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MIC401 - Week 3 of lecture notes

by: Christinee

MIC401 - Week 3 of lecture notes MIC 401

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This is this weeks lecture notes aka the last lectures included on the first exam.
Biomedical Microbiology
Dr. Amy Jacobs
Class Notes
MIC401, Mic 401, Biomedical microbiology, Christian Ahearn, Amanda Bloom, Terry Connell, Darlene Dombroski, Vicki Evancho, Amy Jacobs, Nicole Luke-Marshall, Khan Umaer
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This 12 page Class Notes was uploaded by Christinee on Wednesday February 10, 2016. The Class Notes belongs to MIC 401 at University at Buffalo taught by Dr. Amy Jacobs in Spring 2016. Since its upload, it has received 92 views. For similar materials see Biomedical Microbiology in Microbiology at University at Buffalo.


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Date Created: 02/10/16
Lecture 6 – Antimicrobial Agents *These are additional notes that aren’t included on the slides that were discussed during his  lecture. I. Ideal Antimicrobial Agents (slide 2) – NOTE: he specifically said that this slide was  not as important, however, I took detailed notes anyways for anyone that needs more of  an explanation. Just make sure you get the general idea but you don’t need to completely  memorize. a. Only destroys the offending microbe i. Antibiotics kill offending microbe as well as natural flora – so as of right  now, unable to only just destroy the offending microbe b. Targets microbial professes that aren’t present in the host – like to inhibit  something the bacteria does that humans don’t i. Many antibiotics inhibit the same processes that go on in humans – its just  the bacterial enzymes (ribosomes) are vastly different then human ribosomes – so able to inhibit some ribosomal processes & have a very effective  antimicrobial agent ii. In the case of penicillin and the cell wall active agents, microbes have a cell  wall that’s composed of peptidoglycan – humans don’t make peptidoglycan  & therefore that’s one of the reasons why they’re useful agents  c. Have limited or no toxicity to the host i. Unfortunately, we don’t have any antimicrobial agents that do this  ii. Almost all/ALL the antibiotics currently have some toxicity to the host  iii. There are idiosyncratic reactions, dose related toxicities, or specific side  effects d. Long half life of the drug i. Double edge sword  ii. We like the drug to be around for a very long time so we don’t have to take  it often iii. We now have medication that we are able to take every two weeks II. Downsides of this: 1. Toxcity 2. Allergic reaction to the antibiotic, will stay throughout the course of  the antibiotic ii. Useful when the antibiotic works well, not useful when you have adverse  reactions  b. Not be susceptible to microbial resistance mechanisms i. This will probably never happen ii. Can’t sterilize the host  III. Antibiotics (slide 3) a. For the Exam: remember each classification of antibiotics & where they work IV. Cell wall active agents (slide 4) a. All of these antibiotics act against the formation of the cell wall b. Most important ones to know: penicillins & glycopeptides c. Know the mechanism of action & mechanism of resistance  V. Protein synthesis inhibitors (slide 5) a. All of these antibiotics work on the ribosomes  b. Just like humans, bacteria have large and small ribosomes c. Erythromycin – these days are replaced with azithromycin and clarithromycin  VI. Dna synthesis & maintenance (slide 6) a. Agents that either work on DNA synthesis or the maintenance of the bacterial  DNA b. DNA synthesis: sulfonamides & trimethoprim c. Maintenance: quinolones  VII. Unique microbial targets (slide 8) a. Humans don’t make peptidoglycan b. This makes an absolutely perfect target for an antimicrobial agent VIII. Cell walls (slide 9) a. Gram negative: peptidoglycan in between the inner and outer membrane – pink/red b. Gram positive: large sheets of peptidoglycan that are extensively cross linked –  purple/blue IX. Penicillin (slide 12) a. Penicillin discovered by Alexander Fleming b. He was working on isolating a compound inside of the bacterium called Lysozyme  from Staphylococcus c. Before this, he didn’t believe you could actually make a chemical that could inhibit the bacteria  d. He was trying to find a natural product – thought it was going to be lysozyme  which would supposedly help degrade the cell wall and kill the bacteria  X. Slide 13 a. ­ Circular form – instead of linear form – indicated that there was a compound  diffusing out the media that was killing the Staph Aureus  XI. Transpeptidase reaction (slide 15) a. Determined that the mechanism for the action of penicillin – inhibition of the  transpeptidase reaction b. This happens to be the peptidoglycan of the organism Staph Aureus  c. Notice: side chains of the L­lysines will attach to the next to last D­alanine via this  transpeptidase reaction i. It will kick out the terminal D­alanine & cross links those chains d. Transpeptidation is absolutely essential for bacterial survival i. It establishes a rigid cell wall because bacteria survive in an environment  where they have very high concentrations of solutes inside the cell but very  low outside the cell – so the osmotic pressure is enormous ii. If you can’t cross link the peptidoglycan, then the cell walls will rupture  XII. Penicillins (slide 16) a. Once that molecule is acylated, it is absolutely irriversible & that transpeptidase  can no longer cross link side chains of peptidoglycan – bacteria will die b. Beta­lactam ring – very very reactive  XIII. Cell wall synthesis & penicillin (slide 17) a. Penicillin binding protein 2 b. When it recognizes that D­alanine D­alanine, which triggers that transpeptidase  reaction, if penicillin binds in there it will irriversibly acylate that transpeptidase –  rendering it nonfunctional and will result in the death of the bacterium c. MUST KNOW************ XIV. Stereo chemistry (slide 18) a. ­ They both bind in the same pocket place of the transpeptidase  XV. Beta­lactamases (slide 20) a. IMPORTANT TO REMEMBER****** b. Once hydrolyzed, penicillin can no longer acylate transpeptidase c. Change specificity by just changing one nucleotide  XVI. Humans fight back (slide 21) a. Chemists altered structure of penicillin – so new structure called methicillin would  resist staphylococcus beta lactamase XVII. Bacteria respond (slide 23) a. IMPORTANT TO KNOW b. MRSA  c. Methicillin designed for the degradation of the beta lactamase XVIII. Cell wall synthesis & MRSA (slide 24) a. How MRSA works b. ­  In addition to having PBP 2, it has PBP2A which does the transpeptidation step  c. But PBP2A does not bind methicillin – it’s not the destruction of the methicillin –  it’s the INABILITY of methicillin or any of the common beta lactamase antibiotics to bind to PBP2A d. So bacteria can try to degrade the antibiotic or they can alter their binding site to  render the antibiotic resistant  XIX. Humans respond again (slide 25) a. When we started seeing MRSA, we responded with vancomycin b. VANCOMYCIN IS A GLYCOPEPTIDE c. Glyco = sugar peptide = AA – chain of AA with sugar chains  XX. Vancomycin mechanism (slide 26) a. Intimately involved with the presence of D­alanine D­alanine b. Remember that transpeptidase recognizes D­al D­al and then acylates the  transpeptidase and prevents it from doing the cross linkage c. But what vancomycin does is it covers the site so that once vancomycin is bound to the D­al D­al, it prevents the first step called transglycolation: the addition of the  n­acetylmuramic acid to the n­acetylglucosamine  XXI. Vancomysin resistance (slide 27) a. ­ MUST KNOW: beta lactamases DO NOT DEGRADE vancomycin b. So vancomycin works very well for organisms that have beta lactamases as well as organisms with D­al D­al chains on their peptidoglycan XXII. Slide 28 – continued from previous a. * Do not need to memorize process: just understand general idea b. Penicillins just needed to synthasize a beta lactamase or change binding of  penicillin binding protein – only had to change one target c. For vancomysin resistance: this is not just a change in one gene – you have to  move in an entire set of enzymes that change the structure of the cell wall and  allow for vancomycin resistance to occur d. Very different to resistance of beta lactamase antibiotics XXIII. Protein synthesis inhibitors (slide 29) a. ­ If they did target human ribosomes, would inhibit our protein synthesis  XXIV. protein synthesis inhibition (slide 30) a. In terms of the aminoglycosides, there’s a huge problem with both toxicity – it kills kidneys and hearing & it’s only available intravenously  b. Erythromycins – in the family of drugs called the macrolides – very effective XXV. Resistance to protein synthesis inhibitors (slide 31) a. Two ways of resistance  b. Alter ribosomal binding site – so that the configuration that the antibiotics  recognize is changed  c. Or have efficient efflux pumps: recognizes the antibiotic & pumps it out of the cell  at a rate that it is faster then it can bind to its ribosomal target  XXVI. DNA synthesis & maintenance (slide 32) a. Quinolones work by inhibiting 2 specific targets b. DNA gyrase – allows dna to uncoil c. Topoisomerase – which helps split it into daughter molecules XXVII. DNA synthesis (slide 34) a. DNA synthesis inhibitors b. In bacteria, a bacteria can go from the precursor PABA to dihydrofolic acid  c. Humans don’t synthesize folic acid – bacteria do d. The enzyme dihydropteroate synthase is inhibited by the class of antibiotics  SULFONAMIDES e. Humans convert dihydrofolic acid to tetrahydrofolic acid f. Antibacterial agent or anticancer chemotherapy XXVIII. Sulfonamides a. Allergic reactions Lecture 7 – Sterilization ** These are notes and key points to which she highlighted that are important. Things that are  bolded and/or underlined are key points she repeated, meaning they’re probably going to  be on the exam and are important. Terminology Sterilization- total destruction of all microbes Disinfection – the destruction and removal of most pathogenic microbes from inanimate objects. All microbes are not removed – remaining are nonpathogens and spores of pathogens. A disinfectant is the chemical agent used. Bacteriocidal – an agent that kills bacteria Bacteriostatic – an agent that inhibits bacterial growth Antiseptic – chemicals with low human toxicity that destroy microorganisms capable of causing contamination or disease – no harm to host and apply topically Factors Affecting Sterilization/Disinfection Action 1. time of exposure 2. temperature – most of the time used at room temp; if increase temp, you’re going to have direct proportional increase in lethality*** 3. pH – variable, dependent on disinfectant 4. number of microorganisms 5. types of microorganisms – multiple layers of lipids are hard to kill 6. presence of extraneous matter – is there pus, feces, blood, soil 7. proper exposure 8. concentration of disinfectant or sterilant – usually if you increase concentration of disinfectant, you’re going to decrease killing time (EXCEPTION: ethyl alcohol – most effective at 70% concentration as topical disinfectant, if greater than 90% you’re going to decrease effectiveness) Methods of Physical Control I. Heat – high temperatures inactivate proteins and nucleic acids by breaking their hydrogen bonds – denature the proteins, degrade tertiary structure – unfolding of proteins II. Radiation – causes chemical changes to the nitrogenous bases of nucleic acids **heat & radiation both alter the protein makeup** III. Filtration – physical removal of organisms from the solution that is filtered – exclude or entrap microorganism I. Heat - most widely used method for microbial control****** - heat sterilization parameters – temperature, duration and humidity – determine effectiveness of heat - decimal reduction time or D value – measures on organism’s heat resistance (what you’re looking for) – **D value is when 90% of the microorganisms are killed** Sterility Tests – heat procedures 1. Heat sensitive tape – tape impregnated with chemicals that change color when exposed to a critical temperature – tape turns brownish where dye was impregnated on the tape A. LIMITING FACTOR: looking at only one of the parameters – we know that it reached the appropriate temperature B. In a hospital for accreditation, they would not use this type of heat sensitive tape – they would use a SPORE STRIP 2. Biological monitor – spore strips of Clostridium or Bacillus – the bacterium that is used and impregnated on the filter strip is BACILLUS STEROTHERMOPHILUS – must run once per week in a hospital A. Human pathogens i. Mesophiles – most bacteria are mesophiles; they infect the host; the host is a middle lover 37 degrees C ii. Psychrophiles – are bacteria that prefer to grow in a cold environment iii. Thermophiles – are bacteria that grow in a higher temperature range Heat Treatment Procedures A. dry heat B. moist heat C. boiling water D. pasteurization flash pasteurization Dry heat – generated by an oven/bunsenseburner condition - 160C, two hour – kills spores – SPORICIDAL******* usage – sterilization of laboratory glassware disadvantage – high temperature, chars organic materials Moist Heat – generated by an autoclave (industrial pressure cooker) – more efficient transfer of heat than dry heat 2 condition - 121C, 15 minutes, 15 lb/in – kills spores – HIGH TEMP/HIGH PRESSURE = burst the bacteria usage – sterilization of microbiological media, glassware and commercial canning disadvantage – expensive equipment Boiling water – water bath or chamber – not going to kill spores condition - 100C for 30 minutes – kills all non-spore forming microorganisms usage – hospitals – disinfect bedding/clothing of patients home – boil unsafe drinking water, disinfect baby items (food preparation and utensils) disadvantage – items easily recontaminated when removed from water Pasteurization - process of using mild heat to kill pathogens while preserving the quality and flavor of food – kills most pathogens but doesn’t kill them all Why don’t we want to kill all the pathogens? – if we increase the temperate , you would change the consistency and flavor of the food product When they pasteurize milk, they’re trying to kill the most heat resistant bacteria in milk – coxiella burnetti condition - 62 to 66C, 30 minutes, quick cool Time and temperature conditions required to kill Coxiella burnetti usage – destruction of pathogens in milk, beer and wine – increases shelf life Flash Pasteurization – newer technique – higher temp and shorter minute of cooling condition – 71.7C, 15 seconds, quick cool Bacterial load or burden: acceptable amount of bacteria on food EXAM: bactericidal or sporicidal – killing spores or killing bacteria II. Radiation  U.V. – longer wave length; lower energy – less penetration ability when sterilizing items – limited in what it’s sterilizing or decontaminate  Ionizing – shorter wave length; higher energy – higher penetration capability The emission and propagation of energy through space or through a substance in the form of waves B. Ultraviolet (UV) light (non-ionizing) C. Ionizing radiation A. Ultraviolet light – 100 – 400 nm wavelength Bacteriocidal wavelength – (250-270 nm) DNA absorbs UV light which induces a rearrangement in hydrogen bonding of the DNA strand. These changes result in copying errors which increase the probability of a lethal mutation Germicidal lamp (mercury vapor lamp) – generates radiation at 254 nm usage – disinfection rather than sterilization lamps are placed in air ducts or directly over surface. UV lamps are placed in air supply ducts of operating rooms, food preparation areas, nursing homes and nurseries - sterilization of surfaces & air – bc lower penetration ability effectiveness – reduces the concentration of air-borne microbes by 99%. disadvantage – limited use since UV light is absorbed by solids – damaging to human tissues (skin and eye) – sunburn, wrinkles, cancer and retinal damage B. Ionizing Radiation – shorter wavelength than UV – shorter wavelength: increased energy, energy is absorbed by atoms ions (gain/loss of electrons) Ionization forms primarily free hydroxyl radicals which react with cellular proteins and nucleic acids, inducing chemical alterations that are cidal. *** - inactivates the enzymes or breaks DNA strands usage – 1. sterilizing medical products – drugs, vaccines, medical instruments (especially plastics), syringes, sutures, catheters, surgical gloves and tissues such as bone, skin and heart valves 2. sterilization of meat, fruits and some vegetables advantage – 1. speed 2. high penetration power – ability to penetrate fabrics, plastics, liquids and foods for sterilization disadvantage – radiation exposure **need to know what they’re used for (airducts/surfaces vs. sterilization of heat sensitive products like plastics) IV. Filtration – exclusim & entrapment Sterilization technique for removing microbes, not destroying them. Fluid (air or liquid) is strained through a layer of material with openings (pore sizes) large enough for the fluid to pass through but too small for microorganisms. - viruses (very small, electronmicroscope), bacteria, fungi Filter membranes – cellulose acetate, polycarbonate, variety of plastics (Teflon or nylon) 0.22 m pore size – removes all bacteria 0.025 m pore size – removes all viruses Liquid filtration - used to sterilize liquids that cannot withstand heat – (serum, blood products, vaccines, antibiotics, IV fluids and enzymes) – heat sensitive materials Water purification Air filtration – HEPA (high efficiency particulate air filters) are used in air ducts to critically clean areas HEPA filter – tightly woven fiberglass medium which remove particles as small as 0.3m with 99.9% efficiency – effective in removal of viruses since they’re not alone Usage – HEPA filters are installed in air supply systems to operating rooms, nurseries, intensive care units and biohazard hoods (UV & HEPA filter) ** excluded – too big to filter through, or entrapped bc it’s too big Methods of Chemical Control A. Alcohols – ethyl alcohol, isopropyl alcohol B. Halogens – iodine, chlorine C. Aldehydes – formaldehyde and glutaraldehyde D. Heavy metals – copper, mercury, silver E. Gases – ethylene oxide F. Surface active agents (cationic detergents) – quaternary ammonium compounds QUATS G. Phenols – natural (carbolic acid) – ammonium compounds derivatives (o- phenylphenol and hexachlorophene) H. Oxidizing agents – hydrogen peroxide EXAM:  How do they act: o membrane disruption o Protein denaturation o Enzyme inhibition o DNA alteration  How cidal is it what does it kill? Soluble in water? Low toxicity to host? Low cost? A. Alcohols:  antiseptics for human skin  Low tissue toxicity  Removes oils and lipids on skin & evaporates  Not sporicidal  Bactericidal Ethyl alcohol and Isopropyl alcohol are effective antiseptics when applied as 70-80% aqueous solutions mode of action – alcohols precipitate proteins and solubilize lipids present in the cell walls and cell membranes of bacteria usage – human skin antiseptic effectiveness – kills vegetative cells but are not sporicidal B. Halogens Iodine – effective antiseptic when solubilized in 70% ethyl alcohol (tincture) mode of Action – inactivates proteins and organic molecules by reacting with hydroxyl groups usage - human skin antiseptic, treatment of cuts and abrasions Iodophors – iodines complexed with soap usage – preoperative skin disinfectant advantage – soluble in water and gradually releases iodine disadvantages – not as effective as tincture  Solubilized in soap (iodophor) or alcohol (tincture)  More effective in alcohol  Topical antiseptic (alcohol)  Preoperative wash (soap) Chlorine – Cl 2gas) Cl and H O HCl and HC1O (hypochlorous acid) 2 2 HC1O – strong oxidant mode of action – oxidizes sulfhydral groups of cell proteins Clorox – common household disinfectant contains 5.25% sodium hypochlorite usage – sanitize toilets, dishes, food processing equipment. Common additive to swimming pools. C. Aldehydes Formaldehyde and glutaraldehyde – same mechanism to kill microbes mode of action - both alkylating agents substitute alkyl groups for the H atoms of reactive groups of enzymes, nucleic acids and proteins Formaldehyde – gaseous state usage – fumigant disadvantage – noxious vapors, irritates tissues Formaldehyde solution – formalin - **formaldehyde + water** usage – tissue fixative, inactivator of vaccines, embalming fluid disadvantage – tissue irritant ­ many people disagree using formalin as inactivator of vaccines because: high correlation with autism Glutaraldehyde solution usage – effective cold sterilizing agent – primary use is sterilization of dental equipment disadvantage – irritates tissues, mildly disagreeable odor D. Heavy Metals  All induce a cidal event – kill bacteria Copper, Mercury and Silver – microbicidal mode of action – heavy metals toxicity is due to their ability to combine with active chemical (sulfhydral) groups on proteins Copper sulfate – (CuSO ) 4 usage – algaecide, additive to marine bottom paints – inhibits attachment of mussels and barnacles Mercurials – (mercury containing compounds) usage – additive to ointments and solutions used in the topical treatment of skin infections disadvantage – toxicity, allergic reactions, and neutralization by organic matter - topical antiseptic: mercurochrome – organic mercury bromide to inhibit growth of bacteria & infection – toxic – skin infection Silver nitrate - (AgNO )3 usage – 1% solution of AgNO wa3 used as eye drops for newborn infants to prevent infections by Neisseria gonorrhoeae – don’t use it, tissue irritant – use erthyomycin which is more effective Silver sulfadiazine usage – additive to ointments used to prevent infections in burn patients – very effective - still used E. Gases Ethylene oxide – bactericidal, fungicidal and sporicidal (12 hours, 70C – kills spores) usage – sterilizing agents for plastics – petri dishes, tubes, pipets, syringes and catheters advantage – ability to sterilize at moderate temperatures with no moisture F. Surface Active Agents – (Cationic Detergents)  Detergents & surgery wash Quaternary Ammonium Compounds (Quats) – bacteriocidal for a wide range of vegetative bacteria mode of action – kills cells by disrupting their cytoplasmic membrane usage – disinfect floors, walls and other inanimate objects. Also used for preparing the vagina and other sensitive mucous membrane structures for surgery. advantage – odorless, colorless, tasteless, inexpensive, non-toxic to mammalian tissue, soluble in water, and active in low concentrations disadvantage – readily inactivated by organic or inorganic substances G. Phenols (phenolics – phenol derivatives) - lower toxicity phenolics are effective disinfectants, non-sporicidal  Old disinfecting agent – carbolic acid  Determine effectiveness: action of phenol vs. test compound  Action of phenol: comparative standard used in test laboratories mode of action – denature proteins, and disrupt cell membranes advantage – reasonable cost, compatibility with detergents, resistance to inactivation by organic matter Lysol -79% ethyl alcohol and o-phenylphenol Hexachlorophene – effectively kills staphylococci usage – 1960’s as an antiseptic for bathing newborns disadvantage – absorption through skin linked to brain damage in infants usage today – prescription only 3% hexachlorophene + soap antiseptic skin detergent cleaning product lotion (Hexagerm/pHisoHex) H. Oxidizing Agent Hydrogen Peroxide – bactericidal or sporicidal dependent on concentration: 3-6% kills bacteria, 10-25% kills spores mode of action – oxidizing agent that inactivates essential protein structures usage – a 3% solution is used to cleanse wounds, and disinfect plastic implants, contact lenses and surgical prostheses advantage – non-irritating to tissues


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