Antibacterial Agents

Drugs have been used for the treatment of infectious diseases since the 17th century (e.g. quinine for malaria, emetine for amebiasis) Chemotherapy as a science began with Paul Ehrlich in the first decade of the 20th century. He formulated the principles of selective toxicity and recognized the specific chemical relationships between pathogens and drugs, the development of drug resistance, and the role of combined therapy. Ehrlich's experiments led to the Arsphenamines for syphilis, the first planned chemotherapy.

The current era of antimicrobial chemotherapy began in 1935, with the discovery of sulfonamides. Although antiseptics had been applied topically to prevent the growth of microorganisms, systemic bacterial infections had not as yet responded to any existing agents. In this year, the red azo dye protosil was shown to protect mice against systemic streptococcal infections and to be curative in patients suffering from such infections. It was soon found that protosil was cleaved in the body to release p-aminobenzene sulfonamide, or sulfanilamide.

Compounds (antibiotics) produced by microorganisms were eventually discovered to inhibit the growth of microorganisms (penicillin, streptomycin, tetracycline, … etc). Despite the rapidity with which new chemotherapeutic agents are introduced, bacteria have shown a remarkable ability to develop resistance to these agents. Thus antibiotic therapy will not be the predicted magical cure for all infections, rather it is only one weapon, albeit an important one, against infectious diseases

Fleming and Penicillin 5

Microbial Sources of Antibiotics 6

Definitions Selective toxicity Antimicrobial agents - Antibiotics Vs chemotherapeutic agents Antibacterial spectrum: range of activity - Broad Vs Narrow Bacteriostatic Vs bactericidal Combination therapy - Synergism Vs antagonism MIC and MBC

Mechanisms of Action Inhibition of cell wall synthesis - Beta lactams, Vancomysin, Teicoplanin, Bacitracin, Isoniazid, Ethambutol, and Cycloserine. Alteration of cell membrane function - Polymyxins, Amphotericin B, Imidazoles, Triazoles, Polyenes. Inhibition of protein synthesis - Chloramphenicol, Erythromycin, Lincomycins, Tetracycline, Aminoglycosides. Inhibition of nucleic acid synthesis - Rifampin, Quinolones, Metronidazole, Sulfonamides, Trimethoprim.

Modes of Antimicrobial Action 9

Inhibitors of Cell Wall Synthesis

Beta Lactam Antibiotics Penicillins (6-aminopenicillanic acid) - composed of a beta- lactam ring, a thiazolidine ring and side chains. Cephalosporins (7-amino cephalosporanic acid) - composed of a beta- lactam ring, a dihydrothiazine

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Cephamycin - contains oxygen in place of sulfur in the dihydrothiazine ring. Carbapenems ( Imipenem) Monobactams ( Aztreonam) Beta- Lactamase inhibitors (sulbactam and clavulanic acid)

b-Lactam Antibiotics S R R R R Monobactams Cephalosporins R S S R R . R R R R Monobactams Cephalosporins R S S R R Carbapenems Penicillins

Beta Lactam Antibiotics Bind to proteins known as penicillin binding proteins (PBPs) PBPs are enzymes involved in cell wall synthesis (transpeptidases, transglycosylates, carboxypeptidases, and endopeptidases) Inhibit cell wall synthesis and cause the release of autolytic enzymes that degrade the cell wall.

Penicillins Highly effective with an extremely low toxicity Derivatives have variable pharmacologic properties. Penicillin G, Penicillin V, methicillin, oxacillin, ampicillin, carbenicillin, ticarcillin, piperacillin.

Classes of Penicillins Natural Penicillins: Pen G, Pen V, Benzathine and Procaine forms For Streptococcal infection, syphilis, spirochetes Highly susceptible to beta lactamases Amino Penicillins: Ampicillin, amoxicillin, others… Enhanced Gram negative coverage (due to amination) Antistaphylococcal Penicillins: Methicillin, oxacillin, dicloxacillin… More stable to beta lactamases, due to modifications near beta lactam ring Poor entry into gram negatives Antipseudomonal Penicillins: Carbenicillin, Ticarcilline, Piperacillin… Enhanced Gram negative coverage (due to extensive side chain addition)

Beta lactam and beta lactamase inhibitor combinations All have activity against staphylococci comparable to methicillin Amoxicillin + clavulanate (Augmentin) active against most respiratory tract, and some GI tract gram negative organisms Ampicillin + sulbactam (Unasyn) Very good activity against GI and respiratory tract gram negatives Piperacillin + tazobactam (Zosyn) Excellent activity against GI and respiratory tract gram negatives, including Pseudomonas aeruginosa

Cephalosporins More stable to beta lactamase hydrolysis and have a wider antibacterial spectrum. First generation: E.coli, Klebsiella, gram-positives Second generation: also H-influenza, Enterobacter, Serratia, Citrobacter, and some anaerobes (Bacteriodes Fragilis). Third generation: most enterobacteriacae and P. aeruginosa Fourth generation: enterobacter and citrobacter

Generations of Cephalosporins 1st Generation: Cephalexin, Cefadroxil, Cefazolin Good gram positive, limited gram negative activity 2nd Generation: Cefaclor, cefuroxime, Cefoxitin, cefotetan Good gram positive, moderate gram negative activity Cefoxitin and cefotetan have reasonable anaerobic activity 3d Generation : Ceftriaxone, cefotaxime, ceftazidime, cefoperazone… Only Ceftriaxone has usable activity against S. aureus All have very good activity against gram negatives, BUT activity against pseudomonas is limited to ceftazidime and cefoperazone. 4th Generation: cefepime Very good activity against gram postives, including S. aureus Very good activity against gram negatives, including P.aeruginosa

Semisynthetic Penicillins Penicilinase-resistant penicillins Carbapenems: very broad spectrum Monobactam: Gram negative Extended-spectrum penicillins Penicillins + -lactamase inhibitors

Carbapemems Imipenem Extremely broad activity, including S.aureus and other gram positives , Pseudomonas, and anaerobic organisms. Toxicities: hypersensitivity; seizures Induces b-lactamases in many gram negatives

Resistance to b-lactams Degradation by beta-lactamases Example: nearly all Staphylococcus aureus Decreased permeability Example: most gram negative bacteria refractory to Penicillin G Altered Penicillin binding proteins Examples: Methicillin resistant Staphylococcus aureus Penicillin resistant Streptococcus pneumoniae

Penicillinase (b Lactamase) Figure 20.8 25

Extended-Spectrum -lactamases Plasmid-mediated ESBLs: predominantly K. pneumoniae, K. oxytoca and E. coli but also P. aeruginosa encodes high-level resistance to ceftazidime, cefotaxime, and aztreonam some susceptible to  -lactam/  -lactamase inhibitors, few susceptible to Ciprofloxacin. Treatment with carbapenems recommended. 18 28 18

Inducible chromosomal cephalosporinases: Serretia, P. aeruginosa, Providencia, Acinetobacter, Citrobacter, Enterobacter. induced by exposure to 3rd generation cephalosporins single step mutation in repressor gene leads to continuous high-level resistance to all cephalosporins and -lactam/  - lactamase Inhibitors Carbapenem is first line Rx, but note Cefepime has low affinity for Bush group 1 b lactamases (eg, Enterobacter)

Glycopeptides Vancomycin and Teicoplanin Complex glycopeptides that disrupt cell wall synthesis in growing gram-positive bacteria at a later stage (bridge formation). For methicillin resistant staphylococci, C. difficile and other gram-positives resistant to beta lactams.

Others Bacitracin -A mixture of polypeptides used in topical applications for the treatment of skin infections caused by gram-positive bacteria. Isoniazid, Ethionamide, Ethambutol, and cycloserine - Antimycobacterial (inhibition of mycolic acid synthesis)

Inhibitors of Cell Membrane Function Polymyxins (B, E) - Cationic branched cyclic decapeptides that destroy bacterial cell membranes. - Nephrotoxic: limited to topical use. Imidazoles and polyenes (antifungal) - Oral: ketoconazole, Itraconzole, Fluconazol - Parentelral: Microconazole, Clotrimazole, Econazole

the first cyclic lipopeptide Daptomycin the first cyclic lipopeptide Binds to the bacterial cell membrane, causes rapid membrane depolarization without lysis Indications: Complicated skin and skin structure infections Staphylococcus aureus bloodstream infections, including right-sided endocarditis NOT for pneumonia, has no activity in lung

Inhibitors of Protein Synthesis Aminoglycosides - Amino sugars linked through glycosidic bonds to an aminocyclitol ring - Streptomycin, Neomycin, Kanamycin - Tobramycin, Gentamicin, Netilmicin, Amikacin - Netilmicin is less ototoxic than gentamicin and tobramycin

- Bind to the 30's subunit of ribosomes; which has two effects: 1) Production of aberrant proteins as the result of misreading of mRNA. 2) Interruption of protein synthesis by causing the premature release of the ribosome from mRNA. - Bactericidal because binding is irreversible - Streptococci and anaerobes are resistant to aminoglycosides (entrance is oxygen dependent)

Resistance to aminoglycosides develops in one of three ways: 1) Mutation of the ribosome binding site (uncommon) 2) Decreased uptake of the antibiotic into the bacterial cell (anaerobes) 3) Enzymatic modification of the antibiotic (phosphorylation, adenylation, acetylation of the amino and hydroxyl groups of the antibiotic)

Tetracyclines Broad spectrum Bacteriostatic Inhibit protein synthesis by blocking the binding of aminoacyl tRNA to the 30's ribosome – mRNA complex. Staining of teeth

Resistance to the tetracyclines can stem from - decreased penetration of the antibiotic into the bacterial cell - Active efflux of the antibiotic out of the cell (most common) - Mutation of the chromosomal gene encoding the outer membrane porin protein (OmpF)

Chloramphenicol Broad spectrum Binds to the peptidyl tranferase component of the 50's ribosomal subunit, thus blocking peptide elongation. Chloramphenicol can disrupt protein synthesis in human bone marrow cells and can produce blood dyscrasias such as aplastic anemia (1/24000).

Resistance is observed in bacteria producing chloramphenicol acetyltransferase (plasmid- encolded) It catalyzes the acetylation of the 3-hydroxy group of chloramphenicol. The product is incapable of binding to the 50's subunit. Less commonly, chromosomal mutations alter the outer membrane porin proteins causing decreased permeability.

Macrolides (Erythromycin) Broad spectrum and bacteriostatic Bind to the 50's ribosome (reversible) which blocks polypeptide elongation. Resistance stems from the methylation of the 23's ribosomal RNA which prevents binding of the antibiotic. Other mechanisms include destruction of the lactone ring by an erythromycin esterase and the active efflux from the bacterial cell.

Clindmycin and Lincomycin Block protein elongation by binding to the 50's ribosome. It inhibits peptidyl tranferase by interfering with the binding of the amino acid- acyl -tRNA complex. Inactive against aerobic gram-negative bacteria. Methylation of the 23's ribosomal RNA is the source of bacterial resistance (cross resistance with erythromycin).

Inhibitors of Nucleic Acid Synthesis Quinolones (Nalidixic acid, Norfloxacin, Ciprofloxacin, Ofloxacin, Levofloxacin, Sparfloxacin) Inhibit bacterial gyrases (topoisomerases) which are required to supercoil DNA (binds to alpha subunit) - Resistance develops rapidly in Pseudomonas, Oxacillin -resistant Staphylococci, and Entrococci - Resistance is chromosomal, mutation in alpha subunit or decreased uptake

Rifampin and Rifabutin Bind to transcriptase inhibiting initiation of RNA synthesis Bactericidal to M. tuberculosis and very active against gram positive cocci Resistance develops rapidly due to a mutation in the chromosomal gene encoding the beta subunit of RNA polymerase Decreased uptake by gram negative bacteria

Metronidazole Has no activity against aerobes or facultative anaerobes Its nitrogroup is reduced by bacterial nitroreductase producing cytotoxic compounds that disrupt DNA Resistance due to decreased uptake or elimination of cytotoxic compounds before interacting with DNA

Antimetabolites Sulfonamides Structural analogues of P- aminobenzoic acid thereby preventing folic acid synthesis. It interferes with the ATP –dependent condensation of petridine with PABA to yield dihydropetroic acid which is a substance converted to folic acid Sulfonamides inhibit dihydropetroate synthetase

Trimethoprim Inhibits dihydrofolic acid rductase which reduces dihydrofolate to tetrahydrofolate a stage in the sequence leading to the synthesis of purines (thymidine). Sulfonamides, 5 parts and trimethoprim, 1 part ( Co- Trimoxazole) are synergistic and bacteridical but each alone is bacteriostatic. Resistance is common due to permeability barriers or decreased affinity of the enzyme and the ability to use exogenous thymidine (enterococci)

Measuring Antimicrobial Sensitivity: Disk Diffusion

Measuring Antimicrobial Sensitivity E Test MIC: Minimal inhibitory concentration

Antimicrobial Resistance Relative or complete lack of effect of antimicrobial against a previously susceptible microbe Increase in MIC

Mechanisms of Antibiotic Resistance Enzymatic destruction of drug Prevention of penetration of drug Alteration of drug's target site Rapid ejection of the drug

Antibiotic Selection for Resistant Bacteria

What Factors Promote Antimicrobial Resistance? Exposure to sub-optimal levels of antimicrobial Exposure to microbes carrying resistance genes

Inappropriate Antimicrobial Use Prescription not taken correctly Antibiotics for viral infections Antibiotics sold without medical supervision Spread of resistant microbes in hospitals due to lack of hygiene

Inappropriate Antimicrobial Use Lack of quality control in manufacture or outdated antimicrobial Inadequate surveillance or defective susceptibility assays Poverty or war Use of antibiotics in foods

Consequences of Antimicrobial Resistance Infections resistant to available antibiotics Increased cost of treatment

Multi-Drug Resistant TB

Proposals to Combat Antimicrobial Resistance Speed development of new antibiotics Track resistance data worldwide Restrict antimicrobial use Direct observed dosing (TB) Use more narrow spectrum antibiotics Use antimicrobial cocktails