Antimicrobial Agents (General Considerations) Prof. R. K. Dixit Pharmacology and Therapeutics K.G.M.U. Lucknow dixitkumarrakesh@gmail.com

Objectives After this lecture you will be able to answer What are antimicrobials, antibiotics, chemotherapeutic agents (Terminologies used in antimicrobial treatment) Classification of antimicrobials Chemicals Mechanism Spectrum Mechanisms of action of antimicrobials Resistance development in antimicrobials Multidrug resistant microorganisms

A naturopath tells “One should never take antibiotics Except in Pneumonia, a kidney infection, boils, meningitis, encephalitis, osteomyelitis, occular infections, or other serious illness………………………………………………….”

Allopath is Lucky to have the help of Antimicrobials But This Luck may not last long due to reasons…… Inappropriate use,… Overuse….. Antimicrobial resistance Reduced immunity and worsening of environment patients having co morbid illnesses like diabetes, malnutrition……………….. Less interest of pharmaceuticals in this field Costly new antimicrobials

Antimicrobials , Antimicrobials , Antimicrobials , Antimicrobials, Antimicrobials , Antimicrobials Antimicrobials!!! Penicillin, ampicillin, amoxycillin, ticarcillin, piperacillin, flucloxacillin, dicloxacillin, oxacillin, methicillin, nafcillin, carbenicillin, eryhtromycin, clindamycin, roxythromycin clarithromycin, tetracycline, doxycycline, minocycline, vancomycin, teicoplanin, augmentin, gentamicin, tobramycin, amikacin, streptomycin, azithromycin, aztreonam, cephalexin, cefotaxime, cephamandole, cefepime, ceftriaxone, ceftazidime, cefpirome, imipenem, chloramphenicol, cotrimoxazole, ciprofloxacin, norfloxacin, trimethoprim,……. ………………………………………………………………………………………………………………………………………………….. hundreds of different antimicrobial agents on the market.

Terminology Chemotherapy – Use of drugs to treat infections and malignancy. (Antimicrobials and Antineoplastic agents) Pharmacodynamic agents- Drugs regulating physiological process of body and act on the body cells. Chemotherapeutic agents- Selectively acting against microbes or malignant cells. (Don’t touch body cells) Antimicrobials – Used in treating infectious diseases. Antibiotics – Produced from microbes to inhibit or kill other microbes. (Antimicrobials from microbes) All antibiotics are antimicrobials but all antimicrobials are not antibiotics

Minimum Inhibitory Concentration (MIC)- Bacteriostatic- Stop the growth of bacteria Bactericidal- Kill the bacteria PAE- Post antibiotic effect Minimum Inhibitory Concentration (MIC)- Which stops the growth Minimum Bactericidal Concentration (MBC)- Which kills by 99.99% (Bactericidal -less value of MBC-MIC, Bacteriostatic - more value of MBC-MIC)

Prebiotics- Probiotics- Lactulose, Lactitol, Inulin Stimulate the growth of intestinal commensals and prevent multiplication and establishment of pathogenic bacteria. Lactulose, Lactitol, Inulin Probiotics- Live microbial substances used as supplements to maintain or improve the intestinal bacterial flora. Lactobacilli and Bifidobacteria

Gram positive & Gram Negative Gram positive bacteria have thick cell wall Peptidoglycan directly accessible from environment Gram negative bacteria have Thin cell wall Surrounded by inner and outer membrane Of lipopolysaccharide, phospholipids, and proteins Outer membrane is a barrier to diffusion of antibiotics Limited antibiotics may diffuse through porins

Historical Perspectives Chenopodium- for intestinal worms Mouldy curd – for boils Chaulmoogra oil- for Leprosy Mercury – for Syphilis Cinchona Bark- for Malaria

Historical perspectives Pasteur- (1877) Phenomenon of antibiosis Paul Ehrlich- (1906) Father of Chemotherapy, Coined term chemotherapy Domagk- (1935) Discovery of sulfonamides (Prontosil to sulphanilamide) Fleming, Chain, Florey- Penicillin (1929, 39, 41) from penicillium Waksman- Streptomycin, from actinomycetes, Coined term antibiotic

Introduction of Class of antimicrobial agents (SPECTM) 1935 - Sulphonamides 1941 - Penicillins 1944 - Aminoglycosides 1945 - Cephalosporins 1949 - Chloramphenicol 1950 - Tetracyclines 1952 - Macrolides 1956 - Glycopeptides 1957 - Rifamycins 1959 - Nitroimidazoles 1962 - Quinolones 1968 - Trimethoprim 2000 - Oxazolidinones 2003 - Lipopeptides

Antimicrobial Classification Chemical structure Mechanism of Action Organism type Spectrum of activity Static or Cidal Origin of antimicrobials

Chemical Classification (Public Loves GOOD Quality BATSMAN) Polypeptides- Polymyxin, Colistin, Bacitracin Poyene antibiotics- Nystatin, Amphotericin-B, Hamycin Lincosamide- Lincomycin, Clindamycin Glycopeptides- Vancomycin, Teicoplanin Oxazolidinone- Linezolid Others-----------------Riampicin, Griseofulvin, etc Diaminopyrimidines- Trimethoprim, Pyrimethamine Quinolones- Nalidixic acid, ciprofloxacin Beta-lactam- Penicillins, Cephalosporins, Monobactams, Carbapenems Aminoglycosides- Streptomycin, Gentamycin Tetracyclines- Oxytetracycline, Doxycycline Sulphonamides- Sulfadiazine, Sulfamethoxazole, Macrolides- Erythromycin, Clarithromycin Azoles- Fluconazole, Clotrimazole Nitroimidazoles- Metronidazole, Tinidazole Nicotinic acid derivatives- Isoniazide, Pyrizinamide, Ethionamide Nitrobenzene derivaties- Chloramphenicol Nitrofuran derivatives- Nitrofurantoin, Furazolidone

Organism affected Sources Anti-viral Fungi- Anti-bacterial Anti-fungal Anti-protozoal Anthelmintic Sources Fungi- Penicillin Cephalosporins Griseofulvin Bacteria- Polymyxin B Colistin Bacitracin Actinomycetes- Most common Aminoglycosides, Tetracyclines, Chloramphenicol Macrolides

Spectrum Bacteristatic Bactericidal Narrow Broad Extended Penicillin G Streptomycin Erythromycin Broad Tetracycline Chloramphenicol Extended Ampicillin Amoxicillin Most…….. Bacteristatic Sulfonamides and Trimethoprim Tetracyclines Macrolides (Erythromycin) Chloramphenicol Ethambutol Nitrofurantoin Bactericidal Cotrimoxazol Penicillins Cephalosporins Aminoglycosides Vancomycin, Daptomycin Fluroquinolones (ciprofloxacin) INH, Rifampicin, Pyrazinamide Polymixins, Bacitracin Metronidazole

Spectrum (Narrow, Broad, Extended)

Mechanism of action Cell Wall synthesis inhibition- Beta-lactams, Vancomycin, Cycloserines Cell membrane Leakage- Polypeptides, Polyenes Folate Synthesis inhibition- Sulfonamides, Pyrimethamine, Cotrimaxazole, PAS, Ethambutol DNA gyrase and Topoisomerase inhibition- Fluroquinolones RNA polymerase inhibition- Rifampicin,, Protein Synthesis Inhibition- (ATT) Aminoglycosides, tetracyclines, Chloramphenicol, Macrolides, Clindamycin, Linezolid

Differences between human cells Vs Bacterial Cells (Makes the antibacterial selective) Human cells don’t posses wall (Peptidoglycans = peptides + sugar) Human cell membrane is different ( Bacteria Contain Hypanoids in place of Sterol) Human cells take preformed dihydrofolic acid (no need of PABA in human) Dihydrofolic acid reducatase enzyme is different (thousand time affinity) Topoisomerase II are different (in bacteria IV, DNA Gyrase) DNA dependent RNA polymerase is different Ribosome 60S subunit (in bacteria 50S) Ribosome 40S subunit (in bacteria 30S) 1 2 3 4 5 6 7 8

Cell Wall Beta Lactams

Protein Synthesis Chloramphenicol- Macrolides- Erythromycin, Azithromycin etc. Aminoglycosides. Gentamicin, Amikacin, etc.

DNA gyrase (Gyrase) belongs to DNA topo-isomerases DNA gyrase, referred to simply as gyrase, DNA gyrase also known as DNA topoisomerase IV (In bacteria). In human Topoisomerase II

Enzyme that relieves strain while double-strand DNA is being unwound by helicase. Causing negative super coiling of the DNA. DNA gyrase is particularly unique because it is the only Topoiosmerase that can both relax positively super coiled DNA and introduce negative supercoils into the DNA (most Topoiosmerase only relieve the positively super coiled DNA) This process occurs in prokaryotes (in particular, in bacteria), whose single circular DNA is cut by DNA gyrase and the two ends are then twisted around each other to form super coils

There are three main types of topology: supercoiling, knotting, and  catenation. (Outside of the essential processes of replication or transcription, DNA must be kept as compact as possible, and these three states help this cause. However, when transcription or replication occurs, DNA must be free, and these states seriously hinder the processes. In addition, during replication, the newly replicated duplex of DNA and the original duplex of DNA become intertwined and must be completely separated in order to ensure genomic integrity as a cell divides.)

As a transcription bubble proceeds, DNA ahead of the transcription fork becomes over wound, or positively super coiled, while DNA behind the transcription bubble becomes under wound, or negatively super coiled. As replication occurs, DNA ahead of the replication bubble becomes positively super coiled, while DNA behind the replication fork becomes entangled forming pre-catenanes. One of the most essential topological problems occurs at the very end of replication, when daughter chromosomes must be fully disentangled before mitosis occurs. Topoiosmerase IIA plays an essential role in resolving these topological problems

Quinolones

Purines and Pyrimidines PABA Sulphonamides (PABA analogue and inhibitor of DHFAS) Dihydro-folic acid Synthetase Dihydrofolic acid Dihydro-folic acid reductase Trimethoprim and Pyrimethamine (inhibitor of DHFAR) Tetrahydrofolic acid Purines and Pyrimidines DNA And RNA Quinolones (Inhibitor of DNA gyrase and Topoisomerase IV) DNA unwinding (DNA gyrase) DNA multiplication Threads sepeartion (Topoisomerase IV) DNA dependent RNA Polymerase Rifampicin (inhibitor of DNA dependant RNA Polymerase) Chloramphenicol, Macrolides (50S) Ribosome unit (50S) Protein Synthesis mRNA Ribosome unit (30S) tRNA + Amino Acids Aminoglycosides, Tetracyclines (30S)

3 4 5 6 7 8 PABA Dihydrofolic acid Tetrahydrofolic acid Purines and Pyrimidines DNA And RNA Dihydro-folic acid Synthetase Dihydro-folic acid reductase Sulphonamides (PABA analogue and inhibitor of DHFAS) Trimethoprim and Pyrimethamine (inhibitor of DHFAR) Quinolones (Inhibitor of DNA gyrase and Topoisomerase IV) Rifampicin (inhibitor of DNA dependant RNA Polymerase) Chloramphenicol, Macrolides (50S) Aminoglycosides, Tetracyclines (30S) 3 4 DNA unwinding (DNA gyrase) Threads sepeartion (Topoisomerase IV) 5 RNA Polymerase 6 7 tRNA + Amino Acids Ribosome unit (50S) Ribosome unit (30S) Protein Synthesis mRNA 8

Beta-lactams, Vancomycin, Cycloserines 1 Cell Wall synthesis inhibition- Beta-lactams, Vancomycin, Cycloserines Cell membrane Leakage- Polypeptides, Polyenes 1 2 PABA Dihydrofolic acid Tetrahydrofolic acid Purines and Pyrimidines DNA And RNA DNA unwinding (DNA gyrase) Threads sepeartion (Topoisomerase IV) RNA Polymerase mRNA tRNA + Amino Acids Ribosome unit (50S) Ribosome unit (30S) Protein Synthesis Dihydro-folic acid Synthetase Dihydro-folic acid reductase DNA multiplication Sulphonamides (PABA analogue and inhibitor of DHFAS) Trimethoprim and Pyrimethamine (inhibitor of DHFAR) Quinolones (Inhibitor of DNA gyrase and Topoisomerase IV) Rifampicin (inhibitor of RNA Polymerase) Chloramphenicol, Macrolides (50S) Aminoglycosides, Tetracyclines (30S) 3 4 5 6 7 8

3 2 4 5 6 7 1 8

Dose-dependent (With PAE) ANTIBIOTICS Dose-dependent (With PAE) Time-dependent Antibacterial effect directly depends on their concentrations in the locus of infection (high doses 1-2 times/24h) Aminoglycosides Fluoroqinolones Metronidazol Amphotericin B Effectiveness depends on a period of time, during which concentration in blood overwhelms MIC for a particular causative agent (constant i.v. infusion or 3-6 times/24h) Beta-lactames Glycopeptides Macrolides Tetracyclines Vancomycin

Post-Antibiotic Effect 4/22/2017 The capacity to inhibit the growth of bacteria after removal of the drug from the culture (body) Provides additional time for the immune system to remove bacteria that might have survived antibiotic treatment before they can eventually regrow after removal of the drug.

1 2 2 3 4 5 6 7 8 Antibacterial - Co-trimoxazole Cell mebrane Polypeptides and Polyenes Polymyxin, Colistin, Bacitracin, Nystatin, Amphotericin-B, Hamycin Cell Wall synthesis by acting on cross linking Penicillins, Cephalosporins, Monobactams, Carbapenems, Vancomycin, Teicoplanin, Cell wall synthesis by acting on inhibition of mycolic acid (Long Fatty acid present in mycobacterial family) Isoniazide, Pyrizinamide, Ethambutol Interfering with folic acid metabolism Sulphonamides- Sulfamethoxazole, Sulfadoxine, Diaminopyrimidines- Trimethoprim, Pyrimethamine DNA gyrase and topoisomerase IV inhibitors Quinolones- Nalidixic acid, ciprofloxacin, Ofloxacin, Pfloxacin, Gatifloxacin, Sparfloxacin Inhibition of DNA dependeant RNA Polymerase Rifampicin, Acting on 50S ribosome Macrolides- Erythromycin, Clarithromycin, Azithromycin, Roxithromycin, Chloramphenicol, Lincomycin, Clindamycin, Linezolid Acting on 30 S ribosome Aminoglycosides- Streptomycin, Gentamycin, Kanamycin, Amikacin, Tobramycin Tetracyclines- Oxytetracycline, Doxycycline 1 2 2 3 Antibacterial - Co-trimoxazole Antimalarial- Co-trimazine 4 5 6 7 8

Mechanisms Of Resistance 4/22/2017 Mechanisms Of Resistance Resistance Intrinsic Acquired Mutation Transferred Conjugation Transformation Transduction Not Dangerous/ less clinical importance Dangerous/ clinical importance

Inherent Resistance (Not Much of clinical importance) Bacteria naturally resistant e.g., Gram-negative bacteria resistant to penicillins Genes transferred to the bacterial progeny. Bacteria may be resistant because No mechanism to transport the drug into the cell. Do not contain antibiotic’s target process or protein.

Acquired Resistance Due to exposure of antimicrobials Horizontal evolution: Resistance genes pass from resistant to nonresistant strain, Antibiotics- a selective pressure. Gene transfer mechanisms: Conjugation. Transduction. Transformation.

Cellular Resistance 4/22/2017 ATTACK OF THE SUPERBUGS: ANTIBIOTIC RESISTANCE By Grace Yim, Science Creative Quarterly. Jan 07

Mechanisms of Resistance Enzyme-based resistance– Break down of antimicrobials. Ribosomal modifications– Methylation of ribosome interferes with antibiotic binding. Protein modifications– Mutations leave target protein unrecognizable to antibiotic Metabolic resistance– Overcome competitive inhibition by alternate pathway. Efflux– Pumps antimicrobials out.

Resistance to Antibiotics

Resistance in some antibiotics 4/22/2017 Resistance in some antibiotics Beta-lactams: - Hydrolysis , mutant PBP Tetracycline: - Active efflux from the cell Aminoglycosides- Inactivation by enzymes Sulfonamides- Alternate pathway, Fluoroquinolones- Mutant DNA gyrase Chloramphenicol- Reduced uptake into cell Macrolides - RNA methylation, drug efflux

Factors favoring Resistance Prescription related factors: Overuse Early discontinuation Over continuation Less dose, duration Livestock doping: Animals exposure

Superbugs (Microorganisms with multiple resistance) MRSA - Methicillin-resistant Staphylococcus aureus VISA - Vancomycin intermediate resistant Staphylococcі VRE - Vancomycin-resistant enterococci ESBLs - Extended-spectrum beta-lactamases (microorganisms – resistant to cephalosporins and monobactams) PRSP - Penicillin-resistant Streptococcus pneumoniae MRPA (MDR-PA)- Multidrug resistant Pseudomonas aeruginosa MRAB (MDR-AB) - Multidrug resistant Acinetobacter baumannii

Why worry? MDRO are dangerous Resource-intensive Difficult to treat More virulent Increase mortality and morbidity Resource-intensive More expensive and toxic antibiotics Increase length of hospitalization Increase demand for isolation-facilities

The number of new antibiotics is falling

Thanks