MicroBiology Exam 4 Flashcards
(118 cards)
1
Q
How do patients vary in their response to HIV infection?Genetic Guardians
A
- Stronger immune system~ difference in symptom of AID’s appear
- Disease response Y/N, maybe an individual is exposed to virus, but there is no infection or disease.
- Type of opportunistic infection
2
Q
What were O’brien’s hypotheses and goals?
Genetic Guardians
A
-Maybe variation in response, has a genetic basis.
3
Q
Which genes were examined?
Genetic Guardians
A
- Attachment via host cell receptor. Look for genes for host cell receptors.
- CD4—initial docking, CCR5—docking and entry, CXCR4–
4
Q
How were genes screened for role in resistance?
Genetic Guardians
A
- Look for alleles for receptor genes
- Correlation between alleles and outcomes, divide into groups, does a particular allele occur more often than predicted in different population.
5
Q
Significance of CCR5-DELTA32: A particular version of CCR% gene.
P.1
Genetic Guardians
A
- What is CCR5-DELTA gene:
- Loss of 32 nucleotides
- How does the mutation affect the gene product:
- Big change in shape—protein has been shorten, non-functional product
- How does CCR5 geneotype affect survival:–how can we survive w/ this mutation even if Homozygous, both alleles= DELTA32—no CCR5 proteins.
- Normal function of CCR5. Chemokine (cytokine) receptor for “messenger” that stimulate WBC activity.
6
Q
Significance of CCR5-DELTA32: A particular version of CCR% gene.
P.2
Genetic Guardians
A
-Why can you apparently live without it?
-Many other ways to stimulate this cells. Other cytokine receptors—we have redundancy CCR5-DELTA32
-What is honozygous for DELTA32?
-No CCR5 receptor, no infection by most HIV strains, no AIDs “immune”
-What if heterozygous?
-One normal CCR5 allele, one DELTA32 allele, HIV can still bind—still have ability to make working CCR5
-Vs. homozygous for normal CCR5~ might be at lower risk, might slow the progression of the disease. Can be infected if heterozygous.
-What is the allele frequency of CCR5-DELTA32?
-In European population: 10%
-Genotype frequency in European pop,
-Homozygous: DELTA32- 1%
-Heterozygous: 20%
Other parts of the world population: close to 0%… for allele
7
Q
Does variation in CCR5 explain all cases of resistance?
Genetic Guardians
A
-Are other alleles for other receptor genes that provide some resistance
8
Q
What are some final conclusions?
Genetic Guardians
A
- Possibilities:
- Gene for receptor, receptor is essential for HIV, not essential for us. t have to be functional –Develop a drug to block it—the drug needs to bind to receptor—blocks HIV.
9
Q
Treatments & Possible reason for receptor
Genetic Guardians
A
- Maraviroc—now available, binds to CCR5, blocks HIV attachment. Risk of liver damage. Helps lower chance of replication once contracted.
- Treatment–> cure?
- Infected patient—through radiation destroyed bone marrow, added stem cells for white blood cells. Source of stem cells used homozygous for DELTA32—can’t find HIV in the body…Very risky and expensive.
- Why is CCR5-DELTA32 allele relatively common in European population, in absence of HIV—probably net negative.
- Allele origins—long before HIV in Europe
- Benefits? Maybe protected (in past) people against some other epidemic disease…perhaps against bubonic plague. Big epidemic ~1348
10
Q
Control of Micro-organisms
A
- Disinfection or sanitation
- Sterilization
11
Q
Disinfection or sanitation
Control of Micro-organisms
A
-A significant reduction in # of microbes. But does not mean complete elimination.
12
Q
Sterilization
Control of Micro-organisms
A
-All living things killed, all things that might come to life, (ex. Spores, cysts)
13
Q
General effects of control methods:
Control of Micro-organisms
A
-Physically remove MO and/or alter conditions to reduce or inhibit growth and/or increase death rate (eliminate by killing)
14
Q
Manipulation of temperature:
Control of Micro-organisms
A
- Each bacterial species has temperature growth range-adapted to a particular environment.
- Significant to us, can prevent growth, by setting temperature outside of range.
- Can kill microbes if hot enough
15
Q
Use of Heat
Control of Micro-organisms
A
- Works b/c it denatures proteins and oxidizes (burns) components.
- Response of microbe depends on the type of microbe and length of exposure to temperature (heat)–proteins can re-nature.
16
Q
Pasteurization
Control of Micro-organisms
A
- Louis Pasteur invented to save French wine industry
- Brief exposure to high temperature. Can’t be exposed for too long, temperature changes properties of food/beverage.
17
Q
LTLT & HTST
MIlk methods of pasteurization
A
- LTLT: Low temperature, long time 63C (145F) for 30mins
- HTST: High temperature, short time 72C(160F) for 15sec
-LTLT/HTST methods will not sterilize, but will significantly reduce the # of spoilage microbes.
Ex: Lactobacillus, extends shelf life. Significantly increases safety—kills all common pathogens.
-Salmonella
-Listeria
-Mycobacterium—causes tuberculosis—cows get TB
18
Q
UHT
Milk methods of pasteurization
A
- Ultra high temperature 140C (285F) for 2 sec
- UHT method will sterilize milk, no need to refrigerate to prevent microbial spoilage as long as container is sealed.
19
Q
Autoclaving
Control of Micro-organisms
A
- Uses pressurized steam, at 15-20 PSI, can get steam temperature to 120 degrees C
- Steam= moist heat, hot water molecules
- Results in sterilization if done correctly—kills everything
- Kills by denaturing proteins, disrupts cell membranes
20
Q
Canning
Control of Micro-organisms
A
- Combination of high temperature to kill and sealing to preserve (prevents contamination or re-inoculation) after cooling.
- Invented by Nicholas Appert in first decade of 1800’s—prize offered by Napoleon, needs to feed armies.
- Method—heated food in bottles, sealed bottles, won prize around 1810.
- English switched to tin cans, sealed with lead…
- Main concern in canning is for spore forming Cl. botulinum, caning creates anaerobic environment—could have spores from dust or soil. If anaerobic, can get deadly Botulinum toxin.
21
Q
Temperatures needed for canning depends on the acidity
A
High acid foods pH4.5 120 degrees C
22
Q
High acid foods pH<4.5
Ex: Tomatoes, Strawberries, Citrus fruits
A
-Relatively lower temperatures can be used compared to lower acid foods
~ 100degrees C is usually sufficient for high acid foods
-Can kill all growing vegetative cells.
-Will NOT kill all spores.
-At very low pH—if highly acidic, acids will prevent germination of spores.
-No growth, no germination, no toxins.
23
Q
Low acid foods pH>4.5
Ex: Most Vegetables, Corn, Meats, Mushrooms
A
-Need higher temperature
~need to kill the spores, once cooled spores germinate, not acidic enough
-Use 120 degrees C requires pressure cooker w/ steam.
-Commercial—use big autoclave. Home—stove top pressure cooker.
-Most botulism cases tied to home canning, still < 50 cases/year
24
Q
Use of Cold
Control of Micro-organisms
A
- Refrigeration (0 to 4 degrees C) (32 - 34 degrees F)
- Freeing at -20 degrees C or lower.
25
Refrigeration (0 to 4 degrees C) (32 - 34 degrees F)
- Usually not fatal to microbes, eventually many microbes grow, spoil food—extends shelf life,but not indefinitely.
- Some microbes that might spoil food (Psychrophiles/ Psychrotrophs) prefer cooler temperatures.
- Ex. Lactic acid bacteria, Pseudomones (grows on meat produce greenish slime), Listeria (can tolerate cool)
26
Freeing at -20 degrees C or lower.
- What all water to be solid.
- Denies cells liquid water.
- Stops growth completely & Preserves indefinitely.
- Some microbes will be killed, cells torn apart by ice crystals, but many are NOT killed, can grow again when food is thawed. Once thawed—spoilage begins.
- Freezing used for long-term storage of microbial cultures. Mix cells with glycerol to stop ice crystals formation.
27
Physical Control Methods
- Hand washing
- Quarantine
- Filtration
- Ionizing Radiation
- UV Radiation
28
Hand washing
| Physical Control Methods
Mode of action:
-Physically removes pathogens; depending on chemical composition of the soap, it may also kill pathogens
Application and Value:
-Reduces transmission of infection by direct hand-to-body contact and from touched inanimate surfaces; of increasing importance in clinical settings due to drug-resistant bacteria
29
Quarantine
| Physical Control Methods
Mode of action:
-Prevents transmission by physically removing or isolating source of pathogens
Application and Value:
-Used to prevent person-to-person transmission of pathogens, especially during epidemics and in the context of controlling the spread of drug-resistant and/or highly virulent pathogens
30
Filtration
| Physical Control Methods
Mode of action:
-Physically removes or retains pathogens from air and/or liquids; typically removes all bacteria and may sterilize if pore size small enough to restrict virus passage
Application and Value:
-Used to remove bacteria and viruses from the air to create sterile work areas and to prevent the escape of pathogens from research facilities; used in place of autoclaving to remove microbes from heat-sensitive or heat-labile liquids such as beverages and pharmaceutical solutions
31
Ionizing Radiation
| Physical Control Methods
Mode of action:
-High energy gamma and X-ray radiation can strip electrons from atoms, creating very reactive free radicals which react destructively with proteins and DNA; inactivates proteins, disrupts biochemical organization and mutates DNA
Application and Value:
- Can sterilize a wide variety of solid materials
- Used for some food products like spices and meats; radiation may degrade proteins to amines, and degrade fats to short chain fatty acids, creating off flavors and aromas
- Widely used to sterilize heat-sensitive solids such as plastic Petri dishes and plastic pipettes
32
UV Radiation
| Physical Control Methods
Mode of action:
-Causes mutations when DNA absorbs UV light energy (max absorbance at 260 nm), causing formation of “dimers” by linking adjacent (same strand) thymine bases
Application and Value:
-UV lamps reduce pathogen populations on surfaces such as operating tables, lab benches and in lab hoods; effectiveness is limited by poor penetrating power of the lower energy UV radiation, by the ability of UV light to cause skin cancer and by the UV resistance of endospores
33
Preservatives
- Mostly used with food, drinks, drugs
- Goal: prevent spoilage, extend shelf life of product and prevent transmission of disease
- Dilemma: Need to stop microbial growth, but agent can't be too toxic, product will be consumed. Most preservatives will be less toxic—static agents.
- Organic Acids
- Sugars
- Salts
- Nitrites & nitrates
- Antioxidants
34
Organic Acids
Preservatives
- Example:
- Acetic acid/Acetate
- Lactic acid/Lactate
- Propionic acid/ Propionate
- Benzoic acid/Benzoate
- Sorbic acid/Sorbate
- Mode of Action:
- ALL act to lower pH; most microbes have enzymes adapted to function at near-neutral pH; at low pHs, enzymes are inhibited or denatured
- Some organic acids, like benzoic and sorbic, also act to inhibit the uptake of key amino acids, and so starve cells of nutrients
- Uses:
- Cheese, pickles, meats, bread, mayonnaise, fruit juices and jams
- Comments:
- Many foods are naturally preserved by organic acid-producing fermentations; for example, cheeses are preserved by lactic and propionic acid fermentation
- Tolerance of low pH varies widely among microbes; fungi are usually more tolerant than bacteria, and high acid foods are usually spoiled by fungi
35
Sugars
Preservatives
-Example:
Glucose, fructose, sucrose
-Mode of Action:
-Creates hypertonic environment; water moves out of cells, membranes collapse and metabolism slows and/or stops
-Uses:
-Milk, fruits and fruit products such as jams
-Comments:
-Some microbes are tolerant of hypertonic conditions; fungi are usually more tolerant than bacteria, and high sugar foods are usually spoiled by fungi
-Takes much higher concentration of sugar to have same inhibitory effect as salts due to “additional salt effects” (see salts)
36
Salts
Preservatives
-Example:
-Sodium chloride (table salt)
-Mode of Action:
-Creates hypertonic environment; water moves out of cells, membranes collapse and metabolism slows and/or stops
-Additional salt effects:
-Also disrupts membranes and denatures proteins
-Uses:
Meats & fish
-Comments:
-Effective at lower concentration than sugars due to “additional salt effects”.
-Staphylococcus species can grow in high salt environments due to adaptation to sweaty skin. Might normally be out-competed in food by other microbes, but has advantage in high salt foods.
S. aureus can cause food poisoning.
37
Nitrites & nitrates
Preservatives
- Mode of Action:
- Inhibits enzymes involved in ATP-yielding metabolism
- Uses:
- Common in preserved meats like bacon, bologna, and pepperoni
- Comments:
- Preserves red color by combining with hemoglobin in red blood cells
- Kills microbes, including common food pathogens like Clostridium/Salmonella
- Converted to nitrosamines in the body; nitrosamine are carcinogens, so restrict intake of nitrate and nitrites
38
Antioxidants
Preservatives
- Example:
- BHA and BHT
- Mode of Action:
- Prevent oxidation of fats and oils to short chain fatty acids
- Uses:
- Found in many types of foods
- Comments:
- Added primarily to prevent chemical changes leading to off tastes and aroma, but may also have anti-microbial properties, too
39
Chemical control methods...terms
- static: chemical inhibits growth of microorganism, but not necessarily killing. Cells may grow again when chemical agent is removed
- cidal: kills existing microorganism
- Preservatives
- Disinfectants
- Antiseptics
40
History of Disinfectant/Antiseptics
Ignaz Semmelweis (1840s), was a physician in Vienna, Austria. He noticed when doctored delivered the baby—mothers died-of childbed fever. While midwives delived much lower mortality rates. Often in the hospital—doctors would come from autopsies of childbed fever deaths and deliver babies. Results in mothers death.
Semmelweis deduced that there was something on hands—starts hand washing w/ chlorinated lime (works) even though it worked his ideas were often rejected. No germ theory.
Germ Theory: developed by several people in 1860's, idea—microorganisms cause disease—there is a link between bacteria and specific illnesses (like childbed fever). Work of many people but best known Louis Pasteur and Robert Koch.
Joseph Lister (1870's) attacked problem of post-surgical infection. General anaesthetics existed...used in medicine in the 1840's, surgeons could take more time, attempt new operation. Results in 50% post-surgery mortality. Lister accepts the idea that microbes cause disease—and decides to use carbolic acid (phenol) to deliberately kill microbes to prevent infect. Results in a large reduction in post-surgery mortality. Lister's ideas spread in 1870's, came to U.S. In 1876 talks to doctors. In 1881 this idea was still not totally accepted. July 1881—Garfield was shot and after 2 ½ months, he dies of massive infection because unsterile poking around searching for the bullet. By the end of the 1880's everybody follows Lister. Late 1800's systematic use of many chemicals.
At this time—can prevent infection. But after infection occurs, can't do much.
41
Toxicity and effects of Disinfectants and Antiseptic
- Most are more toxic than preservatives, mostly-cidal agents, will reduce number of microbes.
- Some will produce sterility, if numbers are reduced to 0, only after long contact time
42
Disinfectants
-Used on non-living things/surfaces. Many are quite toxic to living cells.
Ex of uses: lab benches, floors, instruments
43
Antiseptics
-Used on living tissue.
Key points—need to be careful to not kill too many of our cells.
-Used on the outside of the body only or on mucus membranes lining passages to inside.
-Not going to be chemicals that are injected, may be tolerated on skin, but too toxic for internal use.
-Could be same chemical as a disinfectant (Ex. Alcohol), might reduce the concentration if used as an antiseptic.
44
Factors that influence effectiveness
| Of disinfectants and antiseptics
- Type of microorganism that is trying to be controlled. Endospores are highly resistant, while viruses with lipid envelope can be effected easily by attaching envelope.
- Growth stage—cysts or spores
- Initial microbial populations—larger population takes longer to control
- Concentration of chemical—always critical to question of toxicity.
- Time of exposure/contact time
- Temperature—works faster if warmer—killing reactions are chemical reactions, heat speeds up reactions.
- Amount of organic matter that is mixed in with microbes. Organic material may inhibit chemicals. Ex. Feces, blood, vomit
45
Phenol and Phenolics
- Phenol
- Phenylphenol (LysolTM)
- Hexylresorcinol (in SucretsTM)
- Triclosan
- Hexachlorophene (PhisohexTM)
46
Phenol
Phenol and Phenolics
- Mode of Action:
- Denatures proteins, disrupts membranes
- Disinfectant application:
- May be used on inanimate surfaces such as floors, but not widely used; strong odor
- Antiseptic application:
- Lister’s old surgical antiseptic “carbolic acid”; rarely used as an antiseptic today, too toxic
47
Phenylphenol (LysolTM)
Phenol and Phenolics
- Mode of Action:
- Denatures proteins, disrupts membranes
- Disinfectant application:
- Widely used to disinfect surfaces including floors, institutional showers, etc
- Antiseptic application:
- Too toxic for topical use
48
Hexylresorcinol (in SucretsTM)
Phenol and Phenolics
-Mode of Action:
-Denatures proteins, disrupts membranes
-Antiseptic application:
-Used to treat minor skin infections and sore throats;
has topical anesthetic effect
49
Triclosan
Phenol and Phenolics
- Mode of Action:
- Disrupts membrane, and inhibits fatty acid synthesis
- Disinfectant application:
- Sometimes used in cleaning supplies, shower curtains and trash bags; part of MicrobanTM
- Antiseptic application:
- Used in “anti-bacterial” soaps, deodorants and toothpastes
50
Hexachlorophene (PhisohexTM)
Phenol and Phenolics
- Mode of Action:
- Denatures proteins, disrupts membranes
- Antiseptic application:
- Was widely used on skin to treat acne and control Staphy. infections, especially in hospital nurseries, but can be absorbed through the skin and was linked to neurological damage
51
Chlorhexidine
| sometimes classified as a “phenolic”
- Forms:
- HibiclensTM
- Mode of Action:
- Disrupts membranes
- Antiseptic application:
- Used to cleanse minor wounds and to reduce microbial populations on skin prior to surgery (“pre-op scrub”)
52
Alcohols
-Forms:
70 – 75 % ethanol (grain alcohol)
70 – 75 % isopropanol (rubbing alcohol)
-Mode of Action:
-Denatures proteins, disrupts membranes
-Disinfectant application:
-Used to disinfect lab benches and lab or medical instruments
-Antiseptic application:
-Used externally on unbroken skin to reduce pathogen population before hypodermic injections, insertion of IV lines or other skin-breaking methods
53
Chlorine
- Forms:
- Hypochorite (OCl--) solutions, for example, Chlorox bleach (NaOCl)
- Mode of Action:
- Chemically oxidizes many types of cell components and biochemicals
- Disinfectant application:
- Used to disinfect drinking water, swimming pools and treated sewage; used on a variety of surfaces in lab and clinical settings (floors, etc.)
- Antiseptic application:
- Rarely used on skin
54
Iodine
- Forms:
- Tincture (alcohol solution) of iodine
- Iodophores, for example, BetadineTM
- Mode of Action:
- Chemically oxidizes cell components, binds directly to amino acids and inactivates proteins
- Disinfectant application:
- Occasionally used by hikers to disinfect water
- Antiseptic application
- Iodine tinctures are staining, irritating, and may cause allergic reaction; BetadineTM and other iodophores are less like to stain and irritate skin and are used to cleanse minor wounds and to reduce microbial populations on skin prior to surgery (“pre-op scrub”)
55
Aldehydes
- Forms:
- Formalin (35% solution of formaldehyde)
- Glutaraldehyde
- Mode of Action:
- Alkylates, and so, inactivates proteins
- (Alkylate = adds short hydrocarbon chains like –CH3)
- Disinfectant application:
- “High level disinfectant” or “chemical sterilant”; sterilizes with about 3 hours contact time, used to preserve organic tissue, sterilize equipment, kill pathogens for use in vaccines
- Antiseptic application
- NO! Formaldehyde is carcinogenic
56
Ethylene oxide (C2H4O)
-Forms:
Used in gas forms
-Mode of Action:
-Alkylates proteins and DNA, and so, inactivates proteins and mutates DNA
-Disinfectant application:
-“High level disinfectant” or “chemical sterilant”; sterilizes with about 4 – 6 hours contact time, used in “gas chamber” in place of autoclave to sterilize heat- and moisture-sensitive equipment, instruments, linens
-Antiseptic application
-NO! Ethylene oxide is carcinogenic and increases risk of miscarriage
57
Antibiotics
| Historical content
- From 1870's to 1930's have germ theory, have many physical & chemical control methods
- Could understand cause of infection, prevent infection...can't effectively safely treat infection
- Big emphasis on prevention
- Culture—beards go “strictly germproof” (1910's). Permanate (antiseptic), Carbolated soap, sulferated hydrogen, trimming of wiskers, iodoform.
- Before antibiotics—treatments often as toxic as disease
58
Antibiotics
- Can be used to treat internal infections
- Can control microbes after infection
- Due to a property referred to as “selective toxicity”
- Discovery (of antibiotics) changed history. Started in the 1930's with development of sulfa drugs by Domagk—German. Could treat pneumonia. In the 1940's—penicillin.
59
Source of antibiotics
-Many antibiotics from microorganisms usually in soil or sewage (lots and lots of microbial competition)
Genera: Bacterial and Fungi
Other antibiotics are:
-Chemically modified microbial products,
-Chemicals of completely artificial origins
60
Genera -Bacterial
| Source of antibiotics
-Bacillus
Ex. Bacitracin
-*Streptomyces....filamentous prokaryotic—like fungi, most important genus for antibiotics.
61
Genera -Fungi
| Source of antibiotic
-Penicillium
| Ex. Cephalosporium—source of cephalosporins
62
Critical point is selective toxicity
- Lost of microbes make anti-microbial compounds, most compounds are too toxic to use
- Antibiotics in use—usually much more toxic to microbes than to humans
- Can be used internally, find doses that are safe for us and toxic to them
- Can now treat pneumonia, syphilis, TB
63
Mode of action:
- Involved biochemical or structural feature that is either unique to bacteria or different enough
- Anti-bacterials, most work against cell wall, 70S ribosome or may target unique enzyme. Ex. Sulfa drugs, Bacteria have folic acid syn, path, Human—get folic acid in food.
64
Therapeutic index:
- Measure of selective toxicity, T.I. (Concentration (dose) toxic to humans) / (concentration (dose) that harms* microbes) therapeutic dose
* get side effects
* harms inhibit or kill, T.I. (toxic dose) / (therapeutic dose)
-High values are good, take high dose to poison us, take low dose to poison microbes, want multiples higher than 1. Antiseptics—mostly have T.I.'s around 1.0
65
Harm to humans
| Toxicity
- Possible harm to us—side effects, safer if there is a wide range between therapeutic dose and toxic (to us) dose.
- Possible side effects varies w/ specific antibiotics
- Toxicity to humans- some antibiotics may cause some damage to auditory nerves. Some antibiotics may cause allergic reaction—Ex. Penicillin. Damage to organs—liver is the main detox organ, kidney filter out toxins.
66
Effects on Normal flora
antibiotics may also significantly harm normal flora species, normal flora are microbial species that normally live on healthy people. These are beneficial.
67
Reading: “The Challenge of Antibiotics”
"Resistance"
Problem of acquired antibiotics resistance, change in last ~60 years in resistance to specific drugs
Definition of “resistance”: higher and higher concentration (doses) are required to harm microbes.
Major problem—if we need higher doses we start to get more problems with side effects and toxicity to us.
After some time—we can't use drug anymore. Can invent new drug but that takes TIME 10-15 years and takes $$$~$100 million.
While we are inventing—people die of infections that were once curable. Resistance is NOT the same thing as “virulence”.
68
Reading: “The Challenge of Antibiotics”
| "virulence"
Ability to cause disease—degree of severity of disease.
| Resistance—can't treat infection
69
Reading: “The Challenge of Antibiotics”
| Ability to resist—How do cells (bacteria) become more resistant?
- Is product of acquired genes—plasmids through transformation or conjugation (most common), or transduction.
- Result of new mutation or genetic exchange. Spread ^^ and creates new gene. Transfers (by 3 methods) NOT restricted to within species.
70
Reading: “Cross species transfer is the last straw”
MRSA strain of S. aureus. Methicillin. Resist. Staph. Aureus.
Gained vancomycin resistance
71
Reading: “Cross species transfer is the last straw”
How did it gain vancomycin resistance?
What physical object was picked up?
- Most likely through conjugation, could be transduction
- Plasmid –also carried some transposon. Genes on plasmid-- vancomycin resistance and genes for conjugation. Can spread plasmid.
- From Enterococcus-- not from another S. aureus cell.
72
Reading: “Cross species transfer is the last straw”
| What is the significance of Vancomycin resistance MRSA strain?
-MRSA are resistance to multiple antibiotics—could use vancomycin, which is not a preferred antibiotic b/c its relatively toxic, delivered by IV.
Vancomyocin resistance MRSA are hard to treat- most MRSA strains are STILL vancomyocin sensitive.
73
Effects of resistance genes (What do these genes do?)
- Genetic change could elimination of “ports of entry” into cell, gram negative cell wall has porins in outer phosolipid envelope. Porins selectively transport certain molecules could include antibiotics. If the structure of porin is altered, drug may not pass, can't reach target.
-Production of enzymes that in some way degrade or detoxify the antibiotic.
Ex. Penicillin resistance due to beta-lactamase that break open B-lactam.
74
What are the mechanisms of resistance?
-Decreased sensitivity to target structures, mutation changes shape of structure—lowers the affinity of drug for target.
Ex. Change in RNA in ribosome
-Decreased sensitivity of target enzymes alter enzyme shape, drug binding reduces.
Ex. Resistance to sulfa, where there is a change in enzyme for folic acid synthesis. Resistance to Quinolones by Pseudomonas—change shape of gyrase enzyme.
-Drug-removing efflux pumps—resistance is due to an increased ability to pump drug out, if drug is rapidly removed, can't reach toxic concentration. Pumps often relatively non-specific—may pump out many different drugs. Get multiple drug resistance. Known for resistance to Penicillin, Tetracyclines and Quinolones.
75
What are drugs doing to promote emergence, spread of drug resistance or spread of resistant strains in environments
- Drugs NOT causing mutations that create resistance genes.
- Mutations—result of other events prior to or during infection. The drug DOES create selective environment, it favors cells with resistance genes and selects against (or eliminates) cells without resistance genes.
76
Prior to any drug use, frequency of resistance cells are very low, may be 1 in a million.
Resistant cells would remain rare in absence of drug.
When drug is initially used vast majority of cells will be sensitive.
Result: drug cures. Surviving cells often also resistant cells---proportion of resistant cells or freq of resistance genes will be much higher.
-Genes will be passed on—change in the population sensitive pop. Change to resistant.
-If cells reach new host—new infection—antibiotics won't work as well at the original dose
77
Resistance in bacteria population, if antibiotics are used, it is inevitable.
But we can change the rate of spread—buys us time.
What can we do?
Why do doctors prescribe antibacterial for viral infections?
-Figure out if infection is bacterial or viral, if viral using antibacterial drugs select for resistant strains—no value. No effect against virus
-Need education—patient pressure
Exception: prevent pneumonia in flu cases (viral)
78
Taking antibiotics
- Slow the rate of spread of resistance—take full amount of drug for full time prescribed—even if you feel better—its a hassle to take drugs—possible side effects—hording—medical error—expensive.
- If taken as prescribed, maintains high concentration of antibiotic even “mildly” resistant cells, can then be killed. If keep the pathogen population low—give immune system a chance to kill.
79
Use of antibiotics in animal feed—to reduce spread of resistant—need to reduce use in feed. Why using?
To treat specific infection in specific animal. To prevent epizootics in high density population, also to promote growth—even in healthy animals. Result: more animal in less time. Most controversial—could this be reduced? Biggest problem with “CAFO” concentrated animal feeding operation.
80
Use of antibiotics
Creates “selective environ”
| Antibiotic = agent of natural select
81
Immune defense network
- Human being's own internal defense system against microbes
| - System recognizes and attacks microbes/ pathogens (Recognizes and attacks malignant cells, prevents cancer)
82
Collective effort determines “immunity”
- Immunity—relative ability to resist infectious disease—degree of vulnerability to an invading pathogen.
- Divided into two overlapping categories—nonspecific (innate) and specific immunity
83
Nonspecific defenses:
immediate protection generalized, given mechanism NOT directed against specific pathogens
- Physical barriers
- Chemical barriers
84
Physical barriers:
| Nonspecific defenses
- Skin- seals off body. Multi-layer barrier, most microbes can't penetrate w/o break or damage. Infection through skin requires break.
- Mucous membranes-membranes that line passages to outside and body cavity. Mucus keeps lining moist—prevents break/crack in surface. Also traps in mucus.
- Ciliated epithelial cells found in trachea. Cilia trap microbes—sweep up into back of throat-->swallowing microbes (get rid of microbes and sends to stomach) Protects bronchi, alveoli, killed by flu virus, inhibited by smoking
- Fluid flow—saliva, tears, urine in urinary tract,
85
Chemical barriers
| Nonspecific defenses
- Lysozyme: enzyme that can degrade peptidoglycan. Found in tears, saliva, sweat. More effective against gram positive b/c simple cell wall. Ex- Staphylococcus
- Lipids: secreted onto skin by sebaceous glands (onto skin), some fatty acids that are mildly anti-microbial
- Acids: -Hydrochloric acid (in stomach) pH ~ 0 to 1; most microbes are killed. Some microbes can survive the passage through the stomach because they can colonize in the lower digestive tract and into the intestine pH~ 7. Ex: Giardia, Salmonella, E.coli, polio virus, Hep A. Very few species can colonize in the stomach. H. pylori lives in stomach, burrows through non-living mucus layer in stomach—will then colonize in cells lining the stomach. One cause of ulcers. Discovered ulcers could be treated with antibiotics.
- Mild acids: pH 4.5~5.4; surface of skin—fatty acids, lactic acids(produced by bacteria), vaginal tract also protected by lactic acid. Product of Lactobacillus.
86
Normal human flora--”indigenous microbiota”
- Microbial populations mostly bacterial, normally associated with healthy people. Do not cause disease under normal conditions.
- Location of normal flora: skin, mouth, gastrointestinal tract, reproductive tract.
87
Functions of normal flora (benefits):
Synthesis of vitamins which can be absorbed in gut. Mostly B vitamins. Some produce anti-microbial compounds. Mostly take up space—fills up habitat. Prevent attachment of pathogens—out-compete pathogen for nutrients.
88
Value shown with gnotobiotic animals:
“germ-free” animal. W/o normal flora—get poorly developed lymphoid tissue (part of immune system). Require extra vitamins if challenged w/ pathogens -more vulnerable to disease.
89
Babies normal flora
- Flora acquired at and after birth—birth canal has Lactobacillus.
- Picked up from food, water, dust. Infants (1st yr of life) need time to establish normal flora—until then infants can be vulnerable to pathogens –otherwise (if older) not so vulnerable.
- Especially intestinal infections & this is why we don't feed honey to infants. May contain C. botulinum spores.
- Spores will germinate in “open habitat” of infants gut—produce in gut Botulinum toxin. Paralyses muscles. “floppy baby” RARE.
- Protection after about 1 yr—well colonized w/ normal flora. C. botulinum cells can't colonize.
90
Normal flora species some can be opportunistic pathogen's—conditions in which disease colonizes.
-Decline or loss of immune system. Relocation to other body regions, trauma to gut—gut bacteria into blood.
Ex. Ruptured appendix.
-Use of antibiotics kills/inhibits most normal flora if antibiotic resistant strains among normal flora, resistant species were fine, now overgrown, cause disease.
Ex. Enterococcus—found in gut.
Many species are drug resistant
91
Reading: Probiotics: Their tiny worlds under scrutiny
| What are “probiotics”?
Normal flora species, we are using biological produces—pro good bacteria that can out compete the pathogens and protect us. “bacteria”=germs= bad (common belief) Recently promoted for bacterial content ex. yogurt—for good bacteria. “Activia”
92
Reading: Probiotics: Their tiny worlds under scrutiny
| What limits use, limits research?
- Money. Need something to patent, to patent an organism you must alter them. Gut = ecosystem 500 species, limitless # of interactions.
- Hard to predict outcome if introduce species.~ unpredictable.
93
Reading: Probiotics: Their tiny worlds under scrutiny
| Bifidobacteria-
-Digestive tract bacteria, protect against intestinal infections.
-But hard to predict. Use in time of “open habitat”
Ex: after antibiotic use or right after birth
94
Reading: “Stool transplants” for C. difficile colitis
| -Why or when would a doc consider the use of “stool transplant:
-Follows antibiotic use, once infected, antibiotics may be of limited use.
-Chronic diarrhea, chronic inflammation, potentially fatal.
Donors—family
-For the treatment of C. difficile colitis
95
Reading: “Stool transplants” for C. difficile colitis
-How were “stool donors” selected?
-How were the stool transplant done?
What were the results of stool transplant?
- Donors-family, similar flora, to flora present before drug use.
- Dose = 25 cc (1 oz) in through a feeding
- Result of 18 patients, 15 cured ( = 85%), few died—but from other causes. 85% cured after alternatives failed.
96
Reading: “Stool transplants” for C. difficile colitis
| -What are some of the drawbacks and or risks of stool transplantation?
- Not very pleasant, high “Ick-factor”.
- Don't know what is in the “treatment” complex mix of microbial species.
- Could be opportunistic pathogens or if healthy, bad, if already sick.
- Solution “Cocktail” of known species.
97
Non-specific defenses: physical, chemical, normal flora, phagocytic WBC
- Protect by consuming and killing MO's
- Two main types:
- Neutrophils (PMN's)
- Mactrohages (mature monocytes)
- Non-specific in the sense that a given phagocyte can eat many different species.
- Role in specific immunity
98
Specific immunity: recognizes and attacks specific pathogens
- Usually acquired during lifetime following exposure to specific microbes.
- Can recognizes slight biochemical differences. Can identify self vs. non-self & identify one microbe type vs. another MO type
- Memory: remembers past encounters. Re-exposure with same microbe, results in rapid, intense immune response
- Benefits of rapid intense response: can eliminate microbe before disease occurs, before symptoms occur—you are immune to specific pathogen
99
B-lymphocytes (WBC): Produce antibodies
- AB are proteins that are released from B cells, dissolve in blood & other body fluids.
- Structure: roughly Y-shaped, the “stem” has amino acid sequence-very similar in different AB, “Constant region”.
- The “tips” are the place where AB binds antigens—also referred to as the antigen-binding region.
- Tips can be very different in shape among different AB's.
- Each AB has a unique shape at antigen-bind site which fits antigen of unique shape—complementary shape.
100
A given B cell
- Makes AB of just one shape but have a gazillion B cells, so collectively, all of the different B cells together can make millions of different shapes.
- Cells carry receptors on cell surface receptor—attached antibody fits same antigen as free AB.
101
Antigens:
- Usually proteins, glycoproteins or in some cases—carbohydrates. Molecules that provoke AB production. Each AG has a unique 3D shape that fits a specific AB.
- Location: Found on surface of microorganisms OR may be secreted as toxins, marks MO's as “foreign” different from self proteins, glycoproteins.
102
Response of B cells to infection ~ pathogen enters body
- All pathogens carry foreign AG on surface as pathogens multiply—get more AG in the bod
- AG associated with pathogen, eventually encounters B cell w/ receptor that fits.
- Binding of AG to B cell—triggers a series of events. Triggers series of events—including rapid cell division cells with receptors for this particular AG
- All descendent B cells have identical receptors and will make identical Abs. Since descendents are identical, division event called “clonal expansion”
103
Type of clones:
- Plasma B cells
| - Memory B cells
104
-Plasma B cells
-Act as antibody factories—pour out identiccal AB
-All AB bind to stimulating AG. Antigen-binding sites of antibodies, fit antigens that trigger expansion. Lots of AB that will bind to pathogen.
-Get rise in concentration of specific antibody. AKA “Rise in titer” (increase of concentration of a specific antibody, can be measured through titration)
-Value of rise: diagnostic value, AB type will lead to identifying the pathogen. --Detect AB—know pathogen. Activity of AB will lead to elimination of pathogen. Numerous mechanisms-- lots,
Ex: Opsonization. AB bind to AG on microbe surface, AB-coated pathogen is much easier to phagocytize. Enhance macrophage activity.
105
-Memory B cells
-Much longer lived than plasma B cells~ lasts for many years
-All memory B cell clones, carry the same cell surface receptor (AG) that stimulates colonal expansion.
-So memory cells can bind AG associated w/ specific invading pathogen...yrs later
-In future infections, with this specific pathogen, memory B cells bind to antigens.
-Binding triggers clonal expansion, and more plasma and memory cells...but the process will be faster and more intense than the first time.
-Get much more antibody in less time. Rapidity and intensity, key to preventing symptoms and disease. May not realize you were infected~ are “immune”
-Immunity is very specific for particular pathogens.
Ex. Influenza—many strains, frequent mutations change antigens. (Flu shop is trivalent)
106
T-lymphocytes or “T cells”
| Similar to B cells-- (both lymphocytes) they have specific receptors, a given T cell responds to a specific AG.
- Collectively, among all T cells—can find a huge # of different receptor shapes, can respond to many different AG and pathogens (as a collection of T cells)
- Results of AG binding to T cell is clonal expansion. Clones have same receptors. & produce long live memory T cells.
- Main difference b/w B & T, T cells do NOT produce AB, instead they fight infection other ways.
107
-Types of T cells:
- Cytotoxic T cells, effective against virus-infected cells and tumor cells.
- Kill the “target” cells upon contact.
- Helper T cells, stimulated by Ags “presented” by macrophages, macrophages acquire foreign, AG during phagocytosis.
- When stimulated, produces lots of cytokines (cemokines)--chemical messengers produced by WBC—promote/ coordinate immune response. Stimulate B cells, cyto-T cells, macrophages.
- Lose T helpers, get general decline in immunity. These cells are killed by HIV, die of many infections b/c suppressed immune system
108
-Mechanisms of T cell
1. secrete preforins, proteins assemble on surface of target, creates pores, lose membrane integrity.
2. Induce apoptosis, series of events leading to self destruction
109
Vaccination “Immunity”
Increase in relative resistance to specific disease from specific pathogen.
May be acquired naturally by exposure to pathogen—product of activity to memory cells.
110
Problem with natural immune response (with initial infection)
- May take time to develope to point of protection.
- Pathogen has time to multiply, may develope a serious illness or death
- If first encounter is fatal---memory is irrelevant
111
Vaccination “artificial”
- Generates specific immunity to specific diseases by intentional and relatively safe means to antigen of specific pathogen
- Generate immunity w/o need for severe illness or death.
- Process, vaccine is first exposure to antigen associated with pathogen, will take several days for response to peak—delay is not nearly as dangerous as it would be if full strength pathogen.
- So the peak response with this exposure is delayed but memory cells are created
- If later exposed later to real pathogen, it's immune system's second encounter. Thanks to memory cells, rapid/intense response—don't get sick.
112
Risks of vaccination
- Degree of risk depends on the type of vaccine.
- Include allergic reactions, excess inflammation—usually localized. Some vaccines may (rarely) cause the disease itself. Must weigh risks, risk from vaccine compared to risk of lack of immunity.
- If first encounter is with the pathogen—you could get sick or die.
113
```
Weakened (Attenuated) Pathogen
Description
Advantages
Disadvantages
Examples
```
-Pathogen (usually virus) is weakened; pathogen may multiply in the body, but it is easily controlled by the immune system, and so does not cause symptoms
A:-Usually gives very strong immune system stimulation,
so usually gives longer term protection
-Can spread person-to-person to “vaccinate” others in population
D:Pathogen still alive, so tiny but non-zero chance of causing disease; more likely to happen in very young, elderly and immuno-supressed patients
-MMR (Mumps, Measles, Rubella)
-Sabin Oral Polio
-Nasal Flu
-Chicken Pox
-Small Pox
-Rabies
114
```
Killed Pathogen
Description
Advantages
Disadvantages
Examples
```
-Whole pathogenic bacterial cells or viral particles are killed, usually by formalin
A:If vaccine is properly prepared, then vaccine cannot cause infection and disease symptoms, because dead bugs don’t multiply
D: -Whole, but dead, pathogen may not be as stimulating to the immune system as attenuated pathogen, so protection may be as strong or as long-lasting
-If vaccine is not properly prepared, pathogens may be capable of causing disease that we are trying to prevent
Bacterial:
-Perussis or Whooping Cough (Bordetella. pertussis) in DPT vaccine
-Typhoid Fever (Salmonella typhi)
-Cholera (Vibrio cholerae)
Viral:
-Injected Flu
-Salk Injected Polio
115
```
Toxoid
Description
Advantages
Disadvantages
Examples
```
-Bacterial protein toxins altered by formalin treatment so as to make the protein non-toxic but still immune-stimulating or “immunogenic”
A: Not using whole organism, so no chance of infection with pathogen that we are vaccinating against
D: Not using whole organism, so not as stimulating as whole microbe vaccines, protection may be short-term and more frequent boosting* may be required for max effectiveness
*”Booster shots” or “boosting involves additional, repeated injections with the same antigens
-Tetanus (Clostridium tetni) in DPT vaccine
-Diphtheria (Corynebacterium diphtheriae) in DPT vaccine
116
```
Crude Polysaccharide Fraction
Description
Advantages
Disadvantages
Examples
```
-Vaccines usually consist of antigenic polysaccharides from the capsule of certain bacterial species .
-Polysaccharide may be conjugated or joined to a protein to make it more stimulating or immunogenic
A:Not using whole organism, so no chance of infection with pathogen that we are vaccinating against
D:Not using whole organism, so not as stimulating as whole microbe vaccines, protection may be short-term and more frequent boosting* may be required for max effectiveness
*”Booster shots” or “boosting involves additional, repeated injections with the same antigens
-Pneumonia (Streptococcus pneumoniae)
-Meningococcal meningitis (Neisseria meningitidis)
-HiB meningitis(Haemophilus influenza B) –
Most common cause of meningitis in infants
117
```
Purified Proteins
Description
Advantages
Disadvantages
Examples
```
-Vaccines consist of antigenic proteins from the outer surfaces of pathogenic bacteria or viruses (viral proteins would be capsid or envelope proteins); proteins often produced by microorganisms that are genetically engineered to use and express the pathogen protein genes
A:Not using whole organism, so no chance of infection with pathogen that we are vaccinating against
D:Not using whole organism, so not as stimulating as whole microbe vaccines, protection may be short-term and more frequent boosting* may be required for max effectiveness
*”Booster shots” or “boosting involves additional, repeated injections with the same antigens
Bacterial:
-Anthrax (Bacillus anthracis)
Viral:
-Hepatitis B
-HPV (genital warts)
-Most HIV vaccines in development (so far, none have been shown to be effective)
118
Readings: Vaccines: Victims of Their Success?
What is the evidence that vaccines have made a difference in controlling infectious disease?
What are the factors, circumstances and/or beliefs that contribute to anti-vaccine sentiment?
Where do we get information?
- Table from reading—yes they work
- Lack of trust, side effects. Autism? Correlation with time with appearance of autism symptoms and vaccination. Study—no link.
- Internet
- Worked too well and don't see diseases, measles are still out there-still need to use
