10/2/2016. Control of Microbial Growth. Method. Terminology. Disinfectants and Antiseptics

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1 Control of Microbial Growth Disinfectants and Antiseptics 1 Method Three approaches for the control of microbial growth Chemical Disinfectants and antiseptics Physical Heat Ultraviolet Irradiations Mechanical elimination Cleaning Filtration 2 Terminology Cleaning The elimination of visible adherent dirt (blood, proteins and debris), dust or other foreign matter by manual or chemical processes Does not infer the presence or absence of microorganisms Cleanliness Sterility 3 1

2 Disinfection Products aimed at reducing by at least five orders of magnitude (99,999 %) Types: 1) Disinfectants Chemical products used on inanimate objects 2) Antiseptics Chemical products used on living tissues 3) Germicides Chemical products which can be used on either animate (living) or inanimate things Factors which Influence the Efficacy Microbial load Number of microbes Environment Presence of organic matter Concentration of the agent Temperature ph Length of exposure 5 Factors which Influence the Efficacy Microbial characteristics Biofilms Cell wall Resistances Spores 6 2

3 Order of Sensitivity Lipophilic viruses (With lipid bilayer, enveloped virus) Gram positive bacteria (vegetative cells) Gram negative bacteria Fungi Hydrophilic viruses (non enveloped, naked virus) Mycobacteria (Mycobacterium tuberculosis) Bacterial spores More sensitive More resistant 7 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 Modes of Action of Chemical Agents Denaturation of proteins or DNA Mutagenesis of DNA Modification of proteins or of DNA Interference with the plasma membrane Oxidation of functional groups 9 3

4 Number of cells Number of cells Number of cells 10/2/2016 Evaluation of Disinfectant Efficacy Quantitative suspension tests Viable counts are performed on a test microorganism exposed to the chemical agent The number of surviving organisms (B) is counted and compared to the original inoculum size (A) Microbicidal effect (ME) = Log (A) - Log (B) ME = 1 killing of 90% of the initial number ME = 2 99% killed A generally accepted requirement is: ME % of the germs are killed Profile of Mortality Arithmetic Plot Time (Min.) Death is exponential It s therefore impossible to reach zero Established standards: Sterility in the lab: 10-6 cells or spores Sterility for food: cells or spores Logarithmic plot Time (Min.) 11 Parameters of Mortality Decimal reduction time (D) Time required to achieve a reduction of one log or an inactivation factor of 10, therefore the time required to kill 90% of the population Formula: Log (N/N 0 ) =-t/d t: Length of time N: Number of surviving cells N o : Initial number of cells N o /N: Inactivation factor Logarithmic Plot Time (Min.) 12 4

5 Number of cells 1 log 10/2/2016 Decimal Reduction Time 1 X X 10 5 # Bacteria 1 X 10 4 D 120 D =12min X Time (min.) 13 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 log 3 log = 3D 75 Therefore 3D 75 = 18minutes D 75 =6minutes Parameters of Mortality Mortality constant (k) Rate of mortality Negative slope Formula: -kt = ln (N o /N) T: Length of time N: Number of surviving cells N o : Initial number of cells N o /N: Inactivation factor Logarithmic Plot Time (Min.) 15 5

6 Sample Problem A treatment at 100 o C for 1h reduced a bacterial population from 10 8 to 10 2 cells What is the inactivation factor achieved? What is D 100? What is the mortality rate? How much time would be required to reduce the population to 10 2? 16 Control of Microbial Growth In Vivo: Antibiotherapy 17 Antimicrobial Drugs Antibiotic or Antibacterial Against bacteria Antifungal Against fungi Antiviral Against viruses 6

7 Definitions: The Drugs: Antibiotics Literal: Anti (against) biotic (life) Old def.: Any compound synthesized by a microorganism that inhibits or kills 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 (High selective toxicity, why?): Target: cell wall Cyanide (Low selective toxicity, why?): 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 (3 000 mg/kg) Cyanide (15 mg/kg) 3.Low therapeutic dose Concentration of the compound required for the clinical treatment of an infection Penicillin (12.5 mg/kg) Garlic (300 mg/kg) 7

8 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 Vancomycin Plasma membrane Polymixins DNA synthesis Quinolones Metabolism A B Sulfamides Translation Transcription RNA synthesis Macrolides Protein synthesis Aminoglycosides Macrolides Tetracyclines Chloramphenicol 24 8

9 # Modes of Action Time Viable count Direct count Bacteriostatic: Inhibits growth Non-lethal Reversible Bacteriocidal Bacteriolytic Kills Kills Irreversible Cell lysis Irreversible The Beta-Lactams Bacteriolytic Inhibit cell wall synthesis Act only on actively growing bacteria! Penicillines Monobactams They all contain a beta lactam ring Cephalosporins Carbapenems 26 Penicillins & Cephalosporins Natural penicillin penicillin G Narrow spectrum; only acts on Gram positives Aminopenicillin ampicillin and amoxicillin Broad spectrum; acts on Gram positives and negatives Cephalosporins Ex. Cefepime & Ceftazidime Developed to have a broader action spectrum as compared to penicillins 27 9

10 Monobactams & Carbapenems Monobactams Very narrow action spectrum; useless against Gram positives and anaerobes Carbapenems Last generation of beta lactams Very broad action spectrum Acts against Gram positives, negatives, anaerobes and aerobes 28 Quinolones Bacteriocidal Inhibit DNA synthesis Broad spectrum Side effects: Severe gastrointestinal problems Ex. Ciprofloxacin 29 Tetracyclines Bacteriostatic Inhibits protein synthesis Broad spectrum Side effects: Hepatic toxicity Renal toxicity Vitamin deficiency 30 10

11 Macrolides Bacteriostatic Inhibits protein synthesis Narrow spectrum Side effects Diarrhea Hepatic damage Ex. Erythromycin & Clarithromycin 31 Aminoglycosides Bacteriocide Narrow spectrum Inhibits protein synthesis High level of toxicity Side effects: Allergies Renal dammages Deafness Ex. Gentamycin, streptomycin 32 Chloramphenicol Bactericides Narrow spectrum Inhibit protein synthesis Side effects: Only used in extreme cases Hematological toxicity 33 11

12 Glycopeptide Antibiotics Composed of polycyclic amino acids Inhibits cell wall synthesis Acts mostly against Gram Positives Used as a last recourse Ex. Vancomycin 34 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 35 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 12

13 Inhibitory Diameters Vs Conc. 27mm = MIC < 27mm = Conc. > MIC > 27mm = Conc. < MIC 37 E-Test Same principal as the Kirby Bauer assay Makes use of a plastic strip with a predefined gradient of antibiotic concentrations The results are read directly on the strip The intersection point of the zone of inhibition and the strip E Zone of inhibition Bacterial growth 38 E-Test : Example 1 13

14 E-Test : Example 2 40 Determination of Efficacy MIC/MBC MIC: (Minimal Inhibitory Concentration) Cultures with different concentrations of antibiotic MIC=12μg/ml MBC: (Minimal Bacteriocidal Concentration) Sub culture without antibiotics MBC=50μg/ml In Vivo Susceptibility The in vivo concentration is not constant! Influenced by human physiology A range of concentrations is maintained (C Max -C Min ) The concentration at the infection site must be higher than the MIC If <MIC = resistance 42 14

15 Concentration 10/2/2016 Pharmacodynamics of Antibiotics Behavior of antibiotics in vivo Interaction of antibiotics with the bacteria The antibiotic must reach the site where the microbe resides The concentration of the antibiotic at the infection site must be above the MIC The antibiotic must occupy a sufficient number of sites on the target The antibiotic must remain in contact with the target for a sufficient amount of time 43 Pharmacodynamics of Antibiotics C max t trough Dose 2 Dose 3 MIC Time Dose 1 C max : Maximum concentration attained for a given dose C min : Minimal concentration attained between doses t: Time during which the concentration is maintained above the MIC C min 44 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 45 15

16 Example Antibiotic A conc. in vivo = 5-40µg/ml C min : 5ug/ml C max : 40ug/ml Thus: MIC 5 µg/ml = Sensitive MIC 40µg/ml = resistant MIC between 5-40 µg/ml = intermediate sensitivity 46 16