Clinical Bacteriology Lecture Week 3 PDF

CLINICAL BACTERIOLOGY LECTURE / SECOND SEMESTER Mr. Glenford D. Monzon, RMT LESSON 3: MICROBIAL CONTROL AND SAFETY TOPIC 1: FACTORS AFFECTING MICROBIAL GROWTH Oxygen Requirement Ob...

CLINICAL BACTERIOLOGY LECTURE / SECOND SEMESTER Mr. Glenford D. Monzon, RMT LESSON 3: MICROBIAL CONTROL AND SAFETY TOPIC 1: FACTORS AFFECTING MICROBIAL GROWTH Oxygen Requirement Obligate aerobes Vitro: Inside the laboratory Organisms requiring oxygen for growth Vivo: Inside the body (of an organism) Brucella, Franciella, Mycobacterium Nutritional Availability: Exception (Carbon Obligate anaerobes and Oxides) Organisms not requiring oxygen for growth IN VIVO IN VITRO Veilonella, Actimyces, Bifidobacterium Facultative anaerobes Chemotherapy Nutrient Availability Organisms that can live with or without air Anti-microbial Agent Moisture Organisms not requiring air but Antibacterial Agent Temperature enhanced in its presence Enterobacteriaceae Antifungal Agents pH Microaerophile Antiprotozoal Agents Osmolarity and Salinity Organisms requiring small amount of air Campylobacter, Treponema pallidum Antiviral Agents Oxygen and Carbon Dioxide Aerotolerant Concentration Do not grow well but survives in the presence of air Nutritional Requirement Lactobacillus, Propionibacterium acnes Phototroph Organisms that uses light as energy CO2 Requirements source Chemotroph Oxygen Carbon Dioxide Organisms that utilize the energy produced by organic and inorganic Aerobes 15 - 21% 0.03% compound oxidation Anaerobes 0% 5 - 10% Autotroph/Lithotroph Organisms that utilizes inorganic Capnophilic 15% 5 - 10% molecules (CO2) as carbon source Microaerophile 2 - 10% 8 - 10% Heterotroph/Organotroph Organisms that utilizes organic compounds (glucose) for growth and Temperature multiplication Optimal temperature: 35-37oC Saprophytes Yersinia enterocolitica: Blood Bank Infection Organisms that requires decaying or Geobacillus stearothermophilus: Autoclave dead matter as food source QC identification Dying composition Thermos aquaticus: Uses DNA for PRC Jhen Anne D. Abana | 1  Classification Optimum Examples TOPIC 2: FACTORS AFFECTING THE DEGREE OF Temp KILLING o Psychrophile 0 - 20 C ⋄ Listeria Factors that Influence The Degree of Killing monocytogenes ⋄ Yersinia enterolitica Number of Organisms Concentration of Disinfectant o Mesophile 25 - 45 C ⋄ Most pathogenic 70% Alcohol bacteria 10% Bleach solution Thermophiles o 45 - 70 C ⋄ Bacillus Nature of surface to be disinfected stearothermophilus Compatibility of Disinfectant ⋄ Thermos aquaticus pH Some organisms may not work with extremophiles Bacteria ⋄ Bacillus infernus which do not alkaline usually grow Temperature at high temp Will work only at room temperature but can Biofilms withstand exposure to Community of bacteria high temp Glycocalyx: Secrete by bacteria to facilitate attachment to the material or surface pH Requirement Types of Organisms Alkaline Peptone Water: For vibrio Compatibility of disinfectant Acidophilic Disinfectant: For gram negative and Grows between pH 0 - 5.5 gram positive Organisms requiring acidic medium for Broad Spectrum: Kill most bacteria growth Presence of Organic Material Lactobacillus Feces, blood, urine Basophilic/Alkalophilic Contact Time Grows between 8.5 - 11.6 Optimum contact time Organisms requiring alkaline medium for Alcohol: allow it to evaporate for at least growth 20 seconds Vibrio Neutrophilic Different Types of Organisms and Their resistance to Grows between 7.0 - 7.5 Killing Agents (most resistant to least resistant) Most pathogenic bacteria Prions Creutzfeldt-Jakob disease Moisture and Salt Concentration Naked proteins that can infect Moisture is essential for bacterial growth and Higher temperature to contaminate and susceptibility testing. At least 70% moisture is not work on common disinfectant needed to prevent dying Bacterial Spores Halophiles are organisms that grow in Determinant of sterilization and increased concentration of salt contamination (including spores) 6.5%: Enterococci Mycobacteria 7.5%: Staphylococcus aureus Resistant to killing 8 - 10%: Vibrio Fomite Non Lipid Viruses Fungi Bacteria Lipid Viruses Jhen Anne D. Abana | 2 Spaulding Classification Osmotic Pressure Organic Acids Critical: Instruments or devices that are exposed to normally sterile sites Radiation Aldehydes Semi-critical: Instruments that touch mucous membranes may either be sterilized or Gas disinfected Oxidizing Agents Non-critical: Instruments that touch skin or come into contact persons only indirectly can either be cleaned and then disinfected Sterilization with an intermediate-level disinfectant, Moist Heat sanitized with a low-level disinfectant, or Dry Heat simply cleaned with soap and water Thermal Death Point: Lowest temperature within 10 minutes Terminologies Thermal Death Time: Shortest time minimal Sepsis: Refers to microbial contamination length of time in which all bacteria are killed Asepsis: Refers to absence of significant in a given temperature contamination Decimal Reduction Time: Time in minutes in Aseptic Technique: Refers to absence of which 90% of bacteria are killed in a given significant contamination time Decontamination: Reduction in the number of pathogens to a level deemed safe by Moist Heat public health guidelines Autoclaving Sterilization: Removal of all microbial life Steam under pressure Disinfection: Removal of pathogens, except Boiling temperature increases as pressure bacterial spores increases Nonliving: Disinfectant Fastest and simplest method Living: Antiseptic Sterlilize equipment: 121oC, 15 pse, for 15 Antiseptic: Removal of pathogens from minutes living tissue Biohazardous: 132oC, 15 psi, for 30 to 60 Degerming: Removal of microbes for a minutes limited area usually through physical Geobacillus stearothermophilus - at least removal by using detergents 120oC Handwashing Tyndallization Bactericidal: antimicrobials that kills Intermittent / Fractional Sterlilization organisms Uses flow steam Bacteriostatic: Antimicrobial that inhibits Equipment used: Arnold Sterilizer bacterial growth 100oC for 30 minutes for 3 consecutive days TOPIC 3: METHODS OF MICROBIAL CONTROL 1st day: Vegetative 2nd day: Spores 3rd day: Remaining cells Physical Method Chemical Method Pasteurization Heat Phenol Partial sterilization Used to sterilize milk, dairy, and alcoholic Pasteurization Halogens beverages (non sporicidal) Low Temperature: Holding / Batch Filtration Alcohol Method Cold Heavy Metals ⋄ 63oC for 30 minutes High Temperature: Short Time / Flash Desiccation Surface Active Agents pasteurization ⋄ 72oC for 15 seconds Jhen Anne D. Abana | 3  Ultrahigh Temperature: Requires vacuum Dessication / Lyophilization chamber Bacteriostatic: Disruption of bacterial ⋄ 140oC for 3 seconds metabolism Boiling Viable bacteria in dry environment Destroy vegetative cells ⋄ Neiserria gonorrhoeae: 1 hour Temperature does not exceed 100oC at ⋄ Mycobacterium tuberculosis: several sea level months Used to disinfect surgical instruments ⋄ Bacillus and Clostridium: 10 months 100oC for 15 to 30 minutes Osmotic Pressure Inspissation Usage of ultra high concentration of salts Intermittent / Fractional Sterilization and sugar in food to create hypertonic Thickening through evaporation environment Equipment used : Inspissator Causes plasmolysis 75 to 80oC for 2 hours for 3 consecutive days Filtration 1st day: Vegetative Used to sterilize heat sensitive solutions 2nd day: Spores which would be denature by heat 3rd day: Remaining cells Commonly used to sterilize liquid and air in hospital isolation units Dry Heat Depth Filter Oven Heating Solids must pass through a “tortuous Used to sterilize glassware, oil, petroleum, path” or powders Consist of fibrous or granular material Temperature: 160 to 180oC for 1 to 2 hours Examples: Cool the equipment before opening ⋄ Berkefield: diatomaceous earth Indicator: Bacillus atrophaeus (B. subtilis ⋄ Chamberland Filter: Unglazed var. niger) porcelain Direct Flaming ⋄ Asbestos Commonly used for inoculating loops Membrane Filter and needles Traps particles larger that pore size Heat until red hot Consist of variety of polymeric materials Incineration suc as Cellulose acetate, polycarbonate, Efficient and safest method for and polyester sterilization Used to sterilize pharmaceuticals, Most common method of treating ophthalmic solutions, antibiotics, ando oil infectious waste and infected laboratory products animals Examples: 300 to 400oC: Burning into ashes ⋄ Cellulose Nitrate Membrane 870 to 980oC: For hazardous waste ⋄ HEPA Cremation ⋄ Milipore Filter Burning cadaver / body ⋄ Aluminum Oxide Filter Used tocontrol the spread of communicable diseases Radiation When radiation passes through cells, Disinfection hydrogen and hydroxyl radicals and some Low Temperature peroxidase are created. These free radicals Bacteriostatic: Reduces the rate of causes intracellular damage including DNA metabolism damage Exposure to 2 to 8oC for 72 hours is Efficiency is dependent on the wavelength, enough to kill syphilis intensity, and duration (contact time) Ionizing Radiation (Sterilization) Jhen Anne D. Abana | 4  Causes mutation in the DNA and Intermediate Level produces peroxides by ionizing water Germicides used to clean surfaces or Used to sterilize plastic syringes, gloves, instruments which come into contact and catheters with mucous membrane but not invasive 3 types: Electron Beam, Gamma Rays, Usually has tuberculocidal activity but X-ray bot sporicidal Non-ionizing Radiation (Disinfection) Low Level Causes damage to DNA by producing Eliminates only vegetative bacteria, Thymine Dimers fungal cell, and some virus. Used to clean Uses longer wavelength (100nm to materials that touches the skin but not 400nm) mucous membranes Used in exposed surfaces operating rooms and nursery rooms Mechanisms of Antimicrobial Action Agents against cell membrane Chemical Agents Surfactants, Phenolic compound, Used to control growth of microbes on both Alcohols living tissue and inanimate objects Agents that denature proteins Effectiveness of chemical disinfectant Acid / alkalis depend on Alcohols Type of microbe Agents that modify functional groups of Environmental factors protein and nucleic acid ⋄ Concentration of disinfectant Heavy metals, Oxidizing agents, Dyes, ⋄ Organic matter Alkylating agents ⋄ pH ⋄ Time Method of Evaluating Microbial Agent ⋄ Contact with microbiome Phenol Coefficient Old standard test Characteristic of a Disinfectant It is the highest dilution of disinfectant Have a wide spectrum of activity that kills the bacteria after 10 minutes Active at high dilutions and presence of exposure organic matter Test organisms: S. aureus and S. thypi Affective in acid/alkaline media 𝑃𝐶 = 𝐻𝑖𝑔ℎ𝑒𝑠𝑡 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑜𝑓 𝑑𝑖𝑠𝑖𝑛𝑓𝑒𝑐𝑡𝑎𝑛𝑡 𝑡ℎ𝑎𝑡 𝑘𝑖𝑙𝑙𝑠𝑜𝑟𝑎𝑛𝑖𝑠𝑚 𝑎𝑡 𝑎 𝑔𝑖𝑣𝑒𝑛 𝑡𝑖𝑚𝑒 ℎ𝑖𝑔ℎ𝑒𝑠𝑡 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝ℎ𝑒𝑛𝑜𝑙 𝑡ℎ𝑎𝑡 𝑤𝑖𝑙𝑙 𝑘𝑖𝑙𝑙 𝑜𝑟𝑔𝑎𝑛𝑖𝑠𝑚 𝑎𝑡 𝑎 𝑔𝑖𝑣𝑒𝑛 𝑡𝑖𝑚𝑒 Have speedy reaction Result: PC > 1 Disinfectant is more effective than High penetrating power phenol Stable and compatible with other antiseptic and disinfectant Use-Dilution Test Do not corrode metals, cause irritation or Useful in determining the ability of sensitization disinfectants to kill microorganisms dried Will not interfere with healing onto a typical clinical surface Not toxic if absorbed Microbe used Cheap and easily available ⋄ Salmonella choleraesuis Safe and easy to use ⋄ Staphylococcus aureus ⋄ Pseudomonas aeruginosa Categories of Disinfectant Metal cylinders are contaminated then High Level immersed in the disinfectant for 10 Germicide able to approach sterilization minutes. It will then reinoculated to a in effectiveness. Used for items involved culture medium to let surviving bacteria in invasive procedures grow Disk Diffusion Method A method that require less manipulation to judge the efficacy of disinfectants Jhen Anne D. Abana | 5  Filter paper is soaked with disinfectant ⋄ Denaturation of proteins and and then simply placed on the agar dissolution of lipid membranes surface of a petri dish that has been Optimal concentration 70% (60-95). Pure inoculated with a lown of test organism ethanol is ineffective because Presence of zone of inhibition indicates denaturation requires water effectiveness Should be allow to evaporate from the In-Use Test surface to achieve complete antisepsis Used to confirm that the chosen Good microbial activity against tubercle disinfectant has been effective under the bacilli and enveloped virus but has poor conditions of use activity against non-enveloped virus Swabs are taken from objects before and Nonsporicidal after application of disinfectant / Common examples antiseptic ⋄ Ethanol Swabs are the inoculated into a growth ⋄ Isopropanol media and incubated Aldhydes Phenols and Phenolics Mechanism of Action Also known as Carbolic Acid ⋄ Inactivation of protein and nucleic Mechanism of Action acid ⋄ Injuring Plasma ⋄ Commonly used to sterilize medical ⋄ Inactivating Enzymes equipments ⋄ Denaturation of Protein ⋄ Common examples Broad spectrum antiseptic but not 8% formaldehyde sporicidal ⋄ Used to sterilize HEPA filters in BSC Tuberculocidal: 5% phenol with 10 to 30 ⋄ Contact time is at least 30 minutes minutes contact time is effective against ⋄ Not recommended to be routinely mycobacteria used Commonly seen 2% glutaraldehyde ⋄ O-benzyl-p-chlorophenol ⋄ Effective against HIV and HBV when ⋄ O-phenylphenol exposed for 10 minutes Halogens ⋄ Used to sterilize medical instruments Can be used alone or as constituent of made of plastic inorganic / organic compound ⋄ Sporicidal in 3 to 10 hours Common types Quantum Ammonium Compounds Iodine QUATS ⋄ Oldest nad most effective antiseptic Odorless, colorless, nonirritating, and ⋄ Commonly available as tincture of deodorizing iodine / iodophor Cationic, surface-active agents or ⋄ Inactivates enzymes and protein surfactants synthesis by forming complexes Most effective against gram positive Chlorine bacteria ⋄ Used in the for of Hypochlorite. Mechanism of Action Strong oxidizer that prevents cellular ⋄ Disruption of cellular membrane enzyme to function Pseudomonas aeruginosa is resistant to ⋄ Contact time 3 minutes QUATS ⋄ CDC recommends 1:10 dilution of Nonsporicidal nor tuberculocidal NaOCl Examples ⋄ Ineffective in large amount of ⋄ Benzalkonium chloride: Zephiran proteins Heavy Metals Alcohols Slow bacteriocidal, primary Used both as antiseptic and disinfectant bacteriostatic Mechanism of Action Jhen Anne D. Abana | 6  Mechanism of Action Kill pathogens before they migrate and ⋄ Oligodynamic action become resistant ⋄ Alters 3D shape of proteins Examples Terminologies ⋄ 1% AgNO3: gonococcal conjunctivitis Antimicrobial ⋄ ZnCl2: mouthwash ○ Controls infection ⋄ Copper and copper alloys: lining rails Antibiotic and door handles ○ Produces naturally by microorganisms that inhibit or Gases destroy other microorganisms Ethylene Oxide (Gas sterilization) Semisynthetic Most commonly used chemical sterilant ○ Chemically modified drugs Mixed with nitrogen or CO2 before use Synthetic Mode of Action ○ Synthesized in the laboratory ⋄ Alkylation of nucleic acid Narrow Spectrum CONC ○ Effective against limited microbial ⋄ 450 to 700 mg at 55 to 66oC for 2 types hours ○ Targen specific cell component that Peracetic Acid is found only in certain Used in cold sterilization microorganisms Ised in pharmaceutical and medical Broad Spectrum device manufacturing industries ○ Effective against wide variety of H2O2 microorganisms May be used in combination with ○ Target cell component common to peracetic Acid most pathogens Used in pharmaceutical and medical device manufacturing industries Mechanism of Action of Antibiotic Mode of Action: Inhibition for… ⋄ Produces free radicals to damage DNA and Proteins Cell wall synthesis B-lactams Glycopeptides Vancomycin TOPIC 4: ANTIMICROBIAL AGENTS Protein Synthesis Aminoglycosides Antimicrobial Agents tetracyclines macrolides Any chemical or drugs used to treat infectious disease by inhibiting or killing Folic Acid Synthesis Chloramphenicol protein in vivo sulfonamides Antibiotic DNA synthesis Quinolones Substances produced by microorganism that is effective in killing other Cell Membrane Function Polymixin microorganism Penicillin, bacitracin Characteristic of a Good Antimicrobial Agent Kill or inhibit growth of pathogen Cause no damage to the host Cause no allergic reaction to the host Be stable when stored i solid or liquid form Remain in specific tissue long enough to be effective Jhen Anne D. Abana | 7 Inhibition of Cell Wall Synthesis Inhibition of Folic Acid Synthesis Class Representative Remarks Class Representative Remarks Antibiotic Antibiotic Natural Penicillin Penicillin V and G Allergic reaction is Chloramphenicol Chloramphenicol Long term use can common side cause bone effect marrow damage (aplastic anemia) Synthetic Penicillin Methicillin Drug class choice Oxacillin of S. aureus Sulfonamides Sulfamethoxazole Bacteriostatic Ampicillin Trimetophrim Used primarily for Carbenicillin UTI Piperacillin Inhibition of DNA synthesis Cephalosporin Cephalexin Ceftriaxone Cefotoxin Class Representative Remarks Cefepime Antibiotic Carbapenems Imipenem Quinolones Ciprofloxacin Ciprofloxacin is Monobactam Azteonam Levofloxacin used to treat Ofloxacin anthrax Glycopeptide Vancomycin Drug choice for MRSA Inhibition of Cell Membrane Function Inhibition of Protein Synthesis Class Representative Remarks Antibiotic Class Representative Remarks Antibiotic Bacitracin Colistin macrolide Erythromycin Bacteriostatic Polymixin Azithromycin Amphotericin B Clarithromycin Nystatin Tetracycline Tetracycline For intracellular Doxycycline organisms Spirochetes Can stain teeth and abnormal none growth Aminoglycosides Entamycin Nephrotoxic and Amikacin chemotoxic Tobramycin Kanamycin Jhen Anne D. 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