Nursing Microbiology - Exam #3

Inhibitors of ATP Synthase

Diarylquinolines - Inhibits mycobacterial growth - Exact mechanisms are unknown but evidence shows interference with ATP synthase and reducing available ATP/energy for cell - Side effects include: Heart arrhythmia - Drug is reserved for serious cases of tuberculosis

Alkylating Agents ***NEED TO KNOW***

- *** A group of strong disinfection chemicals that act by replacing a hydrogen atom within a molecule with an alkyl group, which inactivates enzymes and nucleic acids ***Formaldehyde - Alkylating agent formaldehyde is commonly used in solution at a concentration of 27% as gaseous disinfectant - *** Has ability to kill bacteria, viruses, fungi, and endospores - *** Cross-links proteins and has been used as chemical fixative - Used for storage of tissue specimens and as an embalming fluid and inactivate infectious agents in vaccine preparation - *** Very irritating to living tissues and is carcinogenic ***Glutaraldehyde - Structurally similar to formaldehyde but has *** two reactive aldehyde groups, which makes it act more quickly - Commonly used at 2% solution of sterilization - *** Used to disinfect surfaces and surgical and medical equipment - *** Irritates skin and is not used as antiseptic o-Pthalaldehyde [OPA] - Has two reactive aldehyde groups, but linked by aromatic bridge - Thought to work similarly to glutaraldehyde and formaldehyde, but much less irritating to skin and nasal passages ***Ethylene Oxide - Type of alkylating agent that is used for gaseous sterilization - *** Highly penetrating and can sterilize items within plastic bags such as catheters, disposable items in labs, and other pieces of equipment - *** Form of cold sterilization - *** Carcinogenic and highly explosive ***β-Propionolactone - Different chemical structure than other chemicals discussed - *** Binds to DNA, which inactivates it - *** Clear liquid with strong odor and has ability to kill endospores; Has been used in liquid and vapor form for sterilization of *** medical instruments

Use-Dilution Test ***NEED TO KNOW****

- *** A method of determining the effectiveness of a disinfectant on an inanimate surface - *** Turbidity = bacterial survival - Cylinder of stainless steal is dipped in a culture of the targeted microorganism and then dried - Cylinder is then dipped into solutions of disinfectant at various concentrations for a specified amount of time - Finally, cylinder is transferred to a new test tube containing fresh sterile medium, which is then incubated

Supercritical Fluids ***NEED TO KNOW***

- *** CO2 with gaseous and liquid properties; Sterilizing applications - *** Works by penetrating cells and forming carbonic acid and thereby lowering the cell pH considerably - *** Technique is effective against vegetative cells and is also used in combination with peracetic acid to kill endospores - Can be augmented with increased temperature or by rapid cycles of pressurization and depressurization, which will result in cell lysis - *** Benefits include nonreactive, nontoxic, nonflammable, preserves the object's integrity and is commonly used for treating foods [spices and juices] and medical devices

Inhibitors of Protein Synthesis

Aminoglycosides - Large, polar molecules that bind to 30S subunit of bacterial ribosome and impair "proofreading" ability; Incorrect amino acids are inserted into cytoplasmic membrane - Ex: Streptomycin, gentamicin, and neomycin; All broad spectrum, but can be toxic to kidneys, neurons and ear Tetracyclines - Bind 30S; Block association of tRNA with ribosome - Bacteriostatic - Broad-spectrum - Side effects: Phototoxicity; Permanently discolor developing teeth, and cause damage to liver and kidneys Macrolides - Binds to 50S subunit - Broad spectrum - Bacteriostatic - Block elongation of proteins by inhibiting peptide bond formation between different combinations of amino acids - Ex: Erythromycin [First natural macrolide to be discovered], azithromycin [Fewer side effects] Lincosamides -Binds 50S subunit - Narrow-spectrum - Bacteriostatic - Similar in mode of action to macrolides by binding to 50S subunit and preventing peptide bond formation - Effective against streptococcal and staphycoccal infections Chloramphenicol - Binds 50S ribosome - First broad-spectrum antibiotic approved by FDA; Although it is natural, it is easily synthesized - Targets mitochondria ribosomes which are similar to prokaryotic ribosomes - First antimicrobial to be mass produced - Broad-spectrum - Bacteriostatic - Rarely used now because of serious side effects; Inhibits bone marrow production, cause anemia Oxizolidinones - MOA: Bind to 50s ribosomal unit and interferes with [initiation complex] association of 30s and other factors - Inhibits translation - Broad-spectrum - Bacteriostatic - Ex: Linezolid

Inhibitors of Metabolic Pathways

Antimetabolites - Competitive inhibitors of enzymes to stop certain pathways Sulfonamides - Sulfa Drug - Halts folic acid synthesis and production of pyrimidines and purines - Often used in combination with Trimethoprim - Broad-spectrum - Bacteriostatic [when alone] - Can be toxic because there can be an allergic reactions Trimethoprim - Inhibits later stage of folic acid synthesis - Sulfamethoxazale & Trimethoprim are commonly used in combination to boost effect; Can treat UTIs, ear infections, and bronchitis - When used together, it creates a lethal level of folic acid in bacterial cells - Folic acid is needed for fetus during pregnancy - Bactericidal Isoniazid - Selectively toxic for mycobacteria to block synthesis of mycolic acid - Used in combination to help treat tuberculosis

Antiprotozoan Drugs

Atovaquone - Inhibits electron transport - Malaria, babesiosis, toxoplasmosis Proquanil - Inhibits folic acid synthesis - Synthetic - Used in combination with Atovaquone to treat malaria Metronidazole - Inhibits DNA synthesis; Creates double strand breaks in low oxygen environments, which inhibits DNA replication - Side effects: Development of cancer in humans - Anti-bacterial - Dysentery, Giardia, trichomoniasis Pentamidine - Cleaves DNA within kinetoplasts; binds tRNA - Side effects: Pancreatic dysfunction and liver damage - African sleeping sickness, leishmaniasis Artemisinin - Unclear, but likely damages target cells by ROS - Antimalarial - Side effects: Hallucinations and cardiac issues Quinolines - Interferes with heme detoxification - Malaria and dysentery - Side effects: Hallucinations and cardiac issues

Inhibitors of Cell Wall Synthesis

Beta-Lactams [Class] - Characterized by presence of lactam ring; similar to peptidoglycan subunit - Pencillin, cephalosporin, monobactam, and carbapenem - Blocks cross-linking of peptide chains in new peptidoglycan; Can block it so well because so similar to peptidoglycan; Penicillin binding protein mistakes it - Drugs in this class target peptidoglycan Penicillin - Derivatives from fungi; Mostly good against G+ and some G- - There can be natural penicillin, but when adding groups such as an amino group, it'll change into other beta-lactam groups that are more effective against certain groups Cephalosporins - Similar to penicillin - Resistance to beta-lactamases, which are enzymes that will render a lot of the penicillins ineffective - More effectiveness against G-'s Carbapenems - Broad spectrum - Against G+/G- - Include a lot of semisynthetic drugs Monobactams - Narrow spectrum - Effectiveness for G- only Vancomycin [Belongs to Class of Glycopeptides] - Natural antibiotic from Antenomyces - Structure is different from beta-lactams - Binds to end of peptide chain to block subunits from adding to peptidoglycan backbone - G+ only Bacitracin [Belongs to Class of B. Subtilis] - Blocks activity of specific cell membrane molecules responsible for movement of peptidoglycan precursors - Effective against bacteria on skin - Can be administered orally or muscularly, but can damage kidneys

What are fomites?

Contaminated inanimate objects [Ex: Doorknobs, toys, or towels]

Alternative Patterns of Cell Division ***NEED TO KNOW***

- Binary fission is the most common pattern of cell division in prokaryotes, but not the only one - Asymmetrical division or production of spores in aerial filaments - *** Fragmentation: Commonly observed in Actinomycetes, process where new cells split from parent filament and float away - *** Budding: Process where other species may form a long narrow extension at one pole, tip of extension swells and forms a smaller cell, eventually detaching from parent; Will only create one daughter cell at a time [Most common in yeast and prosthecate bacteria]

Phenolics ***NEED TO KNOW***

- *** Carbon molecule with benzene ring and -OH group - *** Carbonic acid: First used by Joseph Lister for surgical wounds [Not used anymore because it is a skin irritant] - Lysol: Original formulations - Triclosan: Commonly used in hand soaps; Banned by FDA in 2017 - Thymol and eucalyptol occur naturally in plants - Phenolics tend to be stable, persistent on surfaces, and less toxic than phenol alone - Cresols and o-phenylphenol are active ingredients in various formations of Lysol - o-Phenlyphenol was used in agriculture to control bacteria and fungi growth on harvested crops - Bisphenol hexachlorophene [disinfectant] is active ingredient in pHisoHex, which is a topical cleansing detergent used for handwashing in hospitals - pHisoHex used to be used to wash infants but has been discontinued since hexochlorophrene can lead to neurological problems

Bisbiguanides ***NEED TO KNOW***

- *** Cationic [positively charged] known for their antiseptic properties - Chlorhexidine: broad-spectrum activity against yeasts, gram-postiive bactera, and gram-negative bacteria [Exception of Pseudomonas aeruginose]; *** Disrupts cell membranes and is bacteriostatic at lower concentrations and bactericidal at higher concentrations - *** Chlorhexidine is poorly effective against Mycobacterium tuberculosis and nonenveloped viruses - *** Alexidine: Typically used as a surgical scrub; More persistent than iodophors; May also be used for oral rinses

Alcohols ***NEED TO KNOW***

- *** Disinfectants and antiseptics - *** Denaturing proteins, which inhibits cell metabolism, and by disrupting membranes, which leads to cell lysis - Once denatured, proteins may potentially refold if enough water is present in solution - *** 70% aqueous solution and work better than 100%; This is because alcohol coagulate proteins, which prevents effective penetration - Most commonly used alcohols are ethyl alcohol [ethanol] and isopropyl alcohol [isopropanol, rubbing alcohol] - Alcohol tends to be bactericidal and fungicidal, but can be viricidal for enveloped viruses - Inhibit sporulation and germination

Dosage and Route of Administration *** NEED TO KNOW ***

- *** Dosage: Concentration given in a certain time period; Based on mass fro children, standard is 12+ - *** Optimal dosage has high drug efficiency but low adverse effects - *** Oral is easiest for dosage, but if its a skin infection, it might be best to use a cream - Patients with history of liver or kidney dysfunction may experience reduced drug metabolism or clearance from the body, resulting in increased drug levels that may lead to toxicity and make them more prone to side effects - Half-life of drugs can vary between each drug; Drugs with longer lives are more susceptible to side effects - *** Route of administration: Method used to introduce a drug into the body; Important consideration for drug therapy

Controlling Microbial Growth: Desiccation ***NEED TO KNOW***

- *** Drying, also known as desiccation or dehydration, is used to preserve foods such as raisins, prunes, and jerky; might not kill all microbes or endospores - ALL cells require water for survival - *** Lyophilization: Item is rapidly frozen; "snap-frozen"; placed under vacuum; Combines exposure to cold temps and desiccation; Items that have been lyophilized may be placed in room temp - *** Water activity: Water contents of foods and materials; Can be lowered without physical drying, but instead by adding salts or sugars

Biofilms and Human Health ***NEED TO KNOW***

- *** Layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off infection by pathogens - *** Plaque that forms on teeth is biofilm that can lead to dental and periodontal disease - Can form in wounds and cause infection - Pseudomonas aeruginosa colonizes in airways of patients with cystic fibrosis, causing chronic/fatal infections in lungs **** Biofilms often provide resistance to antibiotics: 1. Cells in deep layers of biofilm are metabolically inactive and may be less susceptible to antibiotic 2. EPS may slow diffusion of antibiotics, preventing it from reaching cells in deep layers of biofilm 3. Provide optimal environment for sharing plasmids

Controlling Microbial Growth: Heat ***NEED TO KNOW***

- *** Most common and oldest forms of microbial control - Can kill microbes by altering their membranes and denaturing proteins *** Thermal Death Point [TDP] - Lowest temp at which all microbes are killed in 10-min exposure - Different microorganisms will respond differently with some being more tolerant [Endospore-formers such as C. Botulinum] *** Thermal Death Time [TDT] - *** Length of time needed to kill all microorganisms in a sample at a given temperature - Parameters often describe sterilization procedures that use high heat [autoclaving] - Boiling is one of the oldest methods of moist-heat control of microbes; Less effective at killing endospores, some can survive 20 hours of boiling; Can be less effective at higher altitudes *** Dry-Heat Sterilization - *** Incinceration; Direct application of high heat [Bunsen burner, oven] - Aseptic technique in lab - Dry heat can be applied for long periods of time [at LEAST 2 hours] at 170 degrees celcius *** Moist-Heat Sterilization - *** Typically more effective b/c penetrates cells better - Includes autoclaves

Controlling Microbial Growth: Pasteurization ***NEED TO KNOW***

- *** Not always practical for other applications apart from medical applications b/c it may alter quality of product - Form of microbial control for food that uses heat but does not render the food sterile - ****** Milkborne organisms killed by pasteurization: E. coli, Mycobacterium tuberculosis, Listeria, Camplyobacter jejuni <- KNOW THIS *** Traditional Pasteurization - Kills pathogens and reduces the number of spoilage-causing microbes while maintaining quality of food - First developed by Louis Pasteur for beer and wine spoilage - Today, most commonly used to kill heat-sensitive pathogens in milk, apple juice, and honey. *** High-Temperature Short-Time [HTST] Pasteurization - *** Exposes milk to temp of 72 degrees celsius in 15 seconds - *** Lowers bacterial numbers while preserving quality of the milk; Will eventually spoil; Requires refrigeration *** Ultra-High-Temperature [UHT] Pasteurization - Milk is exposed to a temperature of 138 degrees celsius for 2 seconds or more - *** This type of pasteurized milk can be stored for a long time in sealed containers without being refrigerated - High temp alter proteins in milk, causing slight changes in taste and smell

Drug Interactions *** NEED TO KNOW ***

- Can be synergistic or antagonistic Synergistic - One antimicrobial may not be enough; Two or more may be needed - When put together, it creates a synergistic effect = better efficacy than drug alone [Ex: Trimethoprim + Bactrim] Antagonistic - Two drugs or more are taken together may cause loss of drug activity, decrease therapeutic levels, or increase toxicity [Rifampin + birth control] - Some drugs better absorbed because of low pH in the stomach are negatively impacted if someone takes a lot of antacids

Oxygen Requirements of Microorganisms ***NEED TO KNOW***

- *** O2 is not always needed or tolerated - *** Many environments do not have O2 - *** Obligate [strict] aerobes are bacteria that cannot grow without an abundant supply of oxygen; Represent large fraction of the microbes in the human gut [Micrococcus Luteus] - *** Obligate anaerobes: Killed by oxygen [Bacteroides] - *** Facultative Anaerobes: Can do both [Staphylococcus spp.] - *** Aerotolerant anaerbobes: Do not use oxygen because they usually have fermentative metabolism; Not harmed by the presence of oxygen [Lactobacilli spp. and streptococci, both oral microbiota] - *** Microaerophiles: Bacteria that require minimum level of oxygen for growth [Camplyobacter spp.] - Faculative anaerobes examples: Staphylococci and Enterbacteriaceae; Staphylococci found in gut and upper respiratory tract but sometimes urinary track - *** Optimal oxygen concentration: Ideal concentration of oxygen for a particular microorganism - *** Minimum permissive oxygen concentration: Lowest concentration of oxygen that allows growth - *** Maximum permissive oxygen concentration: Highest tolerated concentration of oxygen for a microorganism How to study oxygen requirements: - *** Anaerobic chamber: Enclosed box where oxygen is removed - *** Anaerobic jar: Most common approach of culture; Include chemical packs that remove oxygen and release CO2 Fluid Thioglycolate Medium [FTM] - Specialized medium used to observe the different requirements for oxygen by growing them - Aerotolerance is determined. by location of growth

Temperature and Microbial Growth

- *** Optimum growth temperature: Grow rates for bacteria are at the highest - *** Minimum growth temperature: Lowest temperature at which the organism can survive a replicate - *** Maximum growth temperature: The highest temperature at which growth occurs - *** Mesophiles: 20-45 degrees; "middle loving"; Normal human and microbiota and pathogens - *** Psychrotrophs: 4-20 degrees; Prefer cooler environments; Responsible for spoilage of refrigerated food - *** Psychrophiles: <0 degrees ;"cold loving"; do not survive at temperature about 20 degrees; Found in deep waters of the oceans; Important decomposer - *** Thermophiles: 50-80 degrees; "heat loving"; Do not multiply at room temperature; In hot springs, geothermal soils, and manmade environments [Thermus aquaticus and Geobacillus] - *** Hyperthermophiles: 80-110 degrees; Extreme high temperatures; some can survive at 121 degrees [Pyrobolus, Pyrodictium, archaea] How cells are affected: - (Cold) Membranes lose their fluidity and are damaged by ice crystal formation - Chemical reactions and diffusion happen slower - Heat denatures proteins and nucleic acids; Increased fluidity impairs metabolic processes - Proteins in psychrophiles are rich in hydrophobic residues, have increased flexibility, and lower number of secondary stabilizing bonds - In macromolecules that are thermophiles and hyperthermophiles have ratio of saturated polyunsaturated lipids increases to limit fluidity in cell membranes; DNA sequence have higher proportions of guanine-cytosine

First Antimicrobial Drugs *** NEED TO KNOW ***

- *** Paul Ehrlich: Scanned 600 arsenic compounds to find cure for syphilis without harming patient; Compound targeted ***Treponema paliidum [Compound 66] - Ehrlich's assistant, Sahachiro Hata found "magic bullet", which was Treponema pallidum, causative agent of syphilis - ***Josef Klarer, Fritz Mietzsch, and Gerhard Domagk discovered antibacterial activity of synthetic dye, prontosil, that could treat streptococcal and staphylococcal in mice - ***Gerhard Domagk was awarded Nobel Prize in Medicine for his work with prontosil and sulfanilamide; Used treatment on daughter - Sulfanilamide is first synthetic drug created - **** Synthetic antimicrobial: drug that is developed from a chemical, not naturally - *** Alexander Fleming: Credited with discovery of penicillin, the first NATURAL antibiotic - *** Dorothy Hodgkin: Determined structure of penicillin - *** Semisynthetic antimicrobial: Chemically modified derivative of a natural antibiotic - *** Selman Waksman: Discovered several antimicrobials, including *** actinomycin [Source of 50% natural antibiotics] ***, streptomycin, and neomycin

Light ***NEED TO KNOW***

- *** Photoautotrophs: Cyanobacteria and green sulfurs - *** Photoheterotrophs: Purple nonsulfurs - Photosynthetically active radiation [PAR]: The portion of the electromagnetic spectrum that is absorbed by these organisms - Lies within visible light of 400 to 700 nanometers - A number of accessory pigments, such as fucoxanthin in brown algae and phycobilins in cyanobacteria, widen the useful range of wavelengths for photosynthesis and compensate for the low light levels available at greater depths of water - Other microorganisms, such as the archaea of the class Halobacteria, use light energy to drive their proton and sodium pumps. - The light is absorbed by a pigment protein complex called bacteriorhodopsin, which is similar to the eye pigment rhodopsin. - Photosynthetic bacteria are present not only in aquatic environments but also in soil and in symbiosis with fungi in lichens.

Measuring Success of Microbial Control ***NEED TO KNOW***

- *** Physical and chemical methods of microbial control are identified by the suffix -cide or -cidal - *** Bactericides kill bacteria - Viricides kill or inactivate viruses - Fungicides kill fungi - Organisms ending with -stat or -static stop the growth of an organism, but do not kill them; Prevent pathogens from multiplying - *** Bacteriostatic treatments inhibit growth of bacteria; Less toxic to humans and is the better choice - Fungistatic treatments inhibit growth of fungi **** Microbial Death Curve - Describes progress and effectiveness of a particular protocol - When exposed to particular microbial control protocol, fixed percentage of microbes within the population will die - Reduction in microorganisms is typically logarithmic - *** Decimal reduction time [DRT] or D-value: The amount of time it takes to. kill 90%. of population Factors That Contribute to Microbial Control - Length of time of exposure; Longer exposure time kills more microbes - Takes longer to kill high-population load - Concentration of disinfecting agent or intensity of exposure - High temperatures and high concentrations of disinfectants kill microbes more quickly

Controlling Microbial Growth: Sonication ***NEED TO KNOW***

- *** The use of high-frequency ultrasound waves to disrupt cell structures - Ultrasound waves cause rapid changes in pressure within intracellular liquid, *** leading to cavitation, which can disrupt cell structures and cause the cell to lyse - *** Cavitation: Formation of bubbles inside cell - Useful in lab to lyse cells and cleaning surgical equipment AND LENS

Effects of pH on Microbial Growth ***NEED TO KNOW***

- *** pH can affect efficacy of macromolecules; Most vulnerable are proteins - *** Environments with pH values below 7.0 are considered acidic, and above 7.0 are considered basic - Hydrogen bonds holding together DNA break up at high pH - *** Optimum growth pH: Most favorable for the growth of an organism - *** Minimum growth pH: Lowest pH value that an organism can tolerate - *** Maximum growth pH: Highest pH value that an organism can tolerate - *** Neutrophiles: Grow optimally pH ~ 7 [Escherichia coli, staphylococci, and Salmonella spp.] - *** Acidophiles: Microorganisms that grow pH < 5.5 [Sulfolobus spp.; Ferroplasma; Lactobacillus]; Can survive in strong acidic environments - *** Alkaphiles: Microorganisms that grow best at pH 8-10.5 [Vibrio cholerae]

In-Use Test ***NEED TO KNOW***

- ***Determines whether disinfectant is contaminated - Swabs taken from objects before and after application of disinfectant or antiseptic - Swabs inoculated into growth medium and incubated - Medium monitored for growth - Accurate determination of proper strength and application procedure for each specific situation - Growth of 5 or more colonies on a plate suggests that viable microbial cells existed in disinfectant solution and that it is contaminated

Phenol Coefficient ***NEED TO KNOW***

- A numerical expression for the effectiveness of a disinfectant relative to that of phenol - *** Phenol Coefficient: of 1.0 means that the chemical agent has about the same level of effectiveness as phenol; Less than 1.0 is less effective than phenol

Antihelminthic Drugs

- Achieving selective toxicity is challenging because helminths and humans are multicellular eukaryotes Mebendazole - Inhibition of microtubule formation - Binding to betubuline compound in helminths which prevent microtubule formation - Leads reduction in their glucose intake - Broad range Ivermectin - Blocks neuronal transmission in invertebrates causing starvation, paralysis, and death - Treat round worms and parasitic insects like lice and mites - Nontoxic to humans Niclosamide - Inhibit ATP formation under anaerobic conditions - Treat intestinal tape worms Praziguantel - Induce influx of CA into the worm which results in paralysis - Tapeworms, liver flukes, and schistosomiasis [blood flukes] - More likely to be used for tapeworms instead of niclosamide

Sustaining Microbial Growth ***NEED TO KNOW***

- Advantageous to maintain cells in the log phase - Ex: Industries that harvest microbial products; Chemostat is used to maintain continuous culture, controlled amount of air is mixed in for aerobic processes, bacterial suspension is removed at same rate as nutrients flow

Detoxification of Reactive Oxygen Species

- Aerobic respiration constantly generates reactive oxygen species [ROS], which must be detoxified - The three main organisms that break down toxic byproducts: superoxide dismutase, peroxidase, and catalase - Reaction 1 are catalyzed by peroxidase: X−(2H+)+H2O2→oxidized-X+2H2O(1)X−(2H+)+H2O2→oxidized-X+2H2O - Reaction 2 catalyzed by superoxide dismutase: 2O2−+2H+→H2O2+O2(2)2O2−+2H+→H2O2+O2 - Reaction 3 catalyzed by catalase: 2H2O2→2H2O+O2(3)2H2O2→2H2O+O2

Great Oxygenation Event or the Oxygen Revolution

- After cyanobacteria started releasing oxygen as a byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did oxygen levels increase in the atmosphere. - Caused a massive extinction - Most organisms could not survive the powerful oxidative properties of reactive oxygen species [ROS], which are highly unstable ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure when in contact

Nutritional Requirements for Bacteria

- All purpose media: Support growth of a large variety of organisms [Tryptic soy broth (TSB)] - Specialized media are used in identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics - *** Enriched media : Contains special growth factors, vitamins, and other essential nutrients to promote growth of fastidious organisms - **** Fastidious organisms: Organisms that cannot make certain nutrients and require them to be added to the medium - *** Chemically defined medium: When the complete chemical composition of a medium is known - *** Complex media: Contains extracts and digests of yeasts, meat, or plants, the precise chemical composition of medium is not known [Nutrient broth, tryptic soy broth, and brain heart infusion] - *** Selective media: Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competitions [MacConkey Agar, contains bile salts and crystal violet] - *** Enrichment cultures: Promote growth of desired organism; Only represents a fraction present - *** Differential media: Make it easy to distinguish colonies of different bacteria by a change in color of the colonies or the medium color

Bacteriostatic Versus Bactericidal *** NEED TO KNOW ***

- Antibacterial drugs can be bacteriostatic or bactericidal Bacteriostatic ***** - Cause reverse inhibition of growth, with bacterial growth restarting after elimination of the drug - Used with patient with strong immune defenses Bactericidal **** - Drugs kill their bacteria - Used when a patient immunocompromised; Life-threatening infections

Use of Antimicrobial Drugs in Ancient Societies *** NEED TO KNOW ***

- Antimicrobial drugs: Target infectious microorganisms - Dating back to * 350 to 550 AD has shown residue of antimicrobial agent tetracycline in high enough quantities to suggest the purposeful fermentation of tetracycline producing Streptomyces * during beer making process - * Healers of many cultures use fungi from moldy bread to treat wounds

Antibiotic Resistance

- Arises from increased selective pressure [evolution] - Selective pressure increased through: 1. Misuse & inappropriate use of antimicrobials 2. Subtherapeutic dosage 3. Patient noncompliance - Microbes can require these resistant genes through horizontal and vertical transfer *****Mechanisms For Resistance - Enzymatic modification or inactivation of drug - Modification of the antimicrobial target - Overproduction of antimicrobial target - Enzymatic bypass of antimicrobial target - Mimicry of antimicrobial target - Prevent of drug penetration or accumulation ***** Mechanisms For Resistance Examples - DRUG MODIFICATION: Beta-laxctamases hydrolyze lactam bond - TARGET MODIFICATION: LPS structure alteration to affect polymyxins; RNA polymerase alteration to affect Rifampin; Happens often - TARGET OVERPRODUCTION: Vancomycin resistance in S. aureus by decreased cross-linkage of peptide chain in cell wall [Increase in targets] - TARGET MIMICRY: M. Tuberculosis produces pentapeptides to mimic DNA and binds to fluoroquinlones - PREVENT ACCUMULATION: Pathogens produce efflux pump to transport drug out of cell and prevent accumulation; Common in G- pathogens

Antifungal Drugs

- Common MOA: Disruption of sterol synthesis and membrane integrity - Fungal sterol is ergosterol Imidazoles - Disrupt ergosterol biosynthesis - Commonly used in medical and agriculture [keep crops from molding] - Treat infections caused by dermatophytes: Ringworm, tinea pedis [athlete's foot], tinea cruris [jock itch] Triazoles - Inhibit ergosterol biosynthesis - Administered orally or intravenously - Systemic yeast infections: Oral thrush, cryptococcal meningitis - Tends to be selectively toxic - Have fewer side effects Allylamines - Inhibit earlier step in ergosterol biosynthesis - Treat dermatophytic skin infections: Athlete's foot, ringworm, jock itch - Most common medicine is Terbinafine [Lamisil] Polyenes - Bind to ergosterol and create pore in membrane - Used as topical treatment for yeast infections Flucytosine - Interferes with DNA replication and protein synthesis - Associated with bone marrow depression Echinocandins - Inhibit Beta(1-3) glucan synthesis - "Penicillin for fungi" Polyoxins & nikkomycins - Inhibit chitin synthesis - Selective toxicity because not in humans Griseofulvin - Interferes with microtubules involved in spindle formation during mitosis - Anti-fungal agent - Disrupts fungal cell division - One of the first anti-fungal agents used - Orally or topical Atovaquone - Antimetabolite for fungal and protozoal mitochondrial cytochrome - Semisynthetic metabolite - Interferes with cytochrome component of the mitochondria in both microbes - Effects electron transport

Antiviral Drugs

- Common MOA: Inhibiting nucleic acid synthesis - Ex: Acyclovir; Specificity: Viral enzyme activation and affinity for viral DNA polymerase Amantadine & Rimantadine - Treatment of influenza A - MOA: Binds to transmembrane proteins - Blocking escape from endospores prevents viral RNA release into host cells - Increasing resistance for influenza - Side effects: Neurological Oseltamivir [Tamiflu] - MOA: Inhibition of neuraminidase that aids in release of viral particles from host cell - Against influenza virus HIV Hard to treat because... 1. Targets CD4+ WBC [Necessary for normal immune response] 2. Retrovirus; Incorporates into host genome [hides] 3. Rapid development of antiviral drug resistance Reverse Transcriptase Inhibitors - Block viral RNA -> DNA Protease Inhibitors - Block processing of viral proteins, preventing viruses from maturing Integrase Inhibitors - Prevents integration of viral DNA into host chromosome so it can no longer hide Fusion Inhibitors - Prevent binding of virus to host cell and merging of envelope and membrane

Biofilms ***NEED TO KNOW***

- Complex and dynamic ecosystems that form on a variety of environmental surfaces - Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a biofilm *** Structure - *** Filamentous biofilms [streamers] form in rapidly flowing water [freshwater streams, eddies, lab flow cells] - *** Streamers are anchored by a "head" and a "tail" that floats downstream in the current - Mushroom like shape in slow moving water - *** Structure can change based on environmental conditions - Extracellular matrix consists of *** extracellular polymeric substances [EPS] secreted by the organisms in the biofilm [takes up 50-90% of organism] - *** EPS is hydrated gel composed primarily of polysaccharides contain proteins, nucleic acids, and lipids; Makes up most of biofilm; Channels allow movement of nutrients, waste, and gases throughout biofilm - *** EPS shelters organisms in biofilm from predation by other microbes or cells *** Formation - Planktonic Cells: Free-floating microbial cells that live in an aquatic environment - Formation of biofilm involves attachment of planktonic cells to a substrate, where they become sessile [attached to surface] *** Steps of Formation: 1. Reversible attachment of planktonic cells [seconds] 2. First colonizers become irreversibly attached [second, minutes] 3. Growth and cell division [hours, days] 4. Production of EPS and formation of water channels [hours, days] 5. Attachment of secondary colonizers and dispersion of microbes to new sites [days, months] - Some microorganisms establish collabs where the waste product of one organism becomes the nutrient for another [polymicrobial infections] - *** Quorum Sensing: Mechanism by which cells in a biofilm coordinate their activities in response to environment; Occurs between cells of different species in biofilm; Cell to cell communication - *** Autoinducers: Microorganisms can detect their cell density through the release and binding of small, diffusible molecules, which are the autoinducers - When the cell population reaches a critical threshold [a quorum], these autoinducers initiate a cascade of reactions that activate genes associated with cellular functions that are beneficial only when the population reaches a critical density. [Ex: Candida albicans]

Other Methods of Control ***NEED TO KNOW***

- Disinfection: Inactivates most microbes on surface of fomites with heat or antimicrobial chemicals - **** Disinfectants: Should be fast acting, stable, easy to prepare, inexpensive, and easy to use; Used to remove vegetative cells, but endospores may remain; Used on nonliving surfaces [Natural disinfectant= vinegar] - *** Antiseptics: Antimicrobial chemicals safe for use on living skin or tissues, unlike disinfectants - ***Critical items: Must be sterile because they will be used inside the body; Surgical instruments, catheters, intravenous fluids - ****Semicritical items: May contact mucous membranes or nonintact skin but do not penetrate tissues; Do not typically need to be sterilized but need to be disinfected [Gastrointestinal endoscopes] - **** Noncritical items: Contact but do not penetrate skin [bed liners, furniture, crutches, stethoscopes, and blood pressure cuffs]; Need to be cleaned but not highly disinfected - *** Degerming: The act of handwashing; Microbial numbers are significantly reduced by gently scrubbing living tissue, most likely skin - *** Sanitization: Refers to cleansing of fomites to remove enough microbes to achieve levels that are safe for public health [Dishwashers]

"When studying these drugs, know the following..."

- Drug class - MOA (What's targeted) - Broad/narrow spectrum - Which pathogens are targeted

- END OF CHAPTER 13 -

- END OF CHAPTER 13 -

- END OF CHAPTER 9 -

- END OF CHAPTER 9 -

Disk-Diffusion Method ***NEED TO KNOW***

- Evaluates efficacy of chemical agents - *** Measured degree of inhibition - Filter paper disks are soaked in a chemical and placed on a culture - ***As "lawn" of bacteria grows, look for zone of inhibition around disks [clear areas around disk]

Controlling Microbial Growth: Pressure ***NEED TO KNOW***

- Exposure to high pressure kills many microbes - *** Pascalization: High pressure; Is used to kill bacteria, yeast, molds, parasites, and viruses in foods; Like a pressure cooker - *** Usually within 100 to 800 MPa is enough to kill vegetative cells by protein denaturation, but endospores may survive these pressures - In *** hyperbaric therapy, patient breathes pure oxygen at pressure higher than normal so it can treat infections; Helps increase oxygen saturation - *** Increased oxygen levels contribute to formation of toxic free radicals, which can inhibit growth of Clostridium perfringens, a common gas gangrene - *** Hyperbaric oxygen therapy can reduce secretion of bacterial toxin that causes tissue destruction; Rare risks include oxygen toxicity and delicate tissue [eyes, middle ear, and lungs]

The Growth Curve *** NEED TO KNOW ***

- Graph of the number of organisms in growing population over time with no added nutrients and in which waste is not removed - *** Culture density: # of cells per unit volume; In closed environments, culture density is a measure of number of cells in population - Has 4 stages: Lag phase, Log phase, Stationary phase, and Death or decline phase *** Lag Phase - *** Cells are gearing up for the next phase of growth; Number of cells do not change, but they become larger and metabolically active - Synthesize proteins needed to grow within medium - Beginning of growth curve - Represents small number of cells referred to as inoculum, which are added to fresh culture medium - *** Duration of phase is based on genetic make-up, composition of medium, and size of inoculum; Damaged cells might have to go through repair *** Log Phase - *** Cells are actively dividing by binary fission and their number increases exponentially - *** Using up a lot of energy - Referred to as the exponential growth phase - *** Generation time under specific growth conditions is called intrinsic growth rate - Cells show constant growth and uniform metabolic activity - *** Stage where bacteria are most susceptible to disinfectants and antibiotics *** Stationary Phase - *** Waste accumulates and nutrients is gradually used up; Gradual depletion of oxygen begins to limit aerobic cell growth, stalling population growth - The number of new cells created by cell division is now equivalent to the number of cells dying - Culture density in stationary culture is constant - ***. Cells switch to survival mode of metabolism; as growth slows, synthesis of peptidogylcans, proteins, and nucleic acids begins, making this stage less susceptible to antibiotics - *** Undergo sporulation *** Death Phase - Cells die in greater numbers due to the culture medium accumulating toxic waste and nutrients being exhausted - Referred to as the decline phase - *** Persisters: Cells with slow metabolic rate; Antibiotic resistance [Ex: Tuberculosis]

Surfactants ***NEED TO KNOW***

- Group of chemical compounds that lower surface tension of water; Helps lower microbial load - Major ingredients in soaps and detergents ***Soaps - *** Salts of long-chain fatty acids and have polar and nonpolar regions, allowing them to interact with polar and nonpolar regions in other molecules - *** Can interact with nonpolar oils and grease to create emulsions in water, which loosens and lifts away dirt and microbes from surfaces and skin - *** Soap does NOT kill or inhibit microbial growth and are not considered antiseptics or disinfectants - Some soaps add bacteriostatic agents such as triclocarbon/cloflucarban, compounds structurally related to triclosan, that introduce antiseptic or disinfectant properties to the soaps ***Detergents - *** Contain synthetic surfactant molecules with both polar and nonpolar regions that have strong cleansing activity but are more soluble - *** Anionic detergents: Such as the ones for laundry, have negatively charged anion at one end attached to long hydrophobic chain - *** Cationinc detergents: Are apart of a class of disinfectants and antiseptics called *** quaternary ammonium salts [quats], named for the characteristic quaternary nitrogen atom that confers the positive charge - *** Quaternary ammonium salts: Stable, nontoxic, inexpensive, colorless, odorless, and tasteless; endospores unaffected

Halogens

- Halogens iodine, chlorine, and fluorine *** Iodine - *** Works by oxidizing cellular components, including sulfur-containing amino acids, nucleotides, and fatty acids, then destabilizes macromolecules that contain these molecules - *** Iodophor: Compound of iodine complexed with an organic molecule, which increased iodine's stability and efficacy; Usually used right before surgery to disinfect skin - Common iodophor is povidine-iodine, which includes a wetting agent that releases iodine relatively slowly - Betadine is brand of povidine-iodine commonly used for hand scrub by medical personnel before surgery and patient's skin before surgery ***Chlorine - Halogen commonly used for disinfectant - *** When chlorine gas is mixed with water, it produces strong oxidant called hypochlorous acid, which is uncharged and enters cells easily - Chlorine gas is commonly used for municipal drinking water and wastewater treatment plants - *** Sodium hypochlorite is chemical compound of common household bleach; Can be used for swimming pools - Chlorine gas, sodium hypochlorite, and salts [sodium and calcium hypochlorites] are commonly used as disinfectants in food processing and restaurant industries to reduce spread of foodborne diseases - *** Chloramines: Widely used as disinfectants; Are relatively stable, releasing chlorine over long periods of time; Can be used for disinfection of drinking water - Sodium dichloroisocyanurate may also be a good alternative for disinfecting drinking water - Chlorine dioxide can also be used for water and fumigation and sterilization of enclosed areas - Some chlorine products can irritate skin, nose, eyes - Protozoan Cryptosporidium has protective outer shell that makes it resistant to chlorinated disinfectants *** Fluorine - *** Prevention of dental cavities - *** Main active ingredient in toothpaste

Chemical Food Preservatives ***NEED TO KNOW***

- Inhibit microbial growth and minimize food spoilage - Sorbic acid, benzoic acid, and propionic acid, potassium sorbate, sodium benzoate, and calcium propionate, which all are used to control the growth of mold in acidic food - Each preservative is nontoxic and readily metabolized by humans, they are also flavorless and do not compromise flavor of food ***Sorbic and Benzoic Acids - Increased efficacy as the pH decreases - ***Sorbic acid is thought to work by inhibiting various cellular enzymes, including those in citric acid cycle, catalases, and peroxidases - *** Increased efficacy as pH decreases - ***Added preservative in wide variety of foods [Dairy, bread, fruit, and vegetable products] - *** Benzoic acid is found naturally in many types of fruits and berries, spices, and fermented products - *** Preserves fruit juices, jams, ice cream, pastries, soft drinks, chewing gum, and pickles ***Propionic Acid - *** Inhibit enzymes and decreased intracellular pH, similar to benzoic acid - *** More effective at preserving at higher pH than either sorbic or benzoic acid - *** Naturally produced by cheeses and added to other types of cheeses and baked goods to prevent mold contamination ***Sulfur Dioxide - *** Prevents browning of foods and used for preservation of dried fruits; been useful in winemaking - *** Gas dissolves in water readily, forming sulfites, which metabolized by body, can sometimes create allergic reactions ***Nitrites - *** React with iron-sulfur groups which disrupts ETC - *** Added to process meats to maintain color and stop the germination of Clostridium botulinum endospores - *** Reduced to nitric oxide, which reacts with heme groups to create meat's red color

Controlling Microbial Growth: Filtration ***NEED TO KNOW***

- Method of physically separating microbes from samples - High-efficiency particulate air filters [HEPA]: Have effective pore sizes of 0.3 µm, small enough to capture bacterial cells, endospores, and many viruses - HEPA filters are used in clinical settings, cars and airplanes, and in homes [Vacuum cleaners, heating and air-conditioning, air purifiers] *** Membrane Filters - *** Filtration that can be used to remove microbes from liquid samples - Function similarly to HEPA filters for air - *** Used to remove bacteria that have effective pore size of 0.2 µm, which is smaller than average bacterium - Used for removing bacteria from various types of heat-sensitive solutions used in lab [antibiotic solutions and vitamin solutions] Biological Safety Cabinets - Use of HEPA filters - Used to remove particulates in the air either entering cabinet or leaving cabinet - Prevents environmental contaminants from entering cabinet - Three classes of BSCs - Class I BSCs: Protect lab workers and environments from low to moderate risk of exposure to biological agents used in lab; Air is draw into cabinet and then filtered before exiting building - Class II BSCs: Use directional air flow and partial barrier systems to contain infectious agents - Class III BSCs: Working with highly infectious agents like in BSL-4 labs; Gas tight and materials entering and exiting must be passed through double-door system - HEPA also used in hospitals to prevent contamination and spread of airborne microbes

Sterilization ***NEED TO KNOW***

- Most extreme protocols for microbial control aim to achieve sterilization: complete removal or killing of all vegetative cells, endospores, and viruses - Sterilization is usually reserved for lab, medical, manufacturing, and food industry settings - Sterilization can happen through physical or filtration - Sterilants: Chemicals that can be used to achieve sterilization - *** Aseptic technique: Prevent contamination of sterile surfaces; Combination of protocols that maintain sterility [asespsis]; Failure to follow through may cause a patient sepsis [systemic inflammatory response to an infection that results in high fever, increase heart and respiratory rates, shock, and possible death] - Medical procedures that carry risk of contamination must be performed in sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses - *** Commercial sterilization: Food protocol; Uses heat at temp low enough to preserve food but high enough to destroy common pathogens responsible for food poisoning [C. botulinum]; Targets pathogens that cause food spoilage

Osmotic and Barometric Pressure ***NEED TO KNOW***

- Most natural environments tend to have lower solute concentrations than the cytoplasm of most microorganisms - Plasmolysis: The shrinking of the protoplasm away from the intact cell wall; This occurs when there is high osmotic pressure which flows out of the cell; *** Not a lot of protection against high osmotic pressure - *** Halophiles: "salt loving" which require high salt concentrations for growth; Found in oceans - Halobacterium grows in hypersaline lakes such as the Great Salt Lake and is an extreme halophilic microorganism - *** Halotolerant: Do not need high concentrations of salt for growth, will survive and divide in presence of salt; Are important cause of food-borne illnesses because they survive in salty food [S. aureus] - S. aureus, Bacillus cereus, and V. cholerae produce dangerous entertoxins and are major causes of food poisoning - Available moisture is measure as water activity [aw], which is ratio of vapor pressure of medium of interest to the vapor pressure of pure distilled water - *** Barophiles: Microorganisms that require high atmospheric pressure for growth; [Bacteria that live at the bottom of the ocean]

Controlling Microbial Growth: Refrigeration and Freezing ***NEED TO KNOW***

- Not used for sterilization - *** Exposing microbes to low temperatures can be an easy and effective method of microbial control, with the exception of psychrophiles, which prefer cold temperatures - *** Refrigerators used in home kitchens or labs usually have temperature of 0 degrees celsius to 7; helps preserve food in fridge - *** Freezing below -2 degrees celsius may stop microbial growth and even kill susceptible organisms - Only safe way frozen food can be thawed is if its immersed in cold water every 30 minutes - Bacterial cultures and medical specimens require long-term storage at ultra low temps such as -70 degrees celsius or lower; Can be achieved by storing specimen with dry ice

Measurement of Bacterial Growth

- Number of bacteria in sample serves as an indication of the extent of an infection - Ex: Quality control of drinking water, food, medication, cosmetics - Two major approaches are used to measure cells: Direct methods involve counting cells; Indirect methods depend on measurement of cell presence or activity without counting each individual *** Direct Cell Count - Counting of cells in a liquid culture or colonies on a plate -*** Direct microscopic cell count: Involves transferring a known volume of culture to a calibrated slide and counting cells under light microscope - *** Petroff-Hausser chamber: Direct microscopic cell count. Calibrated slide; Similar to hemocytomater used to count red blood cells; *** Can't distinguish live vs. dead cells - Cells in several small squares must be counted and the average taken to obtain a reliable measurement; Not always accurate - *** Fluorescence staining techniques make it possible to distinguish viable and dead bacteria - *** Primary stain [green] can penetrate cytoplasmic membranes, staining both live and dead cells - ***Secondary stain [red] can stain cell only if cytoplasmic membrane is damaged - **** Electronic cell counting device detects and counts changes in electrical resistant in saline solution; *** Does not differentiate between live and dead cells Plate Count - *** Viable plate counts: Count of viable or living cells; Samples are diluted and grown on solid media - Viable cells replicate and give rise to visible colonies when incubated - *** Results expressed through colony-forming units per milimeter because more than one cell may have landed on the same spot - *** Considered a low estimate of actual number of cells b/c some cells are nonculturable but still viable - Count plates usually between *** 30-300 colonies Indirect Cell Counts - *** Measurement of turbidity: Cloudiness of a sample of bacteria in liquid suspension; Light passed through culture and measured on other side; *** Does not determine between live and dead cells; Population increase = turbidity increase - Spectrophotometer is used to measure turbidity - As number of bacteria in suspension increases, the turbidity also increased and causes less light to reach the detector; This is associated with decrease in percent transmission and increase in absorbance - Measuring dry weight of a culture sample is another indirect way - Faster and easier methods measure cell activity, metabolic products, disappearance of reactants, ATP formation, biosynthesis of protein/nucleic acids, and consumption of oxygen

Controlling Microbial Growth: Autoclaves [Heat Sterilization] ***NEED TO KNOW***

- Rely on moist-heat sterilization - *** Are used to raise temperatures about boiling point of water to sterilize items [Surgical equipment, viruses, and endospores] - Is still considered the MOST effective method of sterilization - Increasing amounts of steam trapped within enclosed chamber, which results in increasing interior pressure and temps above boiling point of water - Standard temps for autoclaves is 121 degrees celsius and in some cases 132 - Pressure of 15 to 20 pounds per square inch - Length of exposure is usually 20 min or more - Steam must directly be in contact with items - The two types of autoclaves: Gravity displacement autoclaves and high speed prevacuum sterilizer *** Gravity Displacement Autoclaves - *** Steam is introduced into chamber from top or sides - Air [heavier than steam] sinks to bottom of chamber where it is forced out through vent - Unwrapped glassware *** Prevacuum Sterilizers - *** Air is removed completely from using high speed vacuum before introducing steam into chamber - Air is more eliminated, meaning steam can easily penetrate wrapped items - Packaged instruments ****Heat-Sensitive Autoclave Tape - Internal indicator - *** Has white stripes that turn black when appropriate temperature is achieved - Relatively inexpensive and can be used during every run - Provides no indication of length of exposure, so cannot be used as indicator for sterility ****Biological Indicator Spore Test - Internal indicator - *** Uses either a strip of paper or a liquid suspension of endospores Geobacillus stearothermophilus to determine whether endospores are killed by process - Endospores of Geobacillus stearothermophilus are gold standard used for this purpose because of extreme heat resistance - Biological spore indicators can be used to test effectiveness of sterilization including ethlyene oxide, dry heat, formaldehyde, gamma radiation, and hydrogen peroxide plasma sterilization using G. stearothermophilus, Bacillus atrophaeus, B. subtilis, or B. pumilus spores ****Diack Tube - Third indicator - *** Glass ampule containing temperature-sensitive pellet that melts at the proper sterilization temperature - Spore strips or Diack tubes are using periodically to make sure autoclave is functioning properly

Antibiotic Mechanisms of Action [MOA]

- Selective Toxicity: Inhibiting/killing microbe but not causing harm to host - Can be harder with viruses, fungi, and parasites

Heavy Metals ***NEED TO KNOW***

- Some of the first chemical disinfectants - Kill microbes by binding to proteins, which results in enzymatic activity - Heavy metals are oligodynamic, meaning that very small concentrations show significant antimicrobial activity - Ions of heavy metals bind to sulfur-containing amino acids strongly and bioaccumulate within cells, which allows metals to reach high localized concentrations, leading to proteins to denature - Not selectively toxic to microbial cells - *** Excessive amount in humans can have toxic effects [Too much silver accumulation in body= can result in condition called argyria, which is skin turning irreversibly blue-gray] ***Mercury - *** Heavy metal used to control microbial growth - *** Used for many centuries to treat syphilis - Mercuric chloride [mercury compound] are bacteriostatic and have broad spectrum of activity - Various forms of mercury bind to sulfur-containing amino acids within proteins; Use of compounds has come to a halt because of mercury's toxicity - *** Mercury is toxic to central nervous, digestive, and renal systems at high concentrations - Has negative effect on environment such as bioaccumulation in fish - Topical antiseptics such as mercurochrome, methiolate, and tincture were commonly used but no longer are ***Silver - Drinking water used to be stored in silver jugs - *** Silvadene cream is used to treat topical wounds and helps in preventing infections in burn wounds - *** Silver nitrate drops are used to to apply on newborns eyes to protect from opthalmia neonatorum [eye infections that occur b/c of pathogens in birth canal] - *** Silver often combined with antibiotics, making it more effective - Also used in catheters and bandages ***Copper, Nickel, and Zinc - *** Copper sulfate is common algicide used to control algal growth in swimming pools and fish tanks - Copper linings in incubators help reduce contamination - Use of copper pots for water storage in underdeveloped countries - *** On doorknobs, cabinet hardware, and health-care facilities in attempt to reduce microbes - Zinc chloride is used in mouthwashes - *** Zinc oxide is used in topical antiseptic creams such as calamine lotion, diaper ointments, baby powder, and dandruff shampoo

Most Probable Number ***NEED TO KNOW***

- Statistical procedure for estimating of the number of viable microorganisms in a sample - Used for water and food samples; Evaluates detectable growth by observing changes in turbidity or color due to metabolic activity *** Most probable number: Statistical method used when counts are very low; Used in water and food testing - Typical application is estimation of number of coliforms in sample of pond water; Gram-negative; Coliforms show sign of contamination by fecal matter

Peroxygens ***NEED TO KNOW***

- Strong oxidizing agents which can be used for antiseptics and disinfectants ***Hydrogen Peroxide - ***Most widely known - ***Is used to disinfect surfaces and can be used as gaseous agent - *** Inexpensive skin antiseptics that break down water and oxygen gas, both which are environmentally safe; *** Can be accelerated by light - *** Disadvantage of using hydrogen peroxide is that it can damage skin and delay healing/scarring ***Peracetic Acid - More effective than h2o - Can be used as liquid of plasma - Readily kills endospores; immune to inactivation by catalase and peroxidases ***Benzoyl Peroxide - Present in most acne medications - Very effective against Propionibacterium acnes ***Carbamide Peroxide - Agent in toothpaste that combats biofilms - Don't pose environmental hazard ***Ozone Gas - Used to clean air and water supply Plasma - Hot, ionized gas that is described as the fourth state of matter - Useful for sterilizing equipment because it penetrates surfaces and kills vegetative cells and endospores - More effective than hydrogen peroxide - Examples include: Benzoyl peroxide and carbamide peroxide

Serial Dilution and Plating Methods ***NEED TO KNOW***

- The goal of serial dilution is to obtain plates with CFUs in range of 30-300, involving several dilutions in multiples of 10 - 1 mL is added and mixed with first dilution, containing 9.0 mL [1:10]; This process is then repeated multiple times - Sample is plated with pour plate method or spread plate method *** Pour Plate Method 1. Bacterial sample mixed with warm agar [45-50 degrees] 2. Sample poured onto sterile plate 3. Sample swirled to mix, allowed to solidify 4. Plate incubated until. bacterial colonies grow *** Spread Plate Method 1. Sample [0.1 mL] poured onto solid medium 2. Spread sample evenly over the surface 3. Plate incubated until bacterial colonies grow on the surface of the medium

Laboratory Biological Safety Levels

- There are 4 classification levels called "biological safety levels" [BSLs] - Each level requires a different level of biocontainment to prevent contamination and spread of infectious agents *** BSL-1 - Requires fewest precautions - Include noninfectious bacteria, such as nonpathogenic strains Escherichia coli and Bacillus subtilis - Must wear lab coat, goggles, and gloves if needed *** BSL-2 - Pose moderate risk to lab workers and the community - Include bacteria such as Staphylococcus aureus and Salmonella spp., and viruses such as hepatitis, mumps, and measles - Have restricted areas unlike BSL-1 - Require PPE, including face shield, use of biological safety cabinets - Self-closing doors, eyewash station, an autoclave [special device for sterilizing materials with pressurized steam] *** BSL-3 - Potential to cause lethal infections by inhalation - May be exotic [from foreign location] - Include pathogens such as Mycobacterium tuberculosis, Bacillus anthracis, West Nile virus, and HIV - Restricted areas; Lab workers are under medical surveillance, receiving vaccinations for the microbes they work with - Must wear respirator and work at the cabinet at all times - Requires hands-free sink, eyewash station, two sets of self closing and locking doors, directional airflow - Air cannot be recirculated *** BSL-4 - Most dangerous and fatal; Usually exotic and easily transmitted by inhalation, causing infections for which there are no treatments - Includes Ebola virus and Marburg virus - Lab workers must change their clothes upon entering lab, wear a full-body protective suit, or conduct all work within a biological safety cabinet with HEPA [high-efficiency particulate air] - Suit must have higher air pressure than outside the suit

Generation Time for Eukaryotic Organisms ***NEED TO KNOW***

- Time between the same points of the life cycle in two successive generations [Ex: Typical generation time for human population is 25 years] - Not practical for bacteria - *** Generation time is also referred to as doubling time; Defined as the time it takes for population to double through one round of binary fission - *** Escherichia coli can double in as little as 20 minutes under optimal growth conditions; In harsh environments, it may take several days; Can overlap Calculating population size *** KNOW FORMULA AND COMPONENTS *** - Formula: Nn = No2^n **** - Nn = Number of cells at generation n - n = Number of generations - No = initial number of cells

Controlling Microbial Growth: Radiation ***NEED TO KNOW***

Ionizing Radiation - *** Includes X-rays, gamma rays, and high-energy electron beams - *** Strong enough to pass into the cell, altering molecular structures and cell components [Ex: Introduces double-strand breaks in DNA molecule, causing mutation] - *** X-rays and gamma rays penetrate paper and plastic and can be used to sterilize many packaged materials - *** Gamma irradiation for food preservation; Packaged dried spices; Cannot penetrate thick layers of iron or lead Nonionizing Radiation - Commonly used for disinfection and uses less energy than ionizing radiation - *** Does not penetrate cells or packaging - *** UV light causes thymine dimers to form between adjacent thymines within a single strand of DNA; Leads to mutations and ultimately, death - UV lamps are incorporated into water purification systems - Germicidal lamps are used for surgical suites, safety cabinets, and transfer hoods, emitting UV light at 260 nm

Testing for Efficacy

Kirby-Bauer Disk Difusion Test - Examine degree of susceptibility/resistance - Zone of inhibition: Area of antibacterial activity around drug-impregnated disk - Factors that can change zone of inhibition: What type of drug was used, how it diffuses through agar, the agar itself, how much of drug used - Cannot show bacteriostatic or -cidal Dilution Tests - Determine potency - Minimal inhibitory concentration [MIC]: Lowest concentration of drug that inhibits visible bacterial growth - Minimal bactericidal concentration [MBC]: Lowest drug concentration that kills less than or equal to 99.9% - Etest: Combo of Kirby-Bauer assay and dilution method; Strip with antibacterial gradient placed on agar plate

Inhibitors of Nucleic Acid Synthesis

Metronidazole - Interferes with DNA replication - Not selective toxicity; Targets anaerobic bacteria AND protozoa - Broad spectrum - Semisynthetic - Bactericidal Rifampin - Blocks RNA polymerase activity; Can treat semi-dormant M. Tuberculosis, but can be antagonistic and hepatotoxic - Can be toxic to liver - Narrow-spectrum [Mainly G+, some G-] - Bactericidal Fluoriquinolones - Inhibit DNA gyrase enzyme; Effects the enzyme that helps uncoil the super coils of DNA - Selective toxicity [G+ and G-] - Broad-spectrum - Bactericidal - Usually prescribed for UTIs, respiratory infections, skin infections - Side effects: Phototoxicity, neurotoxicity, toxic to heart, glucose, metabolism, and increased risk for tendon rupture

Special Resistance in Microbes

Multidrug Resistant Microbe - "Superbugs" - 2 million infections per year, 23k deaths - Cross-resistance: One mechanism confers resistance to multiple drugs - ESKAPE pathogens [Enterococcus faecium, Staphylcoccus aureas, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginose, Enterobacter ssp.] - Difficult to treat and cause large # of nosocomial infections Vancomycin - Only effective against G+ - Treat wound and septic infections, endocarditis, and meningitis caused by pathogens resistant to other antibiotics - Last line of defense [including MRSA] - Excessive use in 1970s and 80s - Vancomycin-resistant enterococci [VRE]: Target modification of peptide component in cell wall; prevent binding - Vancomycin-resistant S. aureus [VRSA]: Horizontal gene transfer from patients infected with VRE and MRSA - Vancomycin-intermediate S. aureus [VISA]: Increased in targets, binding to outer cell wall Methicilin-resistant S. aureus [MRSA] - Acquisition of new low-affinity PBP; Resistance to all beta-lactams - Prevents binding - Problem in healthcare settings Extended-spectrum Beta-lactamases [ESBLs] - Resistance to pencillins, cephalosporins, monobactams, beta-lactamase-inhibitors, but NOT carbapenems - Found on plasmids that have other drug resistances Carbapenem-resistant Enterobacteriaceae [CRE] - Produce carbapenemases [beta-lactamases that inactivate all beta-lactams] - Efflux pumps and uptake prevention - Some have developed pan-resistance [resistances to all antibacterials] Multidrug-Resistant Mycobacterium tuberculosis [MDR-TB] - Resistant to both rifampin and isoniazid Extensively drug-resistant Mycobacterium tuberculosis [MDR-TB] - Additionally resistant to any fluorquinolone and at least 1 of 3 others

Spectrum of Activity *** NEED TO KNOW ***

Narrow-Spectrum Antimicrobial *** - Targets only specific subsets of bacterial pathogens - Ex: Some only target gram-positive bacteria - Preferred *** Broad-Spectrum Antimicrobial *** - *** Targets wide variety of bacterial pathogens, including gram positive and negative species - *** Used as empiric therapy to cover a wide range of potential pathogens while waiting on the lab identification of infecting pathogen - *** Also used for polymicrobic infections [mixed infections with multiple bacterial species] - May be selected to treat an infection when narrow-spectrum does not work - Downside is that a super infection could erupt; Could knock out the good microbiota too ***

Inhibitors of Membrane Function

Polymyxins - Lipophilic [detergent like properties] & interact with LPS to disrupt outer and inner membrane of G- bacteria - Target host membranes - Bactericidal - Are not selectively toxic, meaning it can target cells in the nervous system - Polymyxin B has only been used in over the counter antibiotics [Neosporin] Daptomycin - Cyclic lipopeptide that inserts and disrupts G+ membrane

Binary Fission ***NEED TO KNOW***

Process 4 basic steps: **** 1. Growth of cell size and increases all cell components 2. Replication of DNA 3. Division of the cytoplasm [cytokinesis] 4. Septum formation and division of daughter cells - Cell grows and increases its number of cellular components - Replication of DNA starts at a location on the circular chromosome [origin of replication], where chromosome is attached to inner cell membrane - Replication continues in opposite directions along chromosome until terminus is reached - Center of enlarged cell constricts until two daughter cells form, each receiving a copy of the parental genome and cytoplasm division [cytokinesis] - Daughter cells separated by division septum, where all cell's outer layers [cell wall, outer membranes] must be remodeled to complete division Z Ring Assembly **** - Cytokinesis is directed by protein [FtsZ] which then assembles into a Z ring on cytoplasmic membrane - Required for all prokaryotes - Additional proteins for cell division are added to the Z ring to form a structure [divisome] - Divisome activates to produce peptidoglycan cell wall and build a septum that divides daughter cellls