exam 3- final

What is the main source of antibiotic resistant microorganisms? Explain

From meat production, with sub therapeutic treatment of animals leading to increased resistance. This can be seen be comparing Norway (where practice is banned) to other countries where the practice is common

What is the difference between clinical resistance?

Lab resistance is based on how bacteria respond to antibiotics in controlled conditions, while clinical resistance refers to failure of treatment in. a patient, which also depends on factors like drug delivery, metabolism, and immune response

Why is the doctor exaggerating when they comfort a patient by saying that they will give them “a stronger antibiotic”?

There is no such thing as a stronger antibiotic. It can be narrow or broad spectrum, but they are just different antibiotics

A specific antibodies- Magic Bug Zapper - works great in the laboratory. High levels of killing are seen in broth cultures. It is not too toxic for humans to use. You use it to clean your kitchen. Dr. Culture then successfully cultures bacteria from many surfaces of your kitten! Why didnt Magic Bug Zapper work as advertised?

Number of bacteria, Debris on the counter, under stress

mare variety, grooves in the counter

What are the three main mechanisms of drug resistance?

1. Mutate the target: change the target in the pathogen that the antibiotic would use

2. Pump out the drug: the pathogen removes the frug as fast as possible

3. Chew it up: the pathogen breaks the drug into pieces

4. (Fail to activate: some drugs don’t work with certain pathogens)

Small Colony Variant (SCV) Staphylococcus aureus grows very slowly. In the laboratory SCV S.aureus is often found to be resistant to multiple antibiotics without having resistance genes. How did this happen?

The slow growth that is resistant to many antibiotics are most likely in the “Viable but not cultivable phase.” During slow growth inhibitors don’t inhibit enough.

What is a “Resistance Gene Cassette”? Why is this espeically problematic?

It is a mobile DNA element that has insertions sequences around it that also contains antibiotic resistance genes, and it is problematic because it can be easily transferred between bacteria, spreading resistance quickly.

Where are resistance gene cassettes often found in bacteria? How does this facilitate their movement?

Found in:

1. Transposons: jumping gene segments

2. Plasmids: easily moved pieces of DNA

3. Phage: virus DNA, easily moved

gene cassettes are easily lost but can also be maintained in a population. Bacteria that are under stress are also more likely to accept foreign DNA

Why is it significant the Bordatella pertussis adheres to ciliated cells?

Function of ciliated cells is to move mucus, adhere to ciliated cells, and kills them, requires more coughing to clear the mucus and aids in transmission

Explain what a “Two component system” is. What is the role of each of the two components? Use BvgAS as an example.

composed of sensor kinase BvgS and response regulator BvgA, dimerize the response regulator and bind the DNA, turn on/off>200 genes, has 3 states (on, intermediate, off) depending on environment

What is the significance of Bvgi state in the BvgAS system?

at low levels of BvgS activation, some virulence genes are turn on (adhesins) but others are not (toxins). This allows early colonization without full virulence

Why are “avirulent strains” often seen late in infection in children recovering from whooping cough? How do they develop?

after infection bacteria are in altered conditions. These conditions select for bacteria that down regulates virulence factors. The BvgS gene has a frame shift in Bordetella which causes it to be down regulated, therefore causing it to grow slowly and persist, allows prolonged shedding (transmission) for later inefficient transmission

What is the role of FHA in Bordatella pertussis?

Major adhesion of the Brodetella genus. Forms along beta helix and forms filamentous structures on the surface of the bacteria. Secreted by a two-partner secretion system. Mediates adherence to ciliated cells in the respiratory tract; binds to galactose on glycolipids binds to CR3 receptor on neutrophils, and mediates bacterial aggregation

What is the role of tracheal cytotoxin in Bordatella pertussis?

Kill= ciliated epithelial cells, and inhibit neutrophil chemotaxsis

How is tracheal cytotoxin an unusual toxin?

release fragments of peptidoglycan as a toxin, despite the fact that Bordetella is a Gram - . Also N.gonorrheae appears to secrete a TCT that is implicated in fallopian tube death

What is the role of adenylate cyclase toxin in B. pertussis virulence?

The segments of it mediate attachment to target cells and form a pore in the plasma membrane, the toxin causes ionic imbalances in target cells through the pore formation, can survive in Mo (switches them to M2)

What are three roles of pertussis toxin?

1. Functions as an adhesion

2. Inhibits neutrophils and macrophages

3. non-specifically activates T cells

What is the role of the biofilm in B. perussis?

Biofilm formation is induced after the TCT has destroyed epithelial cells to uptake the nutrients given by these cells. The persistence phenotype is upregulated and an increase in mucin is seen. Aids in adherence to respiratory epithelium, protects against immune cells, increase resistance to antibodies, allows long-term colonization of the airway

Nothing is perfect. Use the pertussis vaccine as justification of why generally one should vaccine children

1 in 300,000 get CNS symptoms, versus 1 in 1000

new vaccine has less side effects

The acellular pertussis vaccine is extremely safe. However in the infant baboon model acellular pertussis vaccine does NOT prevent B. pertussis carriage. Why is this a problem?

The bacteria stayed in the baboon’s throat, so it was still contagious even when there are no symptoms. Only reduces symptoms and reduces shedding by 50% then we get carriage states

What are Molecular Koch’s Postulates?

1. Gene must be present in all strains bad

2. Mutate gene

3. Mutant into animal must have reduced virulence

4. Reduction of the gene restores virulence

5. Protective immunity must be protective

How can a metabolic gene sometimes become a virulence factor?

Often during the specific disease processes that are unique metabolic stresses- say a lack of amino acid

How can a loss of a gene lead to increased virulence, why?

Pathogens need to balance disease with transmission, Some virulence genes suppress growth of organism into host, (“Healthy” host transmits disease, “bed ridden”host stops the spread)

You have identified Gene X> You propose in your thesis that it is a virulence factor. However it is not expressed in the animal model. What are two reasons it may be involved in the pathogen success in the host and transmission to a new host

it may be expressed only under specific human conditions not replicated in the animal model, and it may function during transmission or environmental survival rather than during infection in that model.

Explain the concept of a keystone paothgen

pathogen necessary to stat the process. may not”cause” the disease, but causes the events that lead to the disease. If other bacteria are treated, it amy decrease but not stop.

What is dysbiosis? How does it lead to disease?

Complex normal flora is killed by antibiotics leaving a less diverse normal flora, and potentially a badly altered normal flora

An organism is unculturable. How else could you prove it causes the disease?

human experimentation, animal models, tissue models, PCR

What are the main models for studying human disease?

animal models, tissue model, human experimentation

What was the Tuskagee Experiment? What was wrong with it?

USPHS study from 1930-1972 in which 400 black males with syphilis (and 200 control without the disease) were offered “free healthcare” expect no treatment for their syphilis was administered, as multiple available treatments were actively withheld in order to collect data on the progression of this disease. Suffering could have been prevented for these men and their families, yet was consciously permitted. They were used like lab rats rather than as human beings. And race was obviously a factor in this decision.

Who was Jesse Gelsinger? What was wrong with this study?

Jesse Gelsinger had ornithine Transcarboxylase Deficency Syndrome, which is the failure to digest protein correctly that leads to ammonia build up, then death. He had to take meds to remain stable. He was informed of a gene therapy experiment at U Penn and he went to participate when he turned 18. No benefit to participate, and any positive results would only last 4-6 weeks. The virus was infused into his liver at very high levels in spite of previous complications, in otherwise healthy patients. Within increases doses previous patients were having worse and worse complications. Virus infused into Jesse’s liver at high levels and within hours, Jesse went into a coma. He later died

Study population versus Treatment population. Why are results from studies not always generalizable?

Study- select certain conditions -DEFINED- exclusion criteria.

Actual population- once licensed no exclusion criteria exist in reality

What are some issues with human experimentation?

• experimental infections: must be treatable, disease must be modest not permanent

• drug studies: treatment must be compared to correct best practice available, must be likely better than current best

• Disease progression studies: no treatment- only possible for untreatable disease

• Vaccine studies: only possible to test in humans

*human participants are at the mercy of the ethics of the person doing the research

What is the illusion of evidence based medicine?

Almost all large studies of humans are paid for by companies, they make money by selling products not by a running a study. There is failed regulation and commercialization of academia

What are some limitations to using animals as models for human diseases?

1. means of attachment to the host is often different

2. Different types of symptoms in a different host

3. Different types of immune responses are often observed

4. Disease symptoms may develop very slowly

5. Animals cost $$$

What are some practical problems in working with animal models?

Human specific disease is a major challenge often need to use immune compromised animal models, clones of mice that are not the same lead to different results even when it is the same clone because of reproducibility is hard, and animals are expensive thus most experiments are of short duration

What are some advantages of using animal models?

12-16?

Why is the mouse model used so commonly, what are its advantages?

cheap, easy to maintain, easy to clone, allows for a complete control over their diet and activities, similar enough to a human in most cases

What are knock mice? What are knock in mice?

Knock out: large # genes genetically removed,

Knock in: humanized mice, mice w/ humans genes inserted, allows some diseases to be studied in mice

What is meant by this statement about research: Reproducible does not equal Meaningful

A result can be consistently repeated (reproducible) but still lack real biological or clinical significance, meaning it doesn’t actually matter or apply in a meaningful way. Lab conditions are controlled and ideal, real life is not

What is ID50 and LD50? How are they determined?

ID50: Infectious dose, where 50% are infected

LD50: Lethal dose, where 50% are killed

endpoint for experiments in animal models. How many are infected, and how many were killed

What is the disadvantage of using ID50 and LD50?

Crude measures; miss anything subtle. Infection does not equal disease

What is biophotonic imaging?

uses a sensitive camera to detect where fluorescent bacteria (proteins) congregate in an infected mouse (or any moderately thin organism/tissue)

How do competition experiments work? What is the advantage, disadvantage?

Mix 2 bacteria mutants and infect the same animal, measure if they both survive at same rate, sine nay make enough of X so the mutant gets compensates for even if it doesn’t make X

The advantage is seeing if there are subtle difference in survival. The disadvantage is one strain may compensate for another

What is tissue culture? What are the advantages and disadvantages?

Tissue culture is the growth of cells in a controlled lab environment outside the organism. It allows for controlled, reproducible experiments and easy observations, but lacks the complexity of a whole organism (immune systems, interactions), so results may not reflect reality

What is meant by a polarized cell line? Why is the use of such a cell line important?

Polarized cells have a distinct apical and basolaterial surface. Cells grown on extracellular matrix or on special filter disks with media on both sides. These cells are grown in environments more similar to those in living tissue. They can form tight junctions and functional barriers. They offer tissue specific research

What is organ culture? What are the advantages and disadvantage?

Growth on piece of tissue often from deceased, surgical intervention person

What is a gentamicin protection assay? How does it work, is it done in basic terms.

Gentamicin is not absorbed by the host cell; kills extracellular bacteria ONLY; it can determine which bacteria are extracellular and intracellular based on which one dies when a cell is treated with gentamicin. The bacteria are given time to attach to and enter the cell, then it is treated with gentamicin, then the results can be measured

What is plaque assay in bacterial pathogenesis used for?

Used to look for “moth eaten holes” in a monolayer of cells, is used to study bacteriophage in bacteria and bacterial/viruses in animal cells

How can fluorescent compounds/antibodies be used to study pathogens?

To see where bacteria or protein aggregate and what they adhere to in a host, colocalization. etc.

by identifying a fluorescent compound that binds to a protein, specifically antibodies, visualization of anything the antibodies bind to can be achieved. Direct viewing of samples is made possible with this technique, since fluorescent staining is so specific. Wide range of sampling can be tested from dirt to pure culture

How does purification of culture supernatants allow for virulence factor identification?

We can isolate secreted toxins, proteins, etc (virulence factors), and test them easier

You have an avirulent strain that can be genetically modified. How can you identify the virulence genes in your virulent strain using this strain and genetic methods?

Compare the genomes for both the virulent and avirulent strain. Clone the virulence genes of choice from the virulent strain and introduce them to the avirulent strain. Test if the now modified avirulent strain acquires the ability to cause disease in an infection model

How does Random Mutagenesis Experiments allow for virulence factor identification?

Inactivating genes at random usually with a transposon, test the mutants

How does identifying immunogenic proteins allow for virulence factor identification?

The virulence factor can be identified based on the antibodies the host makes to fight the pathogen. These are often surface exposed proteins

You make mutants. You are told to watch polar mutations? What are polar mutations versus non polar mutations?

Polar- a mutation in the chromosomes that can affect more than one gene

Non-Polar- only effect expression of one gene

How does determination of coregulation allow for virulence factor identification? How does one identify genes that are coregulated?

coregulation is the switch that turns on specific genes and once found can be looked for in other genes

• mutate the regulator - look for other genes turned off too

• what conditions genes are expressed in - what turns on at same time

• What sequence the regulator binds to - is it in other promoters

What is reporter expression?

Attaching a tag to a protein, or using a promoterless reporter gene that creates either a visible tag (colorful) or adds a tag that can be easily seen (by adding something like an antibody)

What is immunoprecipitation?

The use of antibodies to bind to Protein/DNA/RNA/Lipids in the cell and pull out the antigen (and other molecules associated with it)

How does plasmid identification allow for virulence factor identification?

often genes are involved in pathogenesis are on extrachromonal DNA, This DNA can be isolated and sequenced

How does identification of gene cassettes allow for virulence factor identification?

pathogenicity islands- genes involved in pathogenesis are clustered in gene cassettes, transposons, or lysogenic phages. Once one gene is identified, neighboring genes may be pursued altered %GC content is a common signature of these cassettes

What is genome screening?

It is a way to see every gene in a bacteria and to ID as many genes as possible, however on average only 70% can be positively identified for what function they perform. We can look for motifs associated with virulence factors

What are some ways of doing genome screening?

Genome subtractive hybridization, SCOTS (Selective capture of transcribed sequences), and microarray analysis

What is SCOTS?

Selective capture of transcribed sequences, where you compare RNA from two samples and look for expressed genes under condition of interest

What is a Microarray? How is it performed?

Availability of the genome allows you to identify short segments of each gene that are unique. Essentially a barcode. Barcodes are printed on a glass slide, thousands of barcodes called oligos. Virulent and Avirulent strains can be compared

What is Signature Tagged Mutagenesis?

make thousands of mutants (each with unique seq= barcode), grow 50-100 mutants at a time and infect animal, collect remaining bacteria for animal and identify bacteria that failed to survive, Go back to original plate and identify mutated gene, Takes A LOT of work, IDs many things that may have otherwise been missed.

What is TN-Seq Technology?

1. Create transposon library

2. Pool high density transposon mutant library

3. Grow transposon library under desired conditions

4. Attach sequencing adaptors and amplify transposon junctions from library DNA

Result: high throughout sequencing of insertion sites in each library

Condition A: in vitro growth; input pool

Condition B: in vivo growth; output pool

5. Map and count reads for each insertion (for conditions to determine any gene expression unique to the output pool)

What is IVET?

In vivo expression technology. If you know a gene is essential for growth in the host, but not the lab, then you can identify genes that are expressed if mutated. Gene identified is not necessarily needed for pathogenesis but is imply highly expressed in the host. Salmonella enterica typhumurium requires genes for purine synthesis- only bacteria expressing purine synthesis will survive in the host

What is the problem with only studying gene RNA levels and not protein levels?

They can be at different concentrations, due to protein degradation/stability, is the protein even active form?

What are protein microarrays?

Bacterial proteins can be spotted on a glass slide. antibodies from patients can be added. Only proteins expressed in vivo should be detected

Where is plague found in the USA?

Western half of USA higher in four corners region

How is Yersinia pestis transmitted?

1. Vector borne- flea

2. Vector borne- lice

3. Hunters- accidental inoculation into wounds while cleaning animals

4. Airborne- extremely contagious, biological weapon

Based on models of plague transmission and the black death- how did plague spread during large outbreaks in 1349-1352?

combination of human ectoparasites, rats, and fleas, and pneumonic plague

What happens during bubonic plague?

Most common form of plague from a lice/flea bite, travels through the lymph node swelling (buboes), when it enters the blood stream causes septicemic plague and necrosis of skin

What happens during pneumonic plague?

results from infection of the lungs either during bubonic or septicemic plague or from inhalation of infected droplets. A rapid infection with death in less than 3 days. mortality rate is 100% anf even with good medical treatment, it is over 50%. Extremely contagious with a transmission rate at 95% via aerosol route

Black death refers to what symptoms?

Necrosis turning the tissue (fingertips) black

Yersinia pestis is clonal. What does this mean?

ALL isolates of Y. pestis are similar, difference is 76 nucleotides which is very little.This means it is fairly recent, treatment wise it doesn’t matter where you catch it.

What allows Yersinia to grow on the proventriculus? What is the effect of growth on this part of the flea? How does this aid in transmission?

Uses Hms genes to produce an extracellular matrix and form a biofilm in the proventriculus. The biofilm clogs the proventriculus so that food cannot be swallowed. This helps transmission because the flea is blocked it cannot properly feed repeatedly bites causing it to regurgitate Yersinia in to the host during feeding

The infectious dose of plague is less than 10 organisms. How does this allow for transmission?

It allows for more chances of successful transmission by needing very few organisms to survive and enter a new host. The flea is releasing a small volume then it is critical that the infectious dose is small.

The infectious does of plague to a flea is 10,000 organisms. What is the implication of this in transmission form blood to flea?

The flea only takes a small blood meal, which means that there needs to be 100 million Yersinia per mL of blood for transmissions to the flea. This much should cause septic shock but doesn’t, so it must evade inflammation. really it just means there is an immense amount of bacteria in the blood the flea consumes in order to spread the pathogen

What is the role of the main genes (Hms and Ymt) turned on at 26 C?

Hms- biofilm formation

Ymt- survival in the midgut

How does Yersinia pestis normally survive in the skin? What is role of the two main virulence genes at this point in time (Pla and Yersiniabactin)?

dissemination from the skin into the tissues via lymph nodes

• Pla (plasminogen activator) degrades complement, allows survival endothelial cells, binding plasmin and prevent blood clots

• Yersiniabactin binds iron

What is the role of the macrophage in Yersinia pestis in going from the skin to the lymph node?

Entry stage lives intracellular in MOs, it isn’t killed by MOs efficiently so it grows in them and uses them for transport. It is killed by Neutrophils efficiently. Once it exits MOs, Y. pestis has had time to express genes needed in mammals

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