi, MSc., MPhil, PhD Senior Lecturer Department of Microbiology Faculty of Medicine AIMST UNIVERSITY

OBJECTIVES The objectives of this lecture are to  Review briefly the history and development of antimicrobials.  Define antimicrobial, antibiotic & related terms.  Describe the classification of antibiotics according to their mechanism of action on bacteria.  Describe the general principles of antibiotic susceptibility testing.

HISTORY AND DEVELOPMENT OF ANTIMICROBIALS  The history of antimicrobials begins with the scientist Pasteur and Joubert who discovered that one type of bacteria could prevent the growth of another organisms.  They did not know at that time that the reason one bacterium failed to grow was that the other bacterium was producing an antibiotic.  The discovery of antimicrobials like Penicillin and Tetracycline paved the way for better health for millions around the world.

History and development of antimicrobials continued….  The word "antibiotics" comes from the Greek anti ("against") and bios ("life"). The noun “antibiotic” was suggested in 1942 by Dr. Selman A. Waksman, soil microbiologist.  The first antibiotic ‘Penicillin’ was discovered by the Scottish physician Sir Alexander Fleming in 1928 in a significant breakthrough for medical science. It was once called as "miracle drug".  Before Penicillin became a viable medical treatment in the early 1940s, no true cure for gonorrhea, strep throat, or pneumonia existed.

History and development of antimicrobials continued….  Alexander Fleming, Howard Walter Florey, and Ernst Chain received the Nobel Prize in 1945 for the discovery and production of Penicillin.  However, with the development of antimicrobials, microorganisms have adapted and become resistant to previous antimicrobial agents. Sources of Antibacterial Agents   Natural - Mainly fungal sources.   Semi-synthetic - Chemically-altered natural compound.   Synthetic - Chemically designed in the lab.

ANTI-MICROBIAL & RELATED TERMS Anti-microbial  An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, virus, fungai or protozoas.  Antimicrobials are classified based on the pathogen such as Antibacterial, Antifungal Antiviral and Antiprotozoan. Antibiotics (Antibacterial)  For ex: Chloramphenicol, Tetracyclines etc.  Antibiotics is a substance derivable from a microorganism or produced by chemical synthesis that kills or prevent the growth of bacteria and cures infections. For ex: Chloramphenicol, Tetracyclines etc.

Anti-microbial & related terms continued.... COMMONLY USED ANTI VIRAL AGENTS For ex: Acyclovir, Amantadine and Ribavirin etc. For ex: Acyclovir, Amantadine and Ribavirin etc. COMMONLY USED ANTI FUNGAL AGENTS For ex: Amphotericin B, Nystatin & Miconazole etc. For ex: Amphotericin B, Nystatin & Miconazole etc.

Anti-microbial & related terms continued.... Anti-microbial & related terms continued.... COMMONLY USED ANTI PARASITIC AGENTS For ex: Mebendazole, Metronidazole & Albendazole etc. For ex: Mebendazole, Metronidazole & Albendazole etc. Chemotherapeutic agent  Chemotherapeutic agents are chemical substances which are primarily refers to the treatment of cancer and it is administered in 'regimens'-one or more cycles, that combine three or more agents.

Anti-microbial & related terms continued.... BACTERICIDAL  Bactericidal agents are able to kill bacteria. For ex: Cephalosporins, Vancomycin etc. For ex: Cephalosporins, Vancomycin etc. BACTERIOSTATIC BACTERIOSTATIC  Bacteriostatic agents only inhibit or prevent the multiplication of bacteria. For ex: Chloramphenicol, Tetracyclines etc. For ex: Chloramphenicol, Tetracyclines etc.

Anti-microbial & related terms continued.... Anti-microbial & related terms continued.... NARROW SPECTRUM DRUGS NARROW SPECTRUM DRUGS  Narrow spectrum drug is effective against a limited number of species. For ex : It acts against either Gram negative agent or Gram positive agent. For ex: Clindamycin, Erythromycin, Vancomycin etc. For ex: Clindamycin, Erythromycin, Vancomycin etc. BROAD SPECTRUM DRUGS  Broad spectrum drug is effective against a wide variety of species. For ex : It acts against both Gram negative agent and Gram positive agent. For ex: Chloramphenicol, Tetracyclines, Streptomycin etc. For ex: Chloramphenicol, Tetracyclines, Streptomycin etc.

CLASSIFICATION OF ANTIBIOTICS  The classification of antibiotics according to their mechanism of action on bacteria can be discussed under four headings; (1) Inhibition of Bacterial cell wall synthesis. (1) Inhibition of Bacterial cell wall synthesis. (2) Inhibition of Cytoplasmic membrane function. (2) Inhibition of Cytoplasmic membrane function. (3) Inhibition of Protein synthesis. (3) Inhibition of Protein synthesis. (4) Inhibition of Nucleic acid synthesis. (4) Inhibition of Nucleic acid synthesis.

MECHANISM OF ACTION OF ANTIBIOTICS DNA mRNA ribosomes DNA topoisomerases quinolones, novobiocin cell membrane polymyxins Protein synthesis tetracyclines aminoglycosides cell wall  -lactams vancomycin bacitracin

Mechanism of action of antibiotics continued……  Inhibition of bacterial cell wall synthesis:-  Injury to the cell wall (eg, by lysozyme) or inhibition of its formation may lead to lysis of the cell wall.  All β -lactam drugs are selective inhibitors of bacterial cell wall synthesis and therefore active against growing bacteria.  The initial step in drug action consists of binding of the drug to cell receptors (Penicillin-binding proteins; PBPs). 

Mechanism of action of antibiotics continued…… The antibiotics which inhibit cell wall synthesis are β – lactam antibiotics such as Penicillin and Cephalosporin etc. The antibiotics which inhibit cell wall synthesis are β – lactam antibiotics such as Penicillin and Cephalosporin etc. This β - lactam antibiotics are inactive against organisms lack of peptidoglycan cell wall such as Mycoplasma. This β - lactam antibiotics are inactive against organisms lack of peptidoglycan cell wall such as Mycoplasma. Inhibition of bacterial Cytoplasmic membrane function :- Inhibition of bacterial Cytoplasmic membrane function :- For ex: Polymyxins. For ex: Polymyxins.

Inhibition of bacterial cell wall synthesis Beta-lactam antibiotics Core structure of penicillin (top) and cephalosporin (bottom). β-lactam ring in red.

Mode of action of antibiotics continued…… Inhibition of bacterial protein synthesis:- Inhibition of bacterial protein synthesis:- For ex: Aminoglycosides, Chloramphenicol and Tetracyclines etc. For ex: Aminoglycosides, Chloramphenicol and Tetracyclines etc. Inhibition of bacterial nucleic acid synthesis:- Inhibition of bacterial nucleic acid synthesis:- For ex: Sulphonamides, Quinolones, Rifamycins and Novobiocin etc. For ex: Sulphonamides, Quinolones, Rifamycins and Novobiocin etc.

GENERAL ANTIBIOTICS FOR GRAM POSITIVE AND GRAM NEGATIVE BACTERIA Commonly used antibiotics for Gram positive bacteria:- Commonly used antibiotics for Gram positive bacteria:- For ex: Penicillin, Cephalolsporin, Erythromycin, Ampicillin, Amoxicillin, Tetracyclines and Vancomycin etc. For ex: Penicillin, Cephalolsporin, Erythromycin, Ampicillin, Amoxicillin, Tetracyclines and Vancomycin etc. Commonly used antibiotics for Gram negative bacteria:- Commonly used antibiotics for Gram negative bacteria:- For ex: Amikacin, Gentamicin, Kanamycin, Cefotaxime, Ofloxacin and Chloramphenicol etc. For ex: Amikacin, Gentamicin, Kanamycin, Cefotaxime, Ofloxacin and Chloramphenicol etc.

PRINCIPLES OF ANTIBIOTIC SUSCEPTIBILITY TEST Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. USES:- USES:- Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo. MIC:- MIC:- The minimum inhibitory concentration (MIC) is defined as the minimum concentration of antibiotic which will inhibit the growth of the microorganism after overnight incubation. The minimum inhibitory concentration (MIC) is defined as the minimum concentration of antibiotic which will inhibit the growth of the microorganism after overnight incubation. There are many ways to measure the MIC, including: Broth dilution, Agar dilution, E-test etc. There are many ways to measure the MIC, including: Broth dilution, Agar dilution, E-test etc.

Antibiotic susceptibility test continued…. Method:- Method:- Testing for antibiotic sensitivity is often done by the Kirby-Bauer method. It is known as disk-diffusion method and most widely used antibiotic susceptibility test. Testing for antibiotic sensitivity is often done by the Kirby-Bauer method. It is known as disk-diffusion method and most widely used antibiotic susceptibility test. A lawn culture is made on Mueller-Hinton agar media with test organism and filter paper discs impregnated with a specific concentration of a particular antibiotic is placed on the medium. A lawn culture is made on Mueller-Hinton agar media with test organism and filter paper discs impregnated with a specific concentration of a particular antibiotic is placed on the medium. The organism will grow the agar plate while the antibiotic inhibits the growth around the disc containing the antibiotic. The organism will grow the agar plate while the antibiotic inhibits the growth around the disc containing the antibiotic.

Antibiotic susceptibility test continued…. Thus a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism. Thus a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism. The measurement is compared to the criteria (standard chart) set by National Committee for Clinical Laboratory Studies (NCCLS). Based on criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible (S). The measurement is compared to the criteria (standard chart) set by National Committee for Clinical Laboratory Studies (NCCLS). Based on criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible (S). The results of antimicrobial susceptibility testing should be combined with clinical information and experience when selecting the most appropriate antibiotic for the patient. The results of antimicrobial susceptibility testing should be combined with clinical information and experience when selecting the most appropriate antibiotic for the patient.

Antibiotic susceptibility test continued….

WHICH IS THE IDEAL ANTI-MICROBIAL? Have the appropriate spectrum of activity for the clinical setting. Have the appropriate spectrum of activity for the clinical setting. Have no toxicity to the host. Have no toxicity to the host. Drug resistance level must be low. Drug resistance level must be low. Not induce hypersensitive reactions in the host. Not induce hypersensitive reactions in the host. Has rapid and extensive tissue distribution. Has rapid and extensive tissue distribution. Has a relatively long life. Has a relatively long life. Be free of interactions with other drugs. Be free of interactions with other drugs. Be convenient for administration. Be convenient for administration. Be relatively inexpensive. Be relatively inexpensive.

LEARNING OUTCOMES  Recall the history and development of antimicrobials.  Define antimicrobial agent, antibiotic & related terms.  Describe with appropriate examples, the classification of antibiotics according to their mechanism of action on bacteria.  Describe the general principles of antibiotic susceptibility testing and apply the knowledge gained in the hands on practical session.

BOOKS FOR REFERENCE  Text book of Microbiology by Ananthanarayan and Paniker’s.  A Text book of Microbiology by P.Chakraborty.  Medical Microbiology by Jawetz, Melnick, & Adelberg's.  Medical Microbiology by Murray.