Micro Extra Credit 2

Microbes talking to each other, get the article for extra credit folder here in the news.

Lecture 20, 10/29 (Dr. Maybruck 5); Microbial Drug Resistance

Audio for lecture 20 on October 29th.

• • Slide 1 Chemotherapy for viral infections (Handout from 10/26)
• Selective toxicity is difficult to achieve with viruses
• Antiviral drugs target points in infectious cycle of viruses
• • Preventing virus entrance into cell
• • Preventing viral replication (duplication of DNA), transcription (synthesis of RNA) and translation (synthesis of proteins).
• Viruses can use our own proteins to make their DNA.
• With our drugs we are trying to prevent the life cycle of the virus. Try to keep the virus from entering the cell.
• • Slide 2
• Different drugs involved in inhibiting viral replication
• • Viral thymine kinase → turns precursor guanine into guanine
• • Acyclovir (“false” guanine) – mimics the precursor guanine. Once it is added to the DNA it prevents extra nucleotides from joining to the strands
• • Azidothymidine (AZT) – mimics thymine. Reverse transcriptase grabs AZT nucleotide and binds it to the Adenine.
• • Nevirapine – non nucelotide reverse transcriptase inhibitor. It bonds to the reverse transcriptase and prevents its function.
• • Slide 3 drug resistance
• Methocillin resistant staphylococcus aureus (MRSA) – developed resistance (“acquired resistance”) to Beta-Lactam drugs. Common hospital infection.
• Why do they have this resistance
• • Some have an Intrinsic resistance – ex. organisms that are responsible for creating the antibiotic.
• Acquired resistance can be looked at two ways:
• • →SPONTANEOUS BENEFICIAL MUTATION
• • Gene is a specific sequence of nucleotides that is going to code for a protein that protein then produces a certain trait.
• • Mutation – a change in the specific nucleotide sequence that will be passed on to the next generation
• 3 types of mutations: lethal, neutral and beneficial.
• Lethal mutation ex. Hexokinase – involved in first step of glucolosis. If it were tainted than glycolosis could not happen resulting in death.
• Neutral – a mutation that results in neither beneficial or lethal. Ex eye color
• Beneficial mutation – increasing that populations ability to reproduce and survive.
• • Point mutation – a change in a few nucleotides
• • Natural selection – picking a trait that benefits the population and passing it on
• • Directional selection – an example of natural selection. This is associated with antibiotic resistance. Environmental pressures cause the organism to begin selecting a trait in the population that will be passed on from one generation to the next. Penicillin is an example of an environmental pressure.
• • →HORIZONTAL GENE TRANSFER
• Conjugation
• • Two microorganisms (of different species) will share information (make a copy of the plasmid) by joining up through conjugation
• • Slide 4 specific mechanisms of drug resistance
• Enzymes are made to inactivate drug
• Beta lactamases – example of an enzyme produced by bacteria that will inactivate the effects of penicillin and cephalosporins. This is one way that MRSA functions (it produces beta lactamase)
• • Slide 5 specific mechanisms of drug resistance
• Impermeability of cell to drug
• Gram negative bacteria
• Active transport pump uses energy to pump out the drug
• • Slide 6 specific mechanisms of drug resistance
• Producing an alteration in the target of the drug.
• Erythromycin
• • Prevents movement of mRNA through the ribosome
• Fungi don’t produce binding substrate at all
• Penicillin binding protein (PBP) – a protein that is used by bacteria to build their peptidoglycan cell walls. This protein readily binds to penicillin and is not a builder of the cell wall. MRSA will alter the attaching sight and the penicillin binding protein will continue to build the wall.
• Alteration of metabolic pathway
• • sulfinimide
  
• changes way the dihydroteric acid is formed
  

Lecture 19, 10/26 (Maybruck 4): Chemotheraputic Drugs

Audio for lecture on 10/26 available here.

• • Slide 1 Antimicrobial target: nucleic acids(handout from 10/4)
• o naladixic acid – inhibits function of DNA gyrase. DNA gyrase unwinds DNA and allows for efficient storage of the chromosome. It also allows for DNA synthesis ot occur
• • slide 2 Antimicrobial target: Protein synthesis
• o protein synthesis involves four primary parts: two ribosomal subunits (50s and 30s → unique to prokaryotes), tRNA and mRNA.
• o Aminoglycosides – attach to 30s ribosomal subunit which causes the tRNA to misread the mRNA. In turn the protein is incorrectly formed and is nonfunctioning.
• o Tetracycline – attaches to 50s ribosomal subunit and prevents tRNA from binding to that sight. (tRNA is bringing amino acids over to ribosomal subunit to create a protein). Considered a semisynthetic drug.
• o Chloramphenicol – prevents bonding of amino acids together. Prevents peptide bond.
• o erithromyacin – binds to 50s portion of subunit and prevents mRNA from sliding through the ribosome. This is where tRNA reads the mRNA and figures out what to bind where and form protein. Because mRNA can’t slide through – protein isn’t made.
• o NOTE: Cells need to be metabolically active in order for the above antibiotics to function
• • Slide 3 Antimicrobial target: Cell membrane
• o These two drugs can work on a cell that is not metabolically active.
• o Polymyxin (broad spectrum) – targets cell membrane. Acts as a surfactant, integrates into cell → disrupts it → cell dies.
• o Chloroquine – little bit more selective then polymyxin. An anti-malarial drug. The causative agent of malaria is the protozoan plasmodium.
• • Life cycle of plasmodium – mosquitoe → to human → to liver → attack red blood cells. Plasmodium brings nutrients (hemoglobin) into food vacuole.
• • Uses enzymes to break down majority of hemoglobin. Doesn’t like the iron cofactor “heme”. Instead it converts iron into hemozoin (Hz). Chloroquine inturupts this function. Plasmodium will be unable to detoxify “heme” and the cell becomes toxified.
• • Slide 4 Antimicrobial target: Folic acid synthesis
• o Folic acid synthesis is unique to bacteria and protozoans.
• o Folic acid is a precursor of DNA RNA and proteins. We get our folic acid from food we consume. Bacteria and protozoans make it for themselves.
• o We prevent folic acid synthesis in a bacteria/protozoans with competitive inhibition of an enzyme.
• o 2 examples:
• • Hexokinase enzyme is responsible for taking glucose, binding it to phosphate and forms glucose 6 phosphate → initial reaction in glycolysis. Take an enzyme that is similar to glucose and present it in a greater concentration. This will take the place of the actual glucose and in turn prevent the process from occurring.
• • Folic acid synthesis forms a dihydroteric acid (which is only formed using Para-aminobenzoic acid (PABA)). PABA binds to pteridine synthetase and forms precurusor to folic acid. We create a “pseudo”PABA that outcompetes the actual PABA. Drugs used are sulfonomides and trimethoprim.
• • Slide 5 antibacterial drug groups: penicillin and related
• o Penicillin (penicillium chrysogenum) – prevents synthesis of the peptidoglycan cell wall.
• o Semi–synthetic antibiotic: what we would be prescribed. It is the modified version of the real thing. Causes it to become broad spectrum
• o Alpha – hemolyses: when bacteria reduces iron or the “heme” group. Cause pnemonia
• o Beta – hemolyses: involved in rupturing red blood cells. Cause strep throat.
• • Slide 6
• o Cephalosporin – Cephalosporium acremonium (similar to penicillin but induces fewer allergic reactions) – removes enteric bacteria (bacteria that colonize intestines) Ex. E. coli.
• o Salmonella is another example → prevents water absorption.
• • Slide 7 animoglycoside tetracycline, and chlorampheniccol
• o See slide 2
• • Antimicrobial Chemotherapy part II
• • Slide 1
• o Fungi are eukaryotic making their removal “tricky” because their functions at a molecular level are similar to ours.
• o Target ergosterol which is in the cell membrane of fungi cells. Ergosterol is similar to cholesterol. Function of ergosterol is to support the cell.
• o Macrolide polyene antibiotics target the ergosterol.
• o It is used to attack Histoplasmosis (fungal infection that causes respiratory problems)
• o Griseofulvin – targets cyctoskeleton of fungi cells. In particular it attacks the microtubules. This keeps the cell from forming properly. Drug is used to treat fungi infections in the hair skin or nails.
• • Slide 2
• o Azoles – prevents ergosterol synthesis. Fights off Cryptococcus meningitis. Meningitis affects the meninges in the spine.
• o Flucytosine
• • Fungi use this flucytosine analog to build the DNA strand. This in turn prevents DNA synthesis.
• • Used to combat candidal cystits (a yeast infection associated with bladder)
• Slide 3 drugs specific to the removal of protozoans
• o Metronidazole – removes anaerobic microorganisms
• • Upon administration to the individual the drug becomes inactive – a prodrug. In turn the protozoans injest this and then it become active in the protozoans. Once in the protozoan it interferes with DNA structure.
• o Entomeaba histolytica amoeba - Fecal contamination is a source of this protozoa. Protozoan in the cyst form is resistant to stomach acid. It then progresses to the large intestine and turns into trophozoite form. In this form it inhibits water absorbtion by the large intestine.

Micro Extra Credit

For those of you who may be behind in your 100 article extra credit thing, here is an interesting article I found today. (I will be posting links to more articles as I find them)

French Dirt May Kill MRSA

Researchers said that a type of French clay can kill several disease-causing bacteria, including M. ulcerans, which is known as a "flesh-eating" bacteria, and the so-called super bug, MRSA.

Lecture 18, 10/24 (Maybruck 3); Control of Microbial Growth

Audio for today's lecture is available here.

• • Slide 1 (handout from 10/22)
• Alcohols characterized as a hydrocarbon group linked to one or more hydroxel groups
• Alcohol has hydrophobic an hydrophilic end. The hydrophilic end is the reactive end - it actively bonds to other molecules.
• Surfactants enter into phospholipids bylayer of a cell membrane. This screws up selectively permeable layer of the cell membrane preventing the cell from functioning properly.
• Alcohol has the surfactant ability at 50% or greater concentration of alcohol.
• Also involved with the denaturing of proteins. If there is low water present then the alcohol will be less effective.
• Viruses with cell envelope are susceptible to alcohol
• • Slide 2
• Oxygen radicals – their metabolism requires oxygen to form energy. The oxygen used sometimes transforms into these oxygen radicals. Highly reactive compounds – create chemical bonds with whatever they can . . . RNA, protein, DNA
• Hydrogen peroxide is an example of oxygen radical
• At 3% can be an effective antiseptic
• At 35% - sterilant
• Oxygen radicals in general are good as a chemical decontaminant for anaerobic organisms.
• • Slide 3
• Surfactants (similar to detergents) – hydrophilic end has an overall positive charge. This is effective as most of our cells have an overall negative charge. This allows the bonding of the surfactant to the cell membrane.
• Soaps - mechanical removal of debris
• • Slide 4
• Main effect of heavy metals is the denaturing of proteins (causes them to unfold) if that metal is not typically associated with that protein.
• Heavy metal compounds
• Mercury, silver, gold, copper, arsenic, zinc
• Cofactor – metal ion that helps enzyme function
• Intermediate activity level
• Anaerobic organisms are susceptible to hydrogen peroxide
• • Slide 5 (he skipped a few)
• Aldehydes –
• Molecules that contain CHO group
• Two types: glutaraldehyde and formaldehyde
• Sugars are characteristic of an aldehyde
• Glutaraldehyde
• Cross link proteins – makes it more toxic. Binds quickly to surface proteins and such. It will also make its way into the cell itself. This messes up function of protein.
• Effective way to kill EVERYTHING. Kills vegetative cells in a few minutes and formaldehyde in a few hours.
• Effective preservitive because it does not lyce the cells. This allows the structure of the organism to be preserved.
• Formaldehyde
• Binds to proteins and nucleic acids
• Effective preservative because it does not lyce the cells. This allows the structure of the organism to be preserved.
• • Slide 1 (handout 10/24/07) Antimicrobial Chemotherapy – using drugs to destroy or remove microorganisms
• What is the perfect drug? (FYI: none exist, so we comprimise)
• Easily administered
• Upon administration it goes straight to the problem spot
• Easily excreted from the body
• Selectively toxic – goes after the bad stuff and not the good stuff
• • Slide 2
• Chemotherapy
• Prophylaxis – drug used to prevent the infection from occurring. (administered to office workers during anthrax scares)
• Antibiotics – chemicals released from microorganisms and fungi. They destroy and kill other microorganisms and fungi.
• Narrow spectrum antibiotic – specific in its target. Narrow range of microorganisms that it can eliminate.
• Broad spectrum antimicrobial – destroys wider range of cells
• • Slide 3
• Primary goal of antimicrobial chemotherapy – creating a drug effective in its removal of microorganisms.
• Microbocidal effect – kill unwanted microorganism
• Selectively toxic
• How is this goal achieved
• Make list of characteristics that allows it (pathogen) to survive
• make list of characteristics that allows our cells to survive
• compare theses two lists and determine the difference.
• Various things that you can target
• inhibit cell wall synthesis
• inhibit nucleic acid synthesis and structure
• inhibit protein syntheses
• alter cell membrane structure
• inhibit folic acid synthesis
• • slide 4: antimicrobial target: cell wall
• cell walls (peptidoglycan) protect cell from osmotic shock. The cell would lyce if it were not protected.
• subslide (drugs that target cell wall synthesis)
• cycloserine – inhibits formation of basic subunits of peptidoglycan.
• vancomycin – prevents elongation of peptidoglycan
• penicillin and cephalosporins – target the peptide bond between glycan sugars. Prevent cross linking of glycan molecules
• those drugs that target the cell wall are narrow in spectrum. This is because the cell wall they are attacking is found in gram (+) bacteria.
• In order for these things to work the organism has to be vegetative. A vegetative organism is always adding to the peptidoglycan layer, if it is not producing peptidoglycan then the drugs are useless (they also have no effect on endospores)
• • slide 5 targeting nucleic acids
• In the context of bacterial microorganisms this is a broad spectrum approach.
• Replication – DNA synthesis
• transcription – formation of RNA
• antibiotic Rifampin – blocks transcription. Binds to RNA polymerase and prevents the RNA polymerase from transcribing.
• Hydroxyurea – prevents formation of nucleotides (found in DNA and RNA: guanine, cytosine, thymine, adenine). It does this by binding to Ribonucleotide reductase.
• • Slide 6
• Mitomycin blocks DNA synthesis → cross linking guanines preventing DNA
• ATGGTCAG
• TACCAGTC

Lecture 17, (Maybruck 2) 10/22; Control of Microbial Growth (cont.)

Audio for lecture.

• • Slide 1 (slide labeled number 12 on the handout from 10/17)
• Sterilization with steam under pressure, this is the only way it can get up to 121ºC
• • Slide 2
• pasteurization does not remove endospore forming bacteria
• removes all vegetative forms of bacteria
• juice, beer, wine, milk are all pasteurized
• two methods used to pasteurize
• • flash method –
• • batch method – exposes microorganisms between 63-66º C for a longer exposure time of 30minutes
• two infections
• • salmonelosis – upon ingestion it will coat the lining of the large intestine (large intestine absorbs extra water) and prevents absorption of extra water.
• • brucellosis – mucus covers are GI tract which aids movement of food. It also traps bacteria and brings it to the stomach in order for it to be destroyed. Brucella escapes this mucis and penetrates to the blood vessels and has a grand ol’ time.
• • Slide 3
• Cold has primarily microbistatic effects
• Food inadequately cooked after being thawed may harbor pathogens
• • Slide 4
• Ionizing radiation (loss or gain of electrons)
• • As the wavelengths decrease the energy associated with that wave increases (enough to break chemical bonds)
• • Ionization causes atoms to lose their electrons and causes them to not be bound to one another anymore.
• • Ionizing radiation can be considered a Sterilant as a removal of endospores
• • Types of radiation that are effective as sterilants are: some UV, x-rays, and gamma rays. (they all attack DNA)
• Ionization radiation acts indirectly and forms oxygen radicals which are very reactive. They are always seeking to make chemical bonds.
• Radiation helps to remove contaminants and prolong shelf life.
• • Slide 5
• Sterilization through filtration (used only on liquid or air)
• Bacteria are .2um – 2um
• Protozoa and algae are 2um – 200um
• Create filter with a pore size less than .2um
• Filtration = sterile
• • Slide 1 (handout from 10/22/07)
• Desirable qualities of chemical antimicrobial agents for decontamination in health professions
• • Rapid action at low concentrations
• • Soluble in water or alcohol
• • Destruction of MOs without harm to animal tissue
• • Penetrates surfaces
• • Resistance to inactivation by organic matter
• • Noncorrosive or nonstaining properties
• • Affordable
• • Slide 2 (list found in book)
• High activity – acts as sterilant
• Intermediate activity – acts as disinfectant
• Low activity – acts as disinfectant to an antiseptic.
• • Slide 3
• How to choose an effective chemical antimicrobial agent
• • Look at characteristics – ex. Are they spore formers or not?
• • Characteristics of surface being treated – ex. Is there organic matter that could interfere. Is the surface porous or nonporous.
• • Initial contamination amount
• • Antimicrobial exposure time
• • Strength of antimicrobial chemical
• • Slide 4
• Halogens (group 17) – non-metals that are readily ionized. They love to gain electrons. When they gain that electron they become halides (the ionized state of a halogen). If iodine is ionized it becomes iodide. The non-ionized state is the more effective antimicrobial state.
• Chlorine is an effective antimicrobial agent.
• • When mixed with water it creates hypochlorous acid which reacts with cystine amino acids (breaks up disulfide bridge)
• • Cystine amino acids have a side chain group that contains sulfur. That sulfur will bind with other cystine amino acids. When they bind they form a covalently bonded disulfide bridge.
• Insulin helps cells take up glucose. Glucose is a carbohydrate we use for energy. Break the disulfide bridge, break up the cell.
• Iodine (another example of halogen) interferes in disulfide bridges and hydrogen bonds.
• Act at intermediate activity level
• • Slide 5
• Phenol – 6 carbon sugar - hydroxel group (OH group)
• Phenolic – any molecule that contains one or more phenol groups
• • Bind to proteins and interferes with their function. They make proteins hydrophobic. Hydrophobic – don’t like water.
• Chlorohexidine is rendered useless by toothpaste as it creates a surfactant
• You can adjust disinfectants by altering their concentrations to turn them into antiseptics.
• • Slide 6
• Alcohols – contain hydrocarbon group with one or more hydroxal groups
• Alcohols act as surfactant. Integrate themselves into cell membrane because they have similar characteristics of the phospholipids. Once there they destabilize membrane and allow things in and out of the cell that shouldn’t be ther.
• Alcohols also Denature proteins
• Decontamination characteristics of alcohol will be effective at >50% concentration.
• • Most effective at 70% alcohol and 30% water
• • It needs water to make it effective. If all the water is removed the proteins will remain stable.
• Alcohol is generally considered as a disinfectant. can be used as an antiseptic but can have adverse affects if it is absorbed through the skin.

Lecture 16, 10/17 (Maybruck 1); Control of Microbial Growth

Dr. Maybruck has slide handouts outlining the lecture.
The audio cut off with about 15-20 minutes left in the lecture. But the first part can be heard here.

• SLIDE 1
• Microbial regulation of organisms that are pathogenic
• Ancient civilizations filtered water and preserved the dead using salts and oils that microorganisms don’t like.
• Epidemic – wide spread disease in a community
• We are interested in regulating pathogens that can cause harm to human health.
• Romans figured out that burning dead bodies kept disease down. Also storage of water in copper and silver kept microbe population down.
• • Slide 2
• Decontamination methods – methods employed that will remove or destroy microorganisms on a surface (including water)
• Physical decontamination method: temp extremes and radiation. mechanical methods: filtration
• Results: sterilization or disinfection (removes all vegetative microorganism)
• Chemical methods
• liquids gases and solids
• results of method: sterilization (kills living and non-living (non-living → like endospores) organisms), disinfection (removes all vegetative microorganism) can only be applied to nonliving surfaces , antisepsis (this can be applied to our skin as it will not kill everything) is usually targeted toward specific microorganisms.
• aqueous chemical decontaminant. Mix of solid or gas with water. If you mix it with a alcohol it is called tincture.
• • Slide 3
• Bacterial endospores (very resistant)
• Two phase life cycle: vegetative (metabolically active and growing) and endospore (keeps them alive a long time and helps them survive in extreme conditions)
• Examples: Bacillus, clostridum, and thermoactinomyces
• How effective is this method of survival? → very effective, these organisms can be considered essentially immortal.
• Layers of sediment is a varve which is an annually deposited layer of sediment. Microorganims will live in these layers and then be able to come back to life once they get the appropriate nutrients.
• • Slide 4
• Antimicrobial agents (physical, mechanical, chemical) fall into two categories:
• Microbicidal agents [“cide”=to kill]
• Bacteriocide, fungacide, virucide and sporicide (could be considered a sterilant)
• Microbistatic agents [-static or –stasis=to prevent growth]
• Used on living tissue, gives our body enough time to get the immune system working
• Bacteriostatic, and fungastatic
• • Slide 5
• The mode of action of antimicrobial agents. More specific=less effective --- less specific=more effective
• Cell wall target – gram positive . . . specific.
• Cell membrane target – this is a less specific way of going about it = more effective.
• Detergents (cell membrane target) - called surfactant. A surfactant is a molecule with hydrophilic and hydrophobic ends. Phospholipids bylayer in cell membrane have a hydrophilic and hydrophobic end as well. The surfactants make their way into the phospholipids bylayer and destabilizes the cell.
• • Slide 6
• The mode of action of antimicrobial agents
• Nucleic acid and protein synthesis prevention
• UV radiation – targets pyrimidine nucleic acids (RNA, DNA). Has greatest effect on DNA. Pyrimidines includes cytosine and thymine. UV radiation “loves” these two nucleotides. For the radiation to occur the pyrimidine bases HAVE to be next to each other. Once hit with UV radiation the thymines will bond to each other – called thymine dimer. This prevents replication which brings about the death of the cell.
• Antibiotic binds to ribosome. Example: Chloramphenicol (an antibiotic) binds to ribosomes in such a way that protein synthesis is inhibited. TRNA cant add amino acids to growing protein strand. Doesn’t inhibit growth of protozoans and fungus.
• • Slide 7
• The mode of action of antimicrobial agents
• Hexokinase helps attach glucose and phosphate
• Alteration in protein conformation
• If pH is altered protein will unfold – called denaturing
• • Slide 8
• Temperature as controller
• Two physical states of heat used
• Moist heat – ex. heat created from boiling water/steam. Keeps some organisms from stabilizing, this is the best way to do mass sterilization.
• Dry heat – ex. Flames used to disinfect loops in lab.
• • Slide 9
• C. botulinum – interferes with nerve connection for muscle contraction
• Practical concerns: thermal death time (TDT)
• Food canning process
• Prevents microbial contamination including spore forming C. botulinum: botulism
• TDT for low-acid foods is 121C for 30 minutes
• • Slide 10
• Sterilization with seen under pressure
• At sea level pressure 15psi will boil water at 100C
• To kill all MOs, pressure at 30 psi and 121C (standard autoclave conditions)
• As pressure is increased temperature is increased

Lecture 15, 10/10; Respiration, Fermentation, DNA replication, etc.

Here is the recording for today's lecture.

• Wednesday Oct 17 dr. maybrook comes in to teach
-Read the chapters – maybrook will follow syllabus
• Submit lecture paper in a week
• Big paper mon nov 19
-Outline
-Abstract
-1 figure (labeled at bottom)
-1 table (labeled at top)
• he will probably return after thanksgiving

This is what I got out of the lecture from 10/10. I would suggest listening to the lecture and checking the book/handouts because there were several parts that I was shady on.

• • Discussing glycolisis (handout 10/5)
• Glucose C6H12O6. Aerobes and anaerobes have to use glycoliysis to get to pyruvate. It is an anaerobic function, happens in the cytoplasm. What does it do? Uses two ATP’s to make energy. Once you get to a diphosphate you will split it into things you can use like reducing power, NADH. Produce four ATP → net 2. This is the two that anaerobic organisms get.
• Glycolosis C6 glucose going to two pyruvates (pyruvates are the central hub of the wheel).
• Krebs cycle (needs oxygen) – takes two C3’s (pyruvate) and breaks it down to 6 CO2’s. It deals with carbon break down. For energetics (p229 in text) you have to have GTP in the same triphosphate. That is why they convert GTP to ATP. Each “turn of the wheel” you get a GTP that is equal to an ATP. We “turn the wheel” twice (two pyruvates). Gives us lots of reducing power: NADH (4 per turn), FADH2 (1 per turn). This reducing power proceeds to the electron transport system.
• E. coli gets 38 ATP’s we get 36.
• • Understand Respiration
• • C6H12O6 + CO2 + 6 Oxygens = 6CO2 + 6H2O (comes off at the end of the cytochrome chain) + Energy (ATP)
• If the organism has oxygen and is an aerobe or a facaltative organism it will do these things for their energy. Both anaerobes and facaltative first get two ATP’s at the glycolosis pathway/substrate level phosphorilation. We get most of our energy from oxidative phosphorilation.
• • fermentation – anaerobic metabolism of sugars (handout 10/5 page 4)
• glucose → pyruvate is glycolosis. All fermentations involve glucose to pyruvate and then pyruvate to acids alcholos and gases.
• yeast or alcoholic fermentation – saccharomyces cerevisiae
• bacterial fermentationts
• • homolactic fermentation – glucose goes to pyruvate and then pyruvate goes to lactic acid. Sour milk organisms → streptococcus lactis (makes sour cream). All streps do this type of fermentation.
• • heterolactic fermentation – produces lactic acid, ethyl alcohol and CO2. Ex. lactobacillus brevis (found around the nipple). Newborns poop goes white to yellow and smells like acid due to this organism. Leuconostoc mesenteroides
• • clostridial fermentation – butyric acid (smells terrible, part of puke).
• • propionic acid fermentation – propionibacterium shermanii. Associated with swiss cheese. The taste, and smell is propionic acid.
• • mixed acid fermentation – Escherichia coli and Enterobacter (found on plants) aergones both do mixed acid fermentation.
• natural fermentation
• • silage – matabiosis=sequential growth. One group dies and then the next starts growing.
• • sourkraut - metabiosis
• chop up cabbage and add 2.5% sodium chloride
• [(?not sure if I got this right?) mixed acid → heterolactic → lactobacillus plantarum → acidic cabbage. ?]
• • protein degradation (p3 of handout 10/5)
• large proteins (gelatin, kasin(sp?)) broken down by extracellular enzymes. Organism produces a protease – if it breaks down gelatin it is a gelintinase and if it breaks down kasin(sp?) it is a kasinase(sp?). B. sub has strong extracellular enzymes that we even use them in detergents.
• After you break the big proteins to amino acids there are organisms that break down individual amino acids like tryptophan (broken down by tryptophanase). Indole smells – rotting meat. – organisms that break down original amino acids like tryptopha.
• Sulfar Containing amino acids → h25. Proteus vulgaris
• decay (aerobic) decomposition of proteins is how sewage treatment works. Putrefaction - anaerobic decomposition of proteins.
• • Applied food microbiology (p5 handout 10/5
• bread
• wine and beer
• vinegar – acetobacter acetii
• cheese – molds ripen cheese (penicilium).
• Curds from producing lactic acid. Organisms responsible: streptococcus lactis and streptococcus cremoris.
• Yogurt
• Soy sauce – double fermentation
• • FUEL
• zymomonas mobilis – produces twice as much ethonal as yeast
• • enzymes – usually come from bacillus or molds
• protease – helps break down proteins
• amylase
• we are currently using penicillium chrysogenum to produce penicillin. It is then altered to get different types of penicillin.
• • P7 of handout from 10/5
• kefir – lightly alchoholic fermented milk drink
• San Francisco sourdough – tastes sour because of the lactic acid. Lactobacillus sanfrancisco
• • last page of handout from 10/5
• bacitracin – made by particular strain of B. sub
• citric acid – used for soda made by Aspergillus and Candida.
• • DNA replication (p6 of handout from 10/5)
• Inside E. coli DNA is coiled over itself many times, this is called supercoiling. To get it uncoiled we use a gyrase, it undoes supercoiling. Once it is straightened out you have got to unzip the DNA which is done by helicase. Make an RNA first by a primase and then start adding on to the RNA primer. Then you take the RNA primer out with polymerase I. With the new strands of DNA you put them together with DNA ligase.
• • Material for exam 2 ends here. -------------------
  

Bioethics Lecture 10/10/07

Bioethics lecture with Professor Gilbert Meilaender. Anderson Chapel, North Park University. October 10, 2007.

Here is the audio of the lecture.

I am not sure if I have spelled his name correctly. If anyone has a different spelling (that they know is correct) then please post it as a comment. (I got my spelling of his name from a google search)

Lecture 14, 10/8; Enzymes

Here is the lecture recording for 10/8.

• Exam2 Monday

• • Aerobes – require air. Pseudomonas aeruginosa, micrococcus luteus, molds.
• • Microaerophiles – require a tiny bit of air
• o Campylobacter jejunii – causes gastrointestinal upset
• • Faciltative can go either way
• o No air – fermentation
• o Air – respiration
• o Ex. E. coli - Faciltative anaerobe, great advantage
• • Anaerobe – absence of air
• o Clostridium. Ex. Tetnus
• • Extra CO2 – capneic
• o Neisseria gonorrhoeae
• • pH requirements – most do well at 7 but some can grow all the way to 14 to lower than 1
• o low pH organism thiobacillus thiooxidans and alcaligenes
• • extrememly high salt means they are an obligate high level halophile - archea’s, halobacteria. 15-20% salt. Low level halophiles living at 3.5-4% salt concentration.
• • halo tolerant organisms – staphylococcus aereus (grows on MSA and causes toxic shock)
• • anaerobiosis (growth in the absence of oxygen) discovered by Pasteur
• • Methods
• o Mixed culture – obligate anaerobe and aerobe together. When the aerobe uses up the oxygen then the anaerobe will grow.
• o overlay media – method that keeps oxygen from diffusing in.
• • chemical removal of oxygen: - gas pak apparatus, biobag allows for a little bit of oxygen “campy pouch”
• • pigments in bacteria
• o intracellular – serratia rudigiensia “prodigiosin”
• o extracellular – Pseudomonas aeroginosa “pyocynan” – blue green. Very resistant to antimicrobials. Pseudomonas fluorescent “fluorascenin” yellow green. Both are typically water organisms.
• • Growth handout (handed out 10/1)
• o 0-4 hours – lag phase or adjustment phase. It is metabolically getting ready to grow. If starch is there it will probably try to produce amylase
• o 4-24 hours - log phase or exponential phase – organisms are the healthiest – they are undergoing binary fission. Time it takes for one cell to become two is called “doubling time”.
• o 24-36 hours – plateau or stationary phase, start to die. Runs out of essential nutrients.
• o Death phase – die at logarithmic rate. Most organisms are usually dead 2-3 days after. Except for those organisms that sporilate (hard to kill spore formers).
• o Archea’s tend to grow faster due to high temperatures.
• o specific times for organisms (back sheet of handout from 10/1)
• • Bacillus Cerius
• • E. coli
• • streps are slower because they only have one type of metabolism – fermentation
• • Mycobacterium tuberculosis – very slow. Need to grow it in Lowenstein jenston. Look for it in about a month
• • Treponema pallidum – grow rabbit testes
• • Mycobacterium leprea – grown in armidillo
• • Saccharomyces cervisiae
• o Evaluating growth
• • Viable count – 30-300colonies is a good count. No more (too numerous) no less (to few to count).
• • Direct count - Hep B is a more common blood bourne illness than AIDS
• • Turbidity – as organisms grow they turn the broth turbid. More turbidity more organisms.
• • Chapter 8
• o Metabolism (all the reactions in the cell) composed of two parts (see handout 10/5)
• • Catabolism – break down of complex to simple. Starch to glucose to CO2. All metabolic reactions are mediated by enzymes.
• • Anabolism – taking something simple (like amino acids) and putting them together into proteins. (needs energy – coupled to catabolism)
• • Nucleotides – used to build up DNA, RNA
• o Pasteur and fermentation – anaerobic
• o Metabolism in a nutshell (handout 10/5) =30:00min in recording
  
• • Produce a Protease – breaks down giant proteins into amino acids.
• • Some organisms require CO2 (autotrophs)
• • Pyruvate – most common intermediate in metabolism. We break down sugar to pyruvate with oxygen. Kreb’s cycle. Then the electrons go to the electron transport chain and energy is produced.
• o Fermentation (without oxygen): classical fermentation – alcohols, acids, gases
• Enzymes (handout 10/5) – biocatalysts of the cell
• • Enzyme + substrate = enzyme substrate complex. It then disacociates to enzyme + product and the enzyme goes back and reacts with another substrate. Usually end in “ase”. Exceptions pepsin, lysozyme, etc.
• • Enzymes have high specificity for the substrate ex. Amylase only acts on starch. They also have temperature and pH optima.
• • Either consititive or indusive
• Constitiuve – always present. Enzymes that are found in common pathways.
• Indusive – only present when substrate is present. You would want indusive because you waste less energy.
• We have a mix of both. Commonly used would be consitiutive, if you don’t see the substrate very often it would be indusive.
• • Intracellular enzymes – made and retained in the cell. Enzymes of the common pathways
• • Extracellular enzymes – made in the cell and secreted to break down large molecules that can be utilized in the cell. Amylase, pepsin, trypsen.
• • Most enzymes are protein derivatives.
• o Waves that we get ATP
• • Substrate level – where the phosphate comes off of the substrate level that is usually found in glycolosis.
• • Oxidative phosphorilation – where we get most of our energy. Associated with Kreb’s cycle and cytochrome system.
• • Direct phos. – energy you get from the Krebs cycle
• • Photo phos. – photosynthetic organism
• o (Table in chap 8) If e. coli grows aerobically it gets 38 ATP’s (we only get 36). If it grows anaerobically (ferments) it only gets 2. Respiration is 19x more efficient than fermentation.

Lecture 13, 10/5; Lab Media

Here is the audio for today's lecture.

• Monday October 15 – exam 2
• No lab practical until he gets back.
• Handout from 10-5 (3 articles inside will be on exam 2)
• Study up on legionair’s disease in your book. He said it will probably appear on exam 2.

• • Metabolism in a nutshell (handout from 10/5/07)
• Glycolosis
• The Krebs cycle
• Electron Transport Chain or System
• Enzymes – biocatylists of the cell
• Fermentation – anaerobic metabolism of sugars
• • Natural fermentations: metabiosis → silage, sourkraut
• Various foods highlighted on page 7 of the handout from 10/5
• • Bacterial Nutrition and Growth Media (handout from 10/1)
• M-9 Important as it gives us an idea of the minimal nutrients for growth in our world.
• • E. coli can make everything it needs on m-9. It can even grow on sugar salts.
• • We would need many more nutrients (for our 20 amino acids) than m-9 has to offer.
• Complex media – don’t know what/how much is in it
• • Nutrient broth – little peptone, beef extract and water
• Peptone: It is a enzymatic digest of protein
• Beef extract: boil beef and extract nutrients
• • To make a nutrient agar add 1.5% agar
• • Nutritional groups
• Autotrophs – use CO2
• • Photo – gets energy from light (plants)
• • Chemo – get energy from oxidation of inorganic substances
• Heterotrophs – use organic carbon
• • Photo – energy from light
• • Chemo – use oxidized organic material for energy (that’s us). Classical bacteria, molds, yeasts, protozoa and E. coli too.
• Hypertrophs – “below metabolism” (don’t have their own metabolism) the category for those that don’t fit in any other groups
• • Viruses, OIP, rickettsia, chlamydia
• • Complex media (continued)
• Peptone – pepsin found in our stomach (enzymatic digest)
• Tryptone – a triptic digest of protein
• Gelatin – a protein
• Beef extract - protein
• Yeast extract – got b vitamins
• Glucose – carbon source, energy
• Agar – complex polysaccharide. Very few organisms can brake it down. It is such a good solidifing agent because it is metabolically inert. Helps it to stay solid. Stable over a wide temperature range – high boiling point and low solidifying point. Agar is hard to melt
• pH indicator – phenol red. Tells us pH of solution
• • pH 7 beautiful red
• • Acid turns it yellow
• • Basic turns it hot pink
• Basic dyes – inhibits gram (+)
• Animal blood – very nutritious. Organisms that grow on this are called – fastidious
• Trace elements – comes from tap water
• • Types of media - Selective and differential media
• Differential – both grow but behave differently therefore you can differentiate them. Best example of differential media is TSI → Triple sugar iron agar.
• Selective – only certain organisms grow. We have seen sab-dex which has a low pH.
• Selective Differential (S/D) media. EMB (eosin methalene blue agar).
• • Selective for gram (-) because of the dye
• • Differential because it has lactose. Organism can either break down lactose or not → lac positive or lac negative.
• • E. coli – lac positive. It produces so much acid from lactose that the colony is dark (metallic green sheen). No breakdown of lactose leaves it clear.
• Entrobacter pyogenes and Klebsiella are also lac positive but don’t form as much acid. Entrobacter has flat fish eye (dark center light perimeter). Klebsiella has gooey fish eye (dark center light perimeter).
• Proteus – lac (-) → clear colony
• MSA mannitol salt agar – also selective and differential. Selective because of salt, differential because of the mannitol and phenol red
• • 7.5% salt media → halo tolerant organisms grow in this media. The staphs are halo (salt) tolerant (halophilic love salt)
• • has mannitol (a sugar alchol) and has phenol red – a pH indicator. These organisms break it down to acid → yellow color.
• Staphylococcus aeureus is mannitol fermenter → changes color
• Staphylococcus epidermidis is a non mannitol fermenter → no color change
• (M-ent) M – enterococcus medium. Highly selective.
• • Highly selective for Enterococcus faecalis – found in poop.
• • In its niche it is non pathogenic. Out of its niche it is!!!
• • Some strains are resistant – (VRE) Vanocomycin resistant enterococci. We would grow MRSA in MSA.
• Enriched Media
• • Blood agar - When in doubt put it on blood agar so that we don’t miss fastidious organisms. Like TSA+5%sheep blood. Human blood is not good to grow because we have lots of antibodies. Sheep blood does not have as many.
• Fastidious: streps, pyogenes, get a sputim for strep pnemonea and grow it on blood,
• • Chocolate agar (looks chocalate)
• If it is real fastidious you grow it here. Made same but agar is at higher temperature when you add the blood which bursts the blood cells and denatures the hemoglobin.
• Extremely fastidious: neisseria gonorrhoeae, neisseria meningitides (sample taken from cerebral spinal fluid), Haemophilus (blood lover) influenza – organism that follows the flu.
• • Considerations in order to grow an unknown organism.
• Nutritional requirements
• • Autotrph – use simple medium because they use CO2 from the air
• • Heterotophs
• • Hypertrphs
• Temperature requirements
• • Thermophiles grow from about 50degrees and up
• Streptococcus thermophiles.
• • Mesophiles – most of the organisms we see are in this category. 15-50degreesC. (we are mesophiles). From the soil we would grow it at 25. If we got it from us we would grow it at 35.
• • Psychorphiles – 1-15C. Optimum growing temp is 10º (that is why stuff still grows in fridge)
• Bacillus psychrophylis
• Oxygen requirements
• • Obligate Aerobes – no oxygen no growth. Ex. Micrococcus luteus, Pseudomonas aeruginosa.
• • Microaerophiles
• • Facultative anaerobes
• • Anaerobes
• pH requirements
• self requirements
• • high level halophiles
• • low level halophiles

Lecture 12, 10/3, Classification, lab media

Listen to today's lecture here.

Sidenotes:
EXTRA CREDIT:

• Probiotics → are they useful in our health in keeping us healthy.
• 3-5 pages
• 3-5 references
• short paper with title page and abstract (summary of paper) in bold.
• due October 10 <-- NOTE THIS IS CHANGED (I had previously written the 18th and meant to write 10th)
  
• (yogurt is a probiotic)
• title page

OTHER NOTES:
• 8 pager needs a snappy title.
• Knipp surgery Oct 12
• Exam 2 will probably have more organisms on it
• Keep up to date on blackboard
• Uncle Julio’s on North and Clyborne is a good place to eat.

• Gram (+) cocci (Section 12)
• Leuconostac – associated with sauerkraut
• Spore forming rods (section 13)
• Bacillus and clostridium
• Section 14
• Lactobacillus – “good guy”
• Yogurt has lactobacillus acidophilus and Lactobacillus bulgaricus – Bulgarian butter milk
• Listeria monocytogenes – food poisening, “tough guy”, found in milk and milk products – unpastuerized cheese.
• Section 15
• Gardnerella
• Arthrobacter – found in soil
• Propioibacterium acnus – the little stuff that comes out of zits is propioinic acid.
• Swiss cheese – made by propioni bacteria named after shermini. Propioni sherminii
• Mycobacteria (section 16) – acid fast
• Tuberculosis
• Leprosea
• Section 18
• Cytophaga – communicate and build things together
• Beggiatoa – found on the bottom of the north branch. It is a a sulfer bacteria
• Section 19
• Chromatium – photosynthetic bacteria
• Rhodospirillum – red photosynthetic bacteria
• Rhodopseudomonas – red photosynthetic bacteria
• Chlorobium – green photosynthetic bacteria
• Methane producers CH4 (section 21)
• halophiles
• Extreme thermophiles
• Section 23
• Gliders – the whole colony moves on a slime. They also form fruiting bodies
• Nitrogen cycle organism (section 24)
• Sulfer cycle organisms
• Streptomyces (section 28)– soil organisms - major antibiotic producing genus
• Streptomyces erythreus – produces erythromyocin antibiotic
• Along with penicillin (mold genus)
• Prokaryota
• Eubacteria –
• true bacteria
• Chlamydia and coxiella – OIP
• Mycoplasmids – no cell wall
• Cyanobacter is split off because it has a eukaryotic type of photosynthesis
• Archae bacteria
• Page 5 of handout from 9/21 is important it summarizes a lot of different thingsthings
• Cultivation
• bacteria grow on cell free media. Ex. TSA agar
• Rickettsia, Chlamydia and viruses – they are OIP’s and need host cell. This can be done with fertile eggs or tissue culture like HELA cell culture – a human culture
• Size
• Bacteria – .5-3 um
• Rickettsia - 400nm (.4um). We can see them with our lab microscope
• Viruses – 150nm (.15um). cant see them in our lab
• Inclusion bodies – a bunch of viruses together – we can see these. Nigre bodies of rabies.
• Visibility
• Filterability
• does it go through a millipore filter?
• Mycoplasma makes it through because it has no cell wall.
• Rickettsia – non filterable except coxiella
• Viruses are all filterable
• Multiplication
• Viruses – complex, no cell wall, strictly host dependent
• Nucleic acids
• Viruses – DNA or RNA, one or the other
• Antibiotics
• Viruses are resistant to antibiotics – they have no cell wall
• Cell wall
• Muramic acid. NAM
• OIP
• Bacteria no. except neisseria, salmonella typhus (typhoid fever), mycobacterium tb,
• Synthesis of ATP
• E. coli gets 38 ATP whereas we get 36.
• Viruses can’t make ATP – they are host dependent
• Ribosomes
• Macromolecular synthesis
• Metabolism (chap 7 in text) Growth Nutrition and Metabolism
• Bacterial nutrition (handout from 10/1)
• In general bacteria is either grown in a liquid broth or a pitri dish (agar)
• All life requires a carbon source. Ex. Glucose
• DNA has a carbon backbone, so does RNA and proteins
  
• Get ATP from oxidation of glucose
• Nitrogen source
• Phosphate source – needed for ATP, DNA, RNA, cell membrane (phospholipids)
• Sulfer source – amino acids; mathyanine - AUG start codon.
• Trace elements – minerals; iron for hemoglobin. Cytochromes – electron transport proteins that form ATP have a central molecule made of iron. Low iron=no energy. Iron deficiency – anemia.
• Magnesium – enzymatic co-factor. Enzymes can’t function without it.
• Media – 2 major groups
• Chemically defined media – we know everything that is in it and the quantities
• Gives a look inside nutrition
• M-9 media
• E. coli grows in M-9 media
• Glucose – provides energy
• Ammonium acid phosphate – provides a phosphate source
• Dipotassium phosphate – forms buffer (buffers maintain pH). Most organisms use a phosphate buffer.
• Magnesium Sulfate – trace minerals
• Sodium Chloride
• Water
• Non chemically defined – a.k.a. complex media

Lecture 11, 10/1; Lab Media, Classification

Listen to today's lecture here.

• Microbes in the news
• Ebola – sweeping through the Congo
• Naegleria fowleri – its in the news. Usually diagnosed in autopsies. It is a brain eating amoeba. On protozoan sheet.
• Dengue Fever -Sweeping through the Caribbean. It is viral and has a mosquito vector
• • Media (more info on handout from 10-1)
• Blood Agar – for fastidious organisms, for those that require a lot of nutrients
• MacConkey agar – for gram (-) rods. E. coli grows on it well.
• Eosin methylene blue – for gram negative rods. Gram (-) rods are the hard ones to identify. That is why there is different media used to identify them.
• SS agar – used to grow the big gram (-) enteric pathogens.
• Triple sugar iron agar – another medium for gram (-), good for differentiation of gram (-) organisms
• Mannitol salt agar – good for telling the two staph’s apart. Staphylococcus aureus medium. This organism is the one that Causes zits, food poisoning, pnemoniae, toxic shock syndrome, and the resistant strain MRSA.
• Litmus milk – used a little bit in the lab.
• • Growth curve (diagram on hadout from 10-1)
• “Ya better know it”
• • Generation times (more info on handout from 10-1)
• E. coli → fast, ideal for genetic engineering
• • 3 generations/hr
• Kochs bacillus (Mycobacterium tuberculosis) is a slow grower. Hits the AIDS patients.
• Yeast buds every two hours
• • Classification Sheet (handed out 9/21)
• Know the archae’s
• Manual of Systematic Bacteriology by Bergey – “The Bacteriologist Bible” --- possible extra credit question?
• Gracilicutes
• • Prokaryotes with thin cell walls, implying a gram-negative type of cell wall
• Firmicutes
• • Prokaryotes with thick and strong skin, indicating a gram (+) type of cell wall
• Tenericutes
• • Prokaryotes of a pliable and soft naure, indicating the lack of a rigid wall.
• Mendosicutes
• • Prokaryotes with faulty cell walls, suggesting the lack of conventional peptidoglycan.
• Aerobic/microaerophilic, motile, helical/vibrioid gram (-) bacteria (Section 2)
• • spirilum volutans – we saw it in lab. Has flagella at both ends. Usually free living.
• • Campylobacter jujunii – causes gastroenteritis. Upset stomach. It is a fecal organism. It has gull wings – two cells that come down. Strict oxygen requirements – microaerophilic.
• • Bdellovibrio – parasitic on e. coli
• gram-negative aerobic rods and cocci (Section 4)
• • Psudemonas aeruginosa – Tough to treat. Blue green puss.
• • Zoogloea – sewage treatment
• • Azotobacter – nitrogen fixer
• • Rhizobium – nitrogen fixer, symbiotic with legumes
• • Agrobacterium – genetic engineering
• • Halobacterium – grows in high salt
• • Acetobacter – Pasteur discovered, makes vinegar (more and more popular)
• • Legionalla – picked up on air conditioners and cooling towers.
• • Neisseria – clap, meningitis
• • Morexella – found in toddlers and old folks that continually rub there eyes → pink eye
• • Brucella – bioterrorism agents. Found in cattle.
• • Bordetalla – whooping cough
• • Francisella – rabbit fever. Tularemia.
• Facultatively Anaerobic Gram (-) rods (Section 5)
• • Escherichia
• • Shigella
• • Salmonella
• • Citrobacter
• • Klebsiella – not quite as pathogenic as the others
• • Entrobacter – associated with plants
• • Erwinia – associated with plants
• • Proteus – they swarm (go across the plate)
• • Morganella – related to proteus
• • Yersinia – the plague
• • Vibrio cholera - used to be called Vibrio comma. Gives us problems in water.
• • Photobacterium – gives off light. Uses same system as fireflies. Oxygen dependent. Usually found in ocean waters. Uses an enzyme called luciferase (?sp.?)
• • Pastuerella multocida – more common than rabies. Get it from a bite.
• • Haemophalis influenzae - Doesn’t cause the flu, the flu is a virus. This had been considered to cause it but as it turns out Haemophalis influenza actually kicks you while you are down with the flu.
• • Chromabacterium - purple pigment
• • Gardnerella - causes vaginitis it is a bacterial vaginitis which is in contrast to yeast vaginitis which is in contrast to protozoan vaginitis (trich)
• Anaerobic Gram (-) straight, curved and helical rods (section 6)
• • Bacteroides
• • Fusobacterium – pointed ends like an arrowhead. Causes trench mouth; real bad breath.
• Section 7
• • sulfides
• The Rickettsia and Chlamydias (Section 9)
• • Rickettsia – obligate intracellular parasites – typically have insect vector
• • Coxiella – q fever, bioterrorism agent, burnetii
• • Chlamydia trachomatis – STD, eye infection
• Mycoplasmids (section 10) - causes walking pnemoniea
• • ureaplasma – causes Urinary Tract Infection
• Gram positive cocci (section 12)
• • Micrococcus luteus – yellow and harmless (nonpathogenic)
• • Staphylococcus epidermidis – safe to work with
• • Staphylococcus aureus – more dangerous
• • Streptococcus pyogenes
• • Streptococcus lactis – responsible for sour milk
• • Enterococcus faecalis - comes from poop, it can be resistant, VRE.