Effects of changes in the microbiota

More and more research groups are seeking to show that changes in the microbiota of a particular site are involved in diseases, such as periodontal disease, inflammatory bowel disease and premature birth. Critics object that showing an association is not the same as demonstrating cause and effect. In the case of the obesity study, scientists tried to do this by inoculating germ-free mice with different variations of the microbiota. Clearly in humans, this would not be possible. How might you prove cause and effect in humans?
Cause and effect is usually proven with experiments and manipulation of variables. However in humans this can be difficult due to ethical considerations. Some have proposed a fifth Koch’s postulate to demonstrate cause and effect by being able to cure or eliminate the disease. Cholera was unable to be re-introduced into an animal model as no animals are susceptible to Cholera, however, by increasing sanitation of water cholera was able to be controlled and in some cases eradicated, proving that the bacteria Vibrio cholerae was responsible for the cause of Cholera. In addition, although Marshall was made infamous when he swallowed a culture of H. Pylori and developed subsequent stomach inflammation and ulcers – skeptics still existed, until the development of antibiotics which were shown to treat and eradicate ulcers. Thus, demonstrating a cause and effect relationship.

Along similar lines, vaccinations that induce immunity against a specific pathogen can also be used to prevent or lessen a disease and again provide evidence of cause and effect. Particularly if the vaccinated individual is introduced to the pathogen and does not subsequently develop disease.

Infants in the first years of life are often more susceptible to certain bacterial infections than older children. How can you explain this? What function of the microbiota does this illustrate?

Members of the microbiota can cause some quite serious diseases. How could a bacterium that normally lives in a benificial or neutral association with it’s human host cause serious disease?

PCR combined with sequencing can provide a quick identification of bacteia. What are the limitations of this approach?
Can be costly.

Resident microbiota provide protection from colonization by some pathogenic bacteria in certain parts of the body. Describe regions of the body where normal microbiota are protective and how they accomplish this protection.

Could you prove cause and effect using something like Koch’s postulates if you could not cultivate the organism you suspect is causing the disease? What type of approach would you use?

What is the advantage of incorporating virulence genes into the chromosome over retaining them on plasmids?
Integration into the chromosome lowers the probability of losing the gene. It circumvents the need for autonomously replicating elements because the genes will be replicated at the same time as the chromosome. This is because plasmids must be under constant selective pressure to be maintained for long periods of time.

Neisseria species are known to undergo both phase and antigenic variation. Define phase versus antigenic variation and describe how each might occur. What advantage do they provide for the pathogen?

What role have bacteriophages plated in pathogen evolution?

Which types of variation in surface antigens would still be seen in a strain of N. Gonorrhoeae that was deficient in the ability to carry out homologous recombination?

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Bacterial cells have the ability to change the set of genes that they express in response to changes in the environment. Explain why the ability to “turn on” only those genes that are needed at a given time is an advantage to the bacterium.

Joe is a sexually promiscuous 22 year old who has had gonorrhea five times in the past two years. Each time he went to the doctor, he was given antibiotics and the infection cleared up. List at least two reasons why N. Gonorrhea was able to cause recurrent infections in Joe.
Changing the surface components allows a certain percentage of the population of bugs to escape the host humoral immune response at any given time (eg. Pilin). This may occur through phase variation or antigenic variation genes. Phase variation of two pilin subunits through the repression of one gene and an inversion of the promoter of another gene which results in the pilin subunit on the cell surface avoiding antibody recognition.

You had to identify a new bacteria but you do not know how virulent the bug is. Design an experiment that allows you to study virulence as well as identify virulence factors.
Model: Infect mice with glowing bug over time using in vivo imager and compare to uninflected mice. This will indicate the peak infection period and if the bug is localized or systemic.

Create a mortality curve comparing infected to non-infected mice OR examine LD50 and/or ID50 which would tell you the lethal dose at 50% of the population.

Using deletion mutants of particular pathogenicity islands or suspected virulence genes, preform a competitive index experiment. If the mutant is less virulent the C1<1 since the WT out competes or has greater fitness than the mutant.

Similarly, you would see less bacteria colonizing via the imaging, and less mortality or less systemic bacteria during the infection.

What are some issues surrounding Koch’s postulates?
1) They assume that all disease symptoms are dependent entirely on the bacterium, yet host susceptibility due to genetic and other factors such as age and proficiency of the immune system, carries in human and animal populations and plays a major role in virulence. Some individuals can be colonized with a potentially disease-causing bacterium yet not develop symptoms of the disease whereas others are severely affected. Not all individuals respond equally to an infectious microbe. In addition, prior infection with one microbe may influence the response of the host to subsequent infections with another microbe.

2) Koch’s postulates assume that a pathogenic bacteria can be readily isolated and cultured. Unfortunately, not all bacteria can be cultured under standard laboratory conditions. Bacteria may change their properties upon being cultured so that they lose some of the traits that allowed them to cause disease (Eg. Biofilm formation.)

3) Koch’s postulates assume that all members of a bacterial species are equally virulent and that a single species causes each disease. This is clearly not the case for all bacterial pathogens. There can be dramatic and distinct differences in disease causing abilities even between closely related bacterial strains.

4) Koch’s postulates require reinoculation into a susceptible host to reproduce the disease symptoms. For humans, this requires either brave volunteers in highly structured settings or, more likely, a good animal model. However an animal model might not be available, and for many diseases non-human animals are only approximate model systems.

What is a modern alternative to detecting and cultivating bacteria in diseased tissues to satisfy Koch’s postulate?
Modern molecular biology approaches such as PCR-based typing. As well as imunohistochemistry to detect bacteria in tissue samples using antibodies against the bacteria that are conjugated either due to an enzyme or that can be used in a colorimetric assay for staining or to a fluorescent dye that could be used for visualization by fluorescent microscopy that would allow bacteria to be detected or visualized in diseased tissue but not in health tissue.

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