01/08/2018. Control of Microbial Growth. Methods. Terminology. Disinfectants and Antiseptics. Three approaches. Cleaning. Chemical.

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1 Control of Microbial Growth Disinfectants and Antiseptics 1 Methods 2 Three approaches Chemical Disinfectants and antiseptics Physical Heat Ultraviolet Irradiations Mechanical elimination Cleaning Filtration Terminology 3 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 1

2 Disinfection 4 The use of chemical or physical agents to kill or inhibit the growth of microorganisms Disinfectants Chemical products used on inanimate objects Germicides Chemical products which can be used on either animate (living) or inanimate things Antiseptics Chemical products used on living tissues Factors that Influence Efficacy 5 Microbial load Number of microbes Environment Presence of organic matter Concentration of the agent Temperature ph Length of exposure Factors that Influence Efficacy 6 Microbial characteristics Biofilms Cell wall Resistances Spores 2

3 Number of cells Disinfectant Efficacy 7 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 Mortality Profile Logarithmic 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 Parameters of Mortality 9 Decimal reduction time (D) Time required to achieve a reduction of one log or an inactivation factor of 10 Formula: Log (N 0 /N) =t/d t: Length of time N: Number of surviving cells N o : Initial number of cells N o /N: Inactivation factor 3

4 Parameters of Mortality 10 Mortality constant (k) Rate of mortality Negative slope Formula: -kt = ln (N o /N) T: Length of time N: Number of surviving cells No: Initial number of cells N o /N: Inactivation factor Relative Resistance 11 Z value Temperature change required to change the D value by 1 log Temperature change required to change the D value by a factor 10 Formula: Z = (T1-T2)/(logD2-logD1) T: Temperature D: Decimal reduction time Sample Problem 12 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 by the same factor at 102 o C if z = 1 o C? 4

5 Control of Microbial Growth In Vivo: Antibiotherapy 13 Antimicrobial Drugs 14 Antibiotic or Antibacterial Against bacteria Antifungal Against fungi Antiviral Against viruses The Drugs: Antibiotics 15 Definitions: Literal: Anti (against) biotic (life) Old def.: Any compound made by a microorganism which inhibits or kills bacteria New def.: Any compound which inhibits or kills bacteria Natural Synthetique Semi-synthetic 5

6 Desired Characteristics 16 High selective toxicity Must kill or inhibit the targeted organism with minimal deleterious effects on the host Penicillin: (High selective toxicity) Targets the cell wall Cyanide: (Low selective toxicity) Targets electron transport of eukaryotes/prokaryotes Desired Characteristics (cont d) 17 High toxic or lethal dose (LD50) Concentration of the agent qui that is toxic for the host Penicillin: (3 000 mg/kg) Arsenic: (15 mg/kg) Low therapeutic dose Concentration of the agent required for the clinical treatment of an infection Penicillin : 12.5 mg/kg Garlic: 300 mg/kg Therapeutic index 18 Toxic dose/therapeutic Dose Want a therapeutic dose which is? High 6

7 Action Spectrums 19 Narrow: Restricted efficacy against some types of microorganisms Broad: Ex. Only acts against Gram - Effective against a wide diversity of microorganisms Ex. Acts on Gram + and - Antibacterial Targets 20 Cell wall synthesis ß-lactams DNA synthesis Quinolones Metabolism A B Transcription Translation RNA synthesis Macrolides Protein synthesis Aminoglycosides Macrolides Tetracyclines Chloramphenicol Modes of Action 21 # Direct count Viable count Bacteriostatic Inhibits growth Non lethal Reversible Time Bactericide Kills Irreversible Bacteriolytic Kills Cell lysis Irreversible 7

8 Beta-Lactams 22 Classe of antibiotics that have a betalactam ring Bacteriolytic Inhibit synthesis of the cell wall Only acts on growing bacteria! Penicillins & Cephalosporins 23 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 Developed to have a broader action spectrum as compared to penicillin Quinolones 24 Bactericides Inhibit DNA synthesis Broad spectrum Side effects: Severe gastrointestinal problems Ex. Ciprofloxacin 8

9 Tetracyclines 25 Bacteriostatic Inhibits protein synthesis Broad spectrum Side effects: Hepatic toxicity Renal toxicity Vitamin deficiency Macrolides 26 Bacteriostatic Inhibits protein synthesis Narrow spectrum Side effects Diarrhea Hepatic damage Ex. Erythromycin & Clarithromycin Aminoglycosides 27 Bactericides Narrow spectrum Inhibit protein synthesis High level of toxicity Side effects: Allergies Renal damages Deafness Ex. Gentamycin, streptomycin 9

10 Chloramphenicol 28 Bactericides Narrow spectrum Inhibit protein synthesis Side effects: Only used in extreme cases Hematological toxicity Sensitivity: Kirby Bauer Assay 29 Medium is inoculated with bacteria to be tested Discs containing antibiotics are deposited on the medium A concentration gradient is established due to the diffusion of the antibiotic in the medium Following the incubation, the inhibition zones are measured The sizes of the zones are compared to those established to determine whether the organism is sensitive or resistant E-Test 30 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 10

11 E-Test 31 Determining Efficacy 32 Minimal Inhibitory Concentration Growth with different concentrations of antibiotic MIC=12μg/ml Subculture without antibiotics MBC=50μg/ml Minimal Bactericide Concentration Diameters of Inhibition Vs Concentration 33 27mm = to MIC < 27mm = Conc. > MIC > 27mm = Conc. < MIC 11

12 Behavior of Antibiotics In Vivo 34 The antibiotic must reach the site where the microbe resides The concentration of the antibiotic at the infection site must be above the MIC Antibiotic concentration fluctuates in vivo between doses C max : Maximum concentration maintained C min : Minimum concentration maintained In Vivo Sensitivity 35 Sensitive pathogen MIC is lower than C min Resistant pathogen MIC is higher than C max Intermediate sensitivity pathogen MIC is between C min and C max A combination of antibiotics is recommended Example 36 In vivo conc. of antibiotic A C min : 5 µg/ml C max : 40 µg/ml Therefore: MIC 5 µg/ml = Sensitive microorganism MIC 40µg/ml = Resistant microorganism MIC between 5-40 µg/ml = Intermediate sensitivity microorganism 12

13 Immunology 37 Non-Self - Antigens 38 Anything that can react with the participants of the immune system Ex. antibodies Epitope: Characteristic of the antigen which allows its recognition as being nonself Ex. Lipids, proteins, lipopolysaccharides The Antigen 39 Epitopes Virus=Antigen 13

14 Immunological Diagnostic 40 Determine the presence of an antigen: An organism A protein A toxin An antibody Dosage by ELISA Used to detect the presence of antibodies or antigens ELISA Detection of Antigen 41 Serum (source of Ag) added to plastic wells Blocking agent added Ab against Ag added Wash Detecting Ab added Wash Substrate added Antigen Present Antigen Absent ELISA Detection of Antibody 42 Target Ag for Ab to be detected added to wells Blocking agent added Serum to be tested added Wash Detecting Ab added Wash Substrate added Ab Present Ab Absent 14

15 Patient 1 Patient 2 Patient 3 Interpretation/Quantification 43 Known concentrations of antigen or antibody used as reference Dilutions 1/4 1/10 1/30 1/50 1/100 1/200 Negative control Abs Choose an absorbance higher than negative control Ex. 1/50: (0.16 ) = 0.14 Determine correlation between abs and concentration Ex. Stock conc. was 2 mg/ml Thus 2 mg/ml X 1/50 = Abs. of mg/ml = Abs. of mg/ml = Abs. of 3.5 Interpretation/Quantification 44 Detection of virus X Ag Abs Dilutions 1/8 1/16 1/32 1/64 1/128 1/256 N.C Abs above NC are positive P1 & P3 are infected P2 is negative P3 has a load that is 6X higher (1.68-)/0.28-) Conc of Ag for P3 Choose value above NC Ex. 1/64 = (0.24-) = 0.22 Undiluted = 0.22 X 64 = Standard: 1.0 mg/ml = Abs. of 3.5 Thus conc. for P3 : 4.0 mg/ml 15