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

Soaps/Detergents Antiseptic/Disinfectant –Sulfate or sodium hydrocarbons –Amphipathic –Emulsifier/surfactant –Dissolves lipid membranes Soap Detergent

The Drugs: Antibiotics 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 Definitions:

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 11 Translation Transcription AB Metabolism Protein synthesis Aminoglycosides Macrolides Tetracyclines Chloramphenicol RNA synthesis Macrolides DNA synthesis Quinolones Cell wall synthesis ß-lactams

Modes of Action Bacteriocidal –Kills –Irreversible Bacteriolytic –Kills –Cell lysis –Irreversible Time Direct count Viable count # Bacteriostatic: – Inhibits growth – Non-lethal – Reversible

The Beta-Lactams Bacteriolytic Inhibit cell wall synthesis –Act only on actively growing bacteria! 13 Penicillines Cephalosporins Carbapenems Monobactams All have the beta lactam ring

Quinolones Bacteriocidal –Inhibit DNA synthesis –Broad spectrum –Side effects: Severe gastrointestinal problems –Ex. Ciprofloxacin 14

Tetracyclines Bacteriostatic –Inhibits protein synthesis –Broad spectrum –Side effects: Hepatic toxicity Renal toxicity Vitamin deficiency 15

Macrolides Bacteriostatic –Inhibits protein synthesis –Narrow spectrum –Side effects Diarrhea Hepatic dammages –Ex. Erythromycin & Clarithromycin 16

Aminoglycosides Bacteriocide –Narrow spectrum –Inhibits protein synthesis –High level of toxicity –Side effects: Allergies Renal dammages Deafness Ex. Gentamycin, streptomycin 17

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 18

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

Determination of Efficacy MIC/MBC Minimal Inhibitory Concentration Cultures with different concentrations of antibiotic Minimal Bacteriocidal Concentration Sub culture without antibiotics MIC=12μg/ml MBC=50μg/ml

Inhibitory Diameters Vs Conc mm = MIC MIC > 27mm = Conc. < MIC Gradient de concentration + -

E-test

In Vivo Susceptibility The in vivo concentration is not constant! –Influenced by human physiology –A range of concentrations is maintained (C1-C2) –The concentration at the infection site must be higher than the MIC If<MIC = resistance 23

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 24

Example Antibiotic “A” conc. in vivo = 5-40µg/ml –Thus: MIC≤ 5 µg/ml = Sensitive MIC≥ 40µg/ml = resistant MIC between µg/ml = intermediate sensitivity 25

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

27 1 X X X X 10 3 # Bacteria Time (min.) D =12min 1 log 120 D

Problem At 75 o C it takes 18 min. to reduce a population of microorganisms from 10 9 to 10 6 What is the value of D 75 ? 18 minutes to go from 10 9 to 10 6 –3 log –3 log = 3D 75 – Therefore 3D 75 = 18minutes D 75 =6minutes