Control of Microorganisms Lecture 8 PDF

Control of microorganisms Introductory microbiology A/Prof. Rebecca Case SCELSE/SBS [email protected] Why do we want to control microorganisms? corrosion plant pathogens human pathogens Food spoilage  Definition of frequently used terms Antis...

Control of microorganisms Introductory microbiology A/Prof. Rebecca Case SCELSE/SBS [email protected] Why do we want to control microorganisms? corrosion plant pathogens human pathogens Food spoilage  Definition of frequently used terms Antisepsis: chemicals (antiseptics) applied to body surfaces to destroy or inhibit pathogens Sanitation: reduction of microbial population to levels deemed safe (based on public health standards) Disinfection: destruction or removal of pathogens, but not bacterial endospores. Usually used on inanimate & objects non-living things Sterilization: The complete removal or destruction of all viable microorganisms. Used on inanimate objects & including endospores Chemotherapy: chemical used to kill or inhibit growth of microorganisms within host tissues Reduction of microbial numbers that arise diff curves can antimicrobial from applying What does Log kill mean? 1 log reduction: 90% kill - there is still quite a lot of microbial completely wiped out 3 log reduction: 99.9% kill not killed not , Number of bacteria in the mouth? Approximately the same as the number of people on Earth: 7.5 Billion or 7.5 x109 3 log reduction: 6.109 > 6 x106 Note: transient reduction  The pattern of microbial death Microorganisms are not killed instantly Population decline (death) occurs exponentially Killing efficiency is measured using decimal reduction time which is the time to kill 90% of population & Persister cells are the hardest to kill log reduct I These cells are viable but nonculturable (VBNC) in which cells are intact, have a low metabolic rate and are not dividing (and can’t form colony forming unit) VBNC cells can recover and regain the ability to reproduce and cause infection with specific conditions & in e case of pathogens  Conditions influencing the effectiveness of antimicrobial agent activity time taken to kill larger popl longer Population size ~ , & pop? bring it to a small larger >time kill >small don't allow Population composition - % cells layer penetrate e.. i slime antibiotics to i biofilm sensitivity differs markedly to antimicrobials Concentration of antimicrobial higher conc. kill more rapidly Duration (time) of exposure longer kills more microbes & time Temperature higher T kills more microbes Local environment e.g. pH, viscosity, organic matter impact effectiveness organisms in biofilms are less susceptible to many W antimicrobial agents Physical control of microbes 1. Heat 2. Radiation  Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Moist heat Very efficient way of killing micro-organisms including viruses, fungi, protists and bacteria ~ higher tema Degrades nucleic acids, denatures proteins and disrupts membranes through hydrolysis Spores are more heat and moisture resistant than vegetative cells e.g. Bacillus spores are killed at a temperature ~40-50ºC higher than replicating (vegetative) cells B. subtilis B692 : 57ºC (veg) vs. 111ºC (spore) how an autoclave works ? take into considerate durat , temp out all e pushes water saturated air which is lower in temp high pressure forces water into molecules  Autoclaving: steam sterilisation Steam sterilisation carried out using an autoclave Performed above 100 oC which requires saturated steam under pressure Saturation requires good penetration so everything must be open ~ steam penetrate cannot they explode them o of high pressure can be Closed containers only heat sterilised Effective against all types of microorganisms including durate spores ~ based I on Quality control - includes strips with Geobacillus stearothermophilus (thermophile) & to resistant he a Magic sterilization formula: 121 oC for 30 min* at 103.5 kPa (15 psi) *Time is increased for large volumes The effect of container size on autocalve sterilization times for liquid solutions 10 ml 15 min 95 ml 15 min 1500 ml 30 min 6750 ml 70 min But it’s not always as possible to follow this guide as when it is over autoclave medium will caramelize ~ It’s important to have an autoclave optimally functioning to keep sterilization time minimal  Why is it the most essential piece of equipment for a microbiologist? Microbiologists can not maintain their organisms or conduct experiments unless sterility is maintained don't ~ want contaminate Aseptic technique and sterilization are the first things you’ll be asked about when applying for an industry job  Pasteurization Invented by Louis Pasteur in 1864. Revolutionary processes that allowed food to be consumed less immediately (no refrigerator at that time): prevented many diseases. Controlled heating to reduce bacteria numberremoval of definit complete ~ : microorganisms Process does not necessarily sterilize, but does kill pathogens present and slow spoilage by reducing the total load of organisms present Applied mainly to the food industry: milk, dairy products, beer and other beverages temp is not super high  Pasteurization Choice of pasteurization method depends on product and goal because pasteurisation can alter food nutrients or taste Traditional Pasteurization: ~ tends to have High-Temperature-Short-Time Treatment (HTST) better P struct e.g. 72°C for 15 seconds. Low-Temperature-Long-Time Treatment (LTLT) e.g. 63°C for 30 minutes. For milk, HTST causes less damage to the nutrient composition and sensory characteristics compared to LTLT  Dry heat sterilization Less effective than moist heat sterilization. Requires higher temperatures and longer exposure times e.g. 160–170oC for 2 to 3 hours Usually not applicable to food industry: oxidizes cell constituents and denatures proteins metal ~ , glass Useful for solids that are effected by moist heat but are unchanged by high temperature  Ionizing radiation gamma radiation penetrates deep into objects destroys bacterial endospores; not always effective against viruses. Non-radioactive used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food (meat, eggs,…)  Ultraviolet (UV) radiation wavelength of 260 is most bactericidal (DNA absorbs) causes thymine dimers preventing replication and transcription Limitation: UV do not penetrate glass, dirt films, water, limited to surface decontamination sterilise surface only - Has been used for water treatment & food industry Sterilisati of water surfaces , Chemical control of microbes  Chemical agents Joseph Lister, 1867 insisted on using carbolic acid, or phenol, to disinfect patients/wounds survivability ↑ Disinfectant must be effective against wide variety of infectious agents at low concentrations Must be effective in the presence of organic matter; should be stable in storage dangerous long term but not short in term Overuse of antiseptics such as triclosan has selected for triclosan- resistant bacteria and possibly antibiotic resistant ↑ microbial in resistance  Phenolics commonly used as laboratory and hospital disinfectants tuberculocidal, effective in presence of organic material, and long lasting act by denaturing proteins and disrupting cell membranes disagreeable odor and can cause skin irritation triclosan is used in hand sanitizers (and toothpastes) Used less due to safety concerns phenol (polychloro phenoxy phenol)  Alcohols among the most widely used disinfectants and antiseptics two most common are ethanol and isopropanol bactericidal, fungicidal, but not sporicidal inactivate some viruses denature proteins and possibly dissolve membrane lipids Most effective when used at 70% with water (wetting agent) & lets alcohol get I into e cell when alcohol is 100 %, , membrane blocks it out& does not allow it to penetrate as well water also allows it stay on a act longer making it longer , more effective , 100 % alcohol evaporates faster  Halogens - iodine skin antiseptic Used a lot in surgery prep oxidizes cell constituents and iodinates proteins at high concentrations may kill spores skin damage, staining, and allergies can be a problem  Halogens - chlorine oxidizes cell constituents important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant destroys vegetative bacteria and fungi chlorine gas is sporicidal - toxid can react with organic matter to form carcinogenic compounds  Heavy metals e.g. ions of mercury, silver, arsenic, zinc, and copper combine with and inactivate proteins; may also precipitate proteins In the past, silver coins used to preserve foods/drinks Silver ointment still in use for skin infection effective but usually toxic & when time increases exposure exposure time is critical ! only for a short time  Quaternary ammonium compounds detergents that have antimicrobial activity and are effective disinfectantsparts ~ non-polar polar and amphipathic organic cleansing agents safe and easy to use, but inactivated by hard water and soap Target integrity of the cell envelop kill most bacteria, but not endospores general structure benzalkonium chloride  Aldehydes commonly used agents are formaldehyde and glutaraldehyde (used to fix cells or bacteria before FACS/microscopy) highly reactive molecules, toxic combine with and inactivate nucleic acids and proteins sporicidal and can be used as chemical sterilant core structure formadehyde  Sterilizing gases used to sterilize heat-sensitive materials they can pole oxygen free have radicals that can activity microbicidal and sporicidal anti-microbial ethylene oxide sterilization is carried out in equipment resembling an autoclave vaporized hydrogen peroxide combine with and inactivate DNA and proteins ethylene oxide Mechanical control of microbes  Filtration Reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms Applicable to air (Hepa filter), food industry (milk, juices), and research labs (filter-sterilize medium) be used to sterilise antibiotics can  Filtering liquids Membrane filters - Porous membranes with defined pore sizes that remove microorganisms primarily by physical screening. Pore size of 0.2 microns adapted to bacteria (not viruses) Use in food industry (milk) than E smaller Use in laboratory to filter thet heat-senstive culture broth a organisms e able through media it Costly technology  Filtering air surgical masks; cotton plugs on culture vessels High-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets and home-filters. Struct of HEPA Biological control of microbes  Biological control of microorganisms Emerging field showing great promise. Natural control mechanisms predation by Bdellovibrio (natural bacteria predator) toxin-mediated killing using bacteriocins (protein, antibiotic-like activity, narrow spectrum, attack membranes) Bacteriophages (bacteria viruses) viral-mediated lysis using pathogen specific bacteriophage lysins  Bacteriophage products Bacteriophages expresses lysins that lyse the bacteria by wall well targeting the peptidoglycan ~ virion-associated peptidoglycan hydrolases: make a hole to allow DNA injection (initial infection) Endolysin: lyse the peptidoglycan at the end of the cycle to release the viruses Both enzymes classes are being developed as antibacterial agents (Gangagen, Lysando, Micreos)  The development of chemotherapy Paul Ehrlich (1904) Developed concept of selective toxicity (cancer and anti-infectives) Identified dyes that effectively treated African sleeping sickness Sahachiro Hato (1910) ~ toxic findto working with Ehrlich, identified arsenic compounds impt therapeutic e that effectively treated syphilis dise Gerhard Domagk, Jacques and Therese Trefouel (1935) discovered sulfonamides and sulfa drugs (antibiotic and anti-cancer drugs)  Penicillin first discovered by Ernest Duchesne (1896), but discovery lost accidentally re-discovered by Alexander Fleming (1928) observed penicillin activity on contaminated plate did not think it could be developed further Effectiveness demonstrated by Florey, Chain, and Heatley (1939) Fleming, Florey, and Chain received Nobel Prize in 1945 for discovery and production of penicillin  Chemotherapeutics Antibiotics include bactericides, fungicides, algicides, and viricides Bactericidal agents: kill bacteria suffix indicating that agent kills kills pathogens and many nonpathogens but not necessarily endospores Bacteriostatic agents: inhibit growth suffix indicating that agent inhibits growth impt General characterisitics of antimicrobial drugs selective toxicity ability of drug to kill or inhibit pathogen while damaging host as little as possible Narrow-spectrum drugs: attack only a few pathogens Broad-spectrum drugs – attack many different pathogens therapeutic dose drug level required for clinical treatment Side effects – undesirable effects of drugs on human almost all antibiotics have e.g. Streptomycin is ototoxic, linezolid induces effects side neutropenia toxic dose drug level at which drug becomes too toxic for patient (i.e. produces side effects) therapeutic index -ideally want far we it to be close it they ratio of toxic dose to therapeutic dose apart dangerous where are , can be General characterisitics of antimicrobial drugs Effectiveness expressed in two ways minimal inhibitory concentration (MIC) stops growth lowest concentration of drug that inhibits growth of pathogen minimal bactericidal/lethal concentration (MBC or MLC) kills pathogen lowest concentration of drug that kills pathogen  3 types of acts of Terminology of chemical control anti-microbial agents preventse growth » -static: a suffix indicating same here that the agent will prevent growth couc , inhibitory was added growth of the type of organism in question (e.g., bacteriostatic, fungistatic) VCC ↓ » -cide: a suffix indicating that the agent will kill the kind of organism in question (e.g., fungicide, bacteriocide) TCCI VCCL  Ability of drug to reach site of infection depends in part on mode of administration oral topical ~ more straight into serious , bloodstream parenteral routes Intravenous (IV) or Intramuscular (IM) An ideal drug can be given orally or intravenously (to act quickly in emergency situation) Drug can be excluded by blood clots or necrotic tissue way of I get in effectiveness of drug reaching site of infect? Determining antimicrobial potency Important for clinical diagnostic dilution susceptibility tests for MIC disk diffusion tests – Kirby Bauer the E-test MIC and diffusion  Dilution susceptibility tests If broth used, tubes showing no growth (MIC) can be spotted on drug-free agar plates to determine cidality (killing) broth from which microbe can’t be recovered is the minimum bactericidal concentration (MBC) do look for ii) where we not see any growth => MIC = 4 Mg/mL - halved natred - - plate on agai inhibit growth to revive it medium try on a where there is no anti-microbial , it grows again no growth  Disk diffusion tests Disks impregnated with specific drugs are placed on agar plates inoculated with test microbe drug diffuses from disk into agar, establishing concentration gradient Observe clear zones (no growth) around disks  Kirby-Bauer method Standardized method for carrying out disk diffusion test Antibiotic spotted on paper discs dispensed on plates containing the bacteria of interest. Incubation - bacteria antibiotic are not largest zone of inhibit having an effect on Antibiotic disc dispenser => most effective antibiotic microorganism , no zone of inhibit  The E test Convenient for use with anaerobic pathogens similar to disk diffusion method, but uses strip rather than disk E-test strips contain a gradient diff antibioticsw is of antibiotic ~ Intersection of elliptical zone of inhibition with strip indicates MIC & where zone of clearance is starting cantibiotics +, more effect on microorganism Microbial control methods How do antibiotics work?  alls many diff basteria have features &: have diffmodes of attack Mechanisms of action Antibiotic target the most fundamental processes molecular target often conserved in human (but still specific) Inhibitors of cell wall synthesis I Protein synthesis inhibitors Nucleic acid synthesis inhibition within basteria calls Metabolic antagonists  Major targets of common antibiotics DNA Fluoroquinolones Novobiocin Nitrofurans Ribosomes Tetracyclines Cell Wall Aminoglycosides b-lactams Macrolides Glycopeptides Chloramphenicol Ethambutol Isoniazid Adapted from: http://amrls.cvm.msu.edu/pharmacology/antimicrobials/mode-of-action 52  Inhibitors of cell wall synthesis Penicillins (b-lactams) Most important antibiotic class Most are 6-aminopenicillanic acid derivatives most crucial feature of molecule is the b-lactam ring essential for bioactivity penicillin resistant organisms produce b-lactamase that hydrolyzes a bond in this ring b-lactam ring  Penicillins Mode of action blocks the enzyme that catalyzes transpeptidation (formation of cross-links in peptidoglycan) Enzymes are called penicillin-binding proteins = DD-transpeptidases prevents the synthesis of complete cell walls leading to lysis of cell ~ dividing acts only on growing bacteria that are synthesizing new peptidoglycan  Strands are crosslinked Amino acids serve to link together the glycan chains in several ways 55  Penicillins naturally occurring penicillins penicillin V and G are narrow spectrum semisynthetic penicillins have a broader spectrum than naturally occurring ones ~1–5% of adults in U.S. are allergic to penicillin allergy can lead to a violent allergic response and death Resistance to b-lactams is a huge issue  Protein synthesis inhibitors many antibiotics bind specifically to the bacterial ribosome binding can be to 30S (small) or 50S (large) ribosomal subunit other antibiotics inhibit a step in protein synthesis aminoacyl-tRNA binding peptide bond formation mRNA reading translocation  Aminoglycoside antibiotics large family which all contain a cyclohexane ring and amino sugars bind to 30S ribosomal subunit and interfere with protein synthesis by directly inhibiting the process and by causing misreading of the messenger RNA resistance and toxicity Antibiotic resistance  Drug resistance An increasing problem once resistance originates in a population it can be transmitted to other bacteria a particular type of resistance mechanism may confer resistance to multiple antibiotics [drug] lowers e.g. efflux pump over-expression ~ Resistance mutants arise spontaneously and are then selected survival ofe fittest & Phenotypic drug resistance microbes in abscesses or biofilms may be growing slowly and resistant to antibiotics  Mechanisms of drug resistance antibiotics out get e.g. b-lactamase of I system degrades B-lactam - alters e antibiotic as soon as it enters e all  Mechanisms of drug resistance 1. Modification of target enzyme (spontaneous mutation) Reduce/inhibit binding of the antibiotic to the target 2. Inactivation of the antibiotic e.g. b-lactamases degrade b-lactams antibiotics (e.g. penicillin) 3. Antibiotic-altering enzymes Antibiotic is metabolized. e.g. 2is gene in Mycobacterium tuberculosis inactivates aminoglycosides by acetylation 4. Drug efflux (pump drug out of cell) Lower antibiotic amount in the cytosol 5. Use of alternative pathways/enzyme e.g. resistance to sulfonamides: horizontal acquisition of dhps variants that are resistant to inactivation by sulphonamides Dhps: dihydropteroate synthase, sulfonamide target The origin and transmission of drug resistance Immunity genes Antibiotic-producing microbes have resistance genes that protect them from their own antibiotics Horizontal gene transfer Transfer of immunity genes from antibiotic producers to non-producing microbes Antibiotic resistance genes are natural. Why?  Copyright © The McGraw-Hill Companies. Permission required for reproduction or display. Horizontal gene transfer (mainly The origin and transmission of drug resistance Resistance genes can be found on: bacterial chromosomes plasmids transposons phages When found on mobile genetic elements they can be freely exchanged between bacteria  Drug resistant “superbug” serious threat to human health methicilin ~ used to repeated exposure treat SA but led to resistance A methicillin-resistant Staphylococcus aureus (MRSA) that developed resistance to vancomycin Vancomycin: potent last resort antibiotic Vancomycin-resistant S. aureus (VRSA) also resistant to most other antibiotics Emerged from foot ulcers on a diabetic patient Acquired from conjugation from vancomycin- resistant enterococci (VRE) (most likely from same patients) overuse of antibiotics increasesa rate of resistance to these antibiotics Drive further antibiotic resistance  Summary There is a strong motivation to control microbes for human health and industry Growth control vs. killing Antimicrobials target essential functions Understanding mechanisms of action allow for targeted drug design Selection pressure for resistance is high Over use and misuse of antimicrobials increases selection for drug resistance