Antimicrobials 1: Origins and modes of action Dr Fiona Walsh

Objectives of lecture Antibiotic discovery Time-line of currently prescribed antibiotics General principles of antimicrobial agents How antibiotics inhibit or kill bacteria Introduction to all antibiotic classes

Definitions Antibiotic is a naturally occurring substance that inhibits or kills bacteria Antibacterial is a natural, semi-synthetic or synthetic substance that inhibits bacteria Antimicrobial agent is a natural, semi-synthetic or synthetic substance that inhibits microbes

Antibiotic discovery 19th Century Louis Pasteur Identified bacteria as causative agent of Robert Koch disease. (Germ theory) Now know what is causing disease, need to find out how to stop it. 1877 Pasteur Soil bacteria injected into animals made Anthrax harmless 1888 de Freudenreich Isolated product from bacteria with antibacterial properties. Toxic and unstable.

Antibiotic discovery 1st antibacterial, only cured syphilis. 20th Century Erhlich Worked with dyes and arsenicals worked against Trypanosomes, very toxic. 1st antibacterial, only cured syphilis. Domagk Research on dyes. 1st synthetic antibacterial in clinical use. Prontosil cured streptococcus diseases in animals. Active component: sulphonamide group attached to dye. Toxic. Sulphonamide derivatives still used. Less toxic.

Antibiotic discovery 20th Century Fleming and Plates left on bench over weekend. serendipity (1928) Staphylococcus colonies lysed/killed. Fungi beside Staphylococcus. Hypothesis: Fungi lysed Staph. Unable to purify in large quantities. No animal or human tests performed.

Antibiotic discovery 20th Century Florey, Chain Purified the penicillin from the fungus. and Heatley (1939) 1940s (World War II) European and US cooperation led to increased scale production of penicillin.

Antibiotic discovery 20th century Waksman (1943) Isolated streptomycin from soil bacteria Streptomyces. Effective against Mycobacterium tuberculosis and gram negatives . Toxic antibiotic. Used until 1950s when isoniazid used due to shorter course of therapy.

General Principles Selective toxicity The essential property of an antimicrobial drug that equips it for systemic use in treating infections is selective toxicity Drug must inhibit microorganism at lower concentrations than those that produce toxic effects in humans No antibiotic is completely safe

General Principles Oral and Parental Oral antibiotics must be able to survive stomach acid Advantage: Ease and reduced cost Disadvantage: Circuitous route, antibiotic passes to lower bowel Parental antibiotics given by i.v. Advantage: Direct route to site of infection Disadvantage: Increased cost and need for qualified staff

General Principles Half-Lives The length of time it takes for the activity of the drug to reduce by half Short half lives require frequent dosing Old antibiotics have short half lives New antibiotics may have half lives up to 33 hours

General Principles Broad and Narrow spectrum antimicrobials Broad spectrum antibiotics inhibit a wide range of bacteria Narrow spectrum antibiotics inhibit a narrow range of bacteria Broad spectrum desirable if infecting organism not yet identified Narrow spectrum preferable when organism has been identified

General Principles Bactericidal or bacteriostatic action Bactericidal antibiotics kill bacteria Bacteriostatic antibiotics inhibit the bacterial growth Bacteriostatic antibiotics may work as well as bactericidal antibiotics if they sufficiently arrest the bacterial growth to enable the immune system to eliminate the bacteria

General Principles Combinations of antibiotics Some antibiotics work better together than alone Combining 2 or more drugs may be required to prevent the emergence of resistance e.g. tuberculosis Combinations should not be given when 1 drug would suffice Antagonistic effects No ability to adjust 1 drug concentration

Modes of action Antimicrobial agents inhibit 5 essential bacterial processes: Protein synthesis Folic acid synthesis DNA synthesis RNA synthesis Cell wall synthesis

Protein synthesis inhibitors DNA mRNA Protein transcription translation Ribosome is a protein factory in bacteria takes mRNA in and produces proteins from them. Bacterial ribosome has 2 parts: 30S binds to mRNA to translate mRNA into amino acids, which form proteins 50S required for peptide elongation 3 phases from mRNA to protein Initiation Elongation Termination

Protein synthesis inhibitors Aminoglycosides Macrolides/Ketolides Tetracyclines Lincomycins Chloramphenicol Oxazolidinones

Protein synthesis inhibitors Bind irreversibly to ribosome Ribosome cannot bind to mRNA to form amino acid chains (30S) or elongate the chains to form proteins (50S) Disruptive effect on many essential bacterial functions leading to cell death

2. Folic acid synthesis inhibitors pterdine + para-amino benzoic acid dihydropterate dihydrofolate tetrahydrofolate DNA/RNA Sulphamethoxazole (Sulphonamides) Structural analogues of PABA Dihydropteroate synthetase Dihydrofolate reductase Trimethoprim (Diaminopyrimidines) Binding

Reasons for combining Trimethoprim and Sulphonamides There is synergy between the two drugs - the combined effect is greater that the expected sum of their activities Individually the drugs are bacteriostatic; however, in combination they are bactericidal The use of two drugs will delay the emergence of resistance

3. DNA synthesis inhibitors Enzymes required for DNA replication Topoisomerase II (DNA gyrase): GyrA and GyrB Topoisomerase IV: ParC and ParE Quinolones interact/bind to the topoisomerases, which stops DNA replication e.g. nalidixic acid, ciprofloxacin

Action of fluoroquinolones GyrA/GyrB DNA gyrase DNA ParC/ParE Topoisomerase IV Quinolones Cell death

DNA synthesis inhibitors Metronidazole Nitro group is reduced by bacterial enzyme Produces short-lived, highly cytotoxic free radicals that disrupt the DNA Similar effect to UV radiation on cell DNA

4. RNA synthesis inhibitors Rifampicin Forms a stable complex with bacterial DNA-dependent RNA polymerase Prevents chain initiation process of DNA transcription Mammalian RNA synthesis not affected as RNA polymerase is much less sensitive to rifampicin

5. Cell wall synthesis inhibitors Vancomycin Bacitracin β-lactams Penicillins Cephalosporins Carbapenems Monobactams β-lactamase inhibitors Clavulanic acid Sulbactam Tazobactam

Action of Cell wall synthesis inhibitors N-acetyl-glucosamine (NAG) Phospho-enol pyruvate Peptidoglycan formation 1. Building Blocks N-acetyl-muramic acid (NAMA) L-alanine D-glutamic acid L-lysine NAMA L-ala-D-glu-L-lys D-ala-D-ala D-ala L-ala NAMA L-ala-D-glu-L-lys-D-ala-D-ala

Action of Cell wall synthesis inhibitors NAMA L-ala-D-glu-L-lys-D-ala-D-ala Lipid carrier NAG Bacitracin inhibits NAMA - NAG L-ala-D-glu-L-lys-D-ala-D-ala 5 gly Vancomycin &Teicoplanin binds, prevents enzyme polymerisation Phospholipid NAMA - NAG L-ala-D-glu-L-lys-D-ala-D-ala 5 gly

Action of Cell wall synthesis inhibitors Polymerisation NAMA L-ala D-glu L-lys D-ala NAG 5 gly NAMA L-ala D-glu L-lys D-ala NAG 5 gly NAMA L-ala D-glu L-lys D-ala NAG 5 gly NAMA L-ala D-glu L-lys D-ala NAG 5 gly NAMA L-ala D-glu L-lys D-ala NAG 5 gly

Action of Cell wall synthesis inhibitors Transpeptidation b-lactams resemble D-ala-D-ala, bind to enzyme, inhibit cross-linking NAMA L-ala D-glu L-lys D-ala NAG 5 gly D-ala D-ala D-ala D-ala D-ala D-ala L-lys L-lys L-lys D-glu D-glu D-glu L-ala L-ala L-ala NAG NAMA NAG NAMA NAG NAMA

Penicillin Binding Proteins Enzymes involved in cell wall formation Reseal cell as new peptidoglycan layers added Penicillins bind to PBPs block enzyme cross-linking chains Weak cell wall Build up osmotic pressure Lysis Rawalpindi 9 Interestingly the bacteria do not acquire resistance by a beta-lactamase - at least not directly. The beta-lactam binds to the target proteins on the surface of the cell, known as the Penicillin Binding proteins. They can be extracted and separated on a Polyacrylamide gel. S. aureus has very characteristic Penicillin Binding Proteins.

Keynote points Recent history of antibiotic discovery General principles of antibiotic action 5 modes of action Examples of each