Drugs, Microbes, Host – The Elements of Chemotherapy Chapter 12 Drugs, Microbes, Host – The Elements of Chemotherapy

Introduction Terminology Bacteriostatic (inhibits growth) vs. Bactericidal (kills organisms) Broad spectrum vs. Narrow spectrum Generic (chemical composition of drug) vs. Trade name (name given to a drug by a manufacture Sources of antimicrobial agents Antibiotics - growth products of organisms Chemosynthetic agents - synthesized in a laboratory Semi-synthetic agents - growth products of organisms that have been chemically altered in laboratory

Colony of Streptomyces, one of nature’s most prolific antibiotic producers

Major targets of drugs acting on bacterial cells

Mode of action of antimicrobial agents: competitive inhibition Antibacterial drugs Involves inhibiting synthesis of a critical metabolite Bacteriostatic in activity Examples Sulfa - competition based on chemical similarity between sulfas and PABA which is needed for folic acid synthesis; folic acid critical in protein and NA synthesis; side effect = possible kidney damage from crystals

Mode of action of antimicrobial agents: inhibition of cell wall synthesis Interfere with one of multiple steps in CW synthesis Greater effect demonstrated against G(+) than G(-) – most act against synthesis of peptidoglycan; many drugs cannot penetrate LPS Generally considered to be bactericidal - cause lysis

Mode of action of antimicrobial agents: inhibition of cell wall synthesis Drugs with beta lactam ring - prevent cross-linking in last step of synthesis Penicillins Original penicillins effective only against G(+) - esp. staph & strep Semisynthetic penicillins developed to overcome penicillinase (beta lactamase) producing strains (i.e.methicillin, oxacillin & nafcillin) and broaden spectrum to G(-) (i.e. ampicillin & carbenicillin) Toxicity involves hypersensitivity

Mode of action of antimicrobial agents: inhibition of cell wall synthesis Cephalosporins Broad spectrum antibiotics Used when individuals are allergic to penicillin Modifications to original drug increased spectrum (generations 1, 2, and 3) Moxalactams Other drugs - interfere at other steps Cycloserine Bacitracin - used primarily as topical (on the skin - not taken internally because of toxicity) Vancomycin

Mode of action of antimicrobial agents: Inhibition of protein synthesis Inhibit one of many steps in protein synthesis Examples Aminoglycosides - includes streptomycin, kanamycin, tobramycin, gentamicin, neomycin, and amikacin; causes misreading of mRNA; bactericidal; synergistic with penicillins Tetracyclines - block binding to tRNA; bacteriostatic; broad spectrum Chloramphenicol - prevent peptide bond formation; bacteriostatic; excellent penetration of CNS (useful for treating meningitis); side effect = aplastic anemia Erythromycin - antimicrobial spectrum similar to penicillin; used especially in penicillin-allergic Lincomycin and clindamycin - bacteriostatic; clindamycin useful for anaerobes; may cause pseudomembranous colitis

Structure of aminoglycoside, tetracycline, chloramphenicol, and erythromycin

Effects of drugs on bacterial cell membrane Act directly on cell membranes (do not need to enter cell to cause damage) Bactericidal - produce irreversible damage to membrane permeability Example = polymyxins and colistin Highly toxic to kidneys and nerves

Antifungal drugs Damage cell membranes Bind or interfere with ergosterol (unique fungal sterol in membrane) Examples Polyenes - nystatin and amphotericin B Imidazoles - clotrimazole, miconazole (topical) and ketoconazole (systemic) Inhibition of nucleic acid synthesis Griseofulvin - interferes with mitosis; selectively binds to keratin in skin, hair & nails; used primarily with fungi classified as dermatophytic 5-fluorocytosine

Antiviral drugs Interference with uptake or uncoating of virus - amantadine (used to prevent influenza A) Inhibition of nucleic acid synthesis Ribavirin - effective in vitro against a wide range of viruses; highly toxic Acyclovir - Herpes-specific (genital herpes, cold sores, chickenpox) Azidothymidine (AZT) - treatment of HIV infections

Antibiotic resistance in bacteria Sites of resistance Membrane transport - LPS layer of G(-) prevent entry of many drugs Targets of antimicrobial agents (e.g. ribosomes) Presence of antibiotic-destroying enzymes (e.g. beta lactamase)

Antibiotic resistance in bacteria Mechanisms of changing resistance Mutations - permanent changes in chromosomes; not caused by antibiotics Acquisition of new genetic information - methods Transformation = naked DNA Transduction = via viruses Conjugation = via sex pili (sexual recombination) – most rapid method Selective pressures of antimicrobial therapy - use of antibiotics select for bacteria that are resistant - sensitive bacteria are destroyed

Antibiotic susceptibility testing Susceptibility no longer predictable Variables affecting outcome of therapy Condition of host (immune status - underlying diseases) Site of infection (can drugs get to site?) Properties of antimicrobial agent Other drugs taken concurrently Susceptibility of organism to drug

The role of antimicrobials in disrupting microbial flora and causing superinfections

Broth dilution methods (Minimal Inhibitory Concentration) – Quantitative Method Procedure Uses decreasing concentrations of antimicrobial agents prepared in 2-fold dilutions of broth that will support growth of test organism Standard inoculum is added to broth containing dilutions of antimicrobial agent, incubated overnight and then examined for growth (turbidity) Lowest concentration of agent that inhibits growth as detected by lack of visible turbidity = Minimal Inhibitory Concentration (MIC) Susceptibility and resistance determined by break point of drug (highest conc. of drug in the blood that can be achieved with maximal therapy); if MIC is lower than breakpoint, organism = susceptible; if MIC is higher than breakpoint, organism = resistant

Minimum Inhibitory Concentration

Disk diffusion (Kirby/Bauer) - Qualitative Advantage - rapid testing of several antibiotics simultaneously Uses antimicrobial agents incorporated into filter paper disks placed on agar media causing drug to diffuse creating a concentration gradient (conc. highest closest to disk) Susceptibility/resistance determined by measuring diameter of zone of inhibition around disk and comparing to established zones for each antibiotic Standardization - Bauer, Kirby, Sherris & Turck (correlated with MIC’s using large numbers strains & regression analysis)

Techniques for preparation and interpretation of disc diffusion tests

Antimicrobial gradient strip method (E test) Disk diffusion method that allows determination of MIC in agar Consists of plastic strip containing gradient of antimicrobial agent along with an interpretive scale Performed similar to disk diffusion Organisms grow in elliptical zone of inhibition around strip relative to concentration of antibiotic along its length MIC determined by reading scale at point where zone of inhibition intersects strip