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Control of microbial growth Antimicrobial Classes • Disinfectants – Products aimed at reducing by at least five powers of 10 (99,999 %) the number of microorganisms/virus present on inanimate objects • Antiseptics – Products aimed at reducing by at least five powers of 10 (99,999 %) the number of microorganisms/virus present on live tissue Antimicrobial Classes (Cont’d) • The drugs – Antibiotic or Antibacterial • Against bacteria – Antifungal • Against fungi – Antiviral • Against viruses Disinfectants and Antiseptics • Ideal characteristics – Broad action spectrum – Powerful; low amounts required for a high efficacy – Low toxicity in humans – Not corrosive – Stable – Hydrophilic and hydrophobic – Low surface tension – No odor or pleasant odor The Drugs: Antibiotics Definitions: • Literal: Anti (against) biotic (life) • Old def.: Any compound synthesized by a microorganism that inhibits or kills other bacteria • New def.: Any compound that inhibits or kills bacteria Desired Characteristics 1. High selective toxicity: – Must kill or inhibit the target organism with a minimum of deleterious effects on the host • Penicillin: – Targets cell wall • Cyanide: – Target: electron transport of eukaryotes/prokaryotes Desired Characteristics (Cont’d) 2. High toxic dose (LD50) – Concentration of the compound that is toxic to the host • Penicillin • Cyanide 3.Low therapeutic dose (MIC or MBC) – Concentration of the compound required for the clinical treatment of an infection • Penicillin • Table salt The Therapeutic Index • Toxic Dose/Therapeutic dose – Want a therapeutic index that is? Action Spectrum • Narrow: – Efficacy restricted to only a few types of microorganisms • Ex. Acts only on Gram - • Broad: – Efficacy is good for a wide variety of microorganisms • Ex. Acts on Gram + and - Antibacterial targets Cell wall synthesis ß-lactams DNA synthesis Quinolones Transcription RNA synthesis Macrolides Metabolism A B Translation Protein synthesis Aminoglycosides Macrolides Tetracyclines Chloramphenicol 10 Modes of Action Viable count # Direct count Time • Bacteriostatic: – Inhibits growth – Non-lethal – Reversible • Bacteriocidal • Bacteriolytic – Kills – Irreversible – Kills – Cell lysis – Irreversible The Beta-Lactams • Bacteriolytic • Inhibit cell wall synthesis – Act only on actively growing bacteria! Penicillines They all contain a beta lactam ring Cephalosporins Monobactams Carbapenems 12 Quinolones • Bacteriocidal – Inhibit DNA synthesis – Broad spectrum – Side effects: • Severe gastrointestinal problems – Ex. Ciprofloxacin 13 Tetracyclines • Bacteriostatic – Inhibits protein synthesis – Broad spectrum – Side effects: • Hepatic toxicity • Renal toxicity • Vitamin deficiency 14 Macrolides • Bacteriostatic – Inhibits protein synthesis – Narrow spectrum – Side effects • Diarrhea • Hepatic dammages – Ex. Erythromycin & Clarithromycin 15 Aminoglycosides • Bacteriocide – – – – Narrow spectrum Inhibits protein synthesis High level of toxicity Side effects: • Allergies • Renal dammages • Deafness • Ex. Gentamycin, streptomycin 16 Antimicrobial Therapies • Empirical – The infectious agent is unknown – Broad spectrum antibiotic is recommended • Definitive – The infectious agent has been identified – A specific therapy is chosen – Narrow spectrum antibiotic is recommended • Prophylactic – Prevent an initial infection or reinfection 17 Kirby-Bauer Disc Diffusion Assay • Agar is inoculated with test bacteria • Antibiotic impregnated discs are laid on the agar • The antibiotic diffuses in the medium creating a gradient • Following the incubation the zones of inhibition are measured • The sizes of the zones of inhibition are compared to those established to determine whether the organism is sensitive or resistant Inhibitory Diameters Vs Conc. 27mm = MIC < 27mm = Conc. > MIC > 27mm = Conc. < MIC 19 Determination of Efficacy MIC/MBC • Minimal Inhibitory Concentration Cultures with different concentrations of antibiotic 100 50 25 MIC=12μg/ml Sub culture without antibiotics MBC=50μg/ml • Minimal Bacteriocidal Concentration 12 6 3 0 E-test In Vivo Susceptibility • The in vivo concentration is not constant! – Influenced by human physiology – A range of concentrations is maintained • (CMax-CMin) – The concentration at the infection site must be higher than the MIC • If <MIC = resistance 22 Sensitivity In Vivo • Sensitive pathogen – MIC is lower than the lowest conc. maintained in vivo • Resistant pathogen – MIC is higher than the highest concentration maintained in vivo • Intermediate sensitivity pathogen – MIC is between the lowest and the highest concentration maintained in vivo • A combination of antibiotics is recommended 23 Example • Antibiotic “A” conc. in vivo = 5-40µg/ml – Thus: • MIC≤ 5 µg/ml = Sensitive • MIC≥ 40µg/ml = resistant • MIC between 5 -40 µg/ml = intermediate sensitivity 24 Decimal Reduction Time • D value – Time required to kill 90% of microorganisms – Time required to reduce the population by a factor of 10 – Time required to reduce the population by one log10 Decimal Reduction Time 1 X 106 # Bacteria 1 log 1 X 105 1 X 104 D 120 D 100 =12min 1 X 103 5 10 15 20 25 Time (min.) 30 35 40 26 Problem • At 75oC it takes 18 min. to reduce a population of microorganisms from 109 to 106 • What is the value of D75 ? • 18 minutes to go from 109 to 106 – 3 log – 3 log = 3D75 – Therefore 3D75 = 18minutes D75=6minutes