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BL222 Medical Microbiology

Fall 2017
Exam 1 (Chapters 1-3; 5-6) Take Home Portion

This exam must be uploaded to Canvas as a Google Doc no later than Friday, September 8th at
11:30 AM. Due to the generous time allotted for completion, any exam received after this
deadline is subject to a 50% grading penalty. 40 points.

Please completely and directly answer the following questions as a work group. In class notes,
supplemental postings to Canvas, and your textbook are excellent resources to help get you
started, but you will need to perform outside research to provide quality answers for most of
these questions. Your outside sources must be:
Scholarly and credible; this includes other textbooks, journal articles, content from
reputable organizations (CDC, WHO, Pasteur Institute, etc.). Use of non-credible sources will be
factored into your grade.
Properly cited via in-text citations and a formatted reference page (APA or Council of
Science Editors format preferred). No direct quotes are permitted in your answers; all material
should be appropriately paraphrased.

Please feel free to consult with me at any time if you have any questions about guidelines,
formatting, or clarification of material. Given the nature of this exam, I am unable to tell you
ahead of time if you have enough material to answer the question, if your answer is correct, etc.
I also strongly suggest that you develop a teamwork contract as soon as possible to help you
fairly divide the workload across your group (provided on next page). Avoid dividing questions
among group members instead of collaborating on this assignment-students who divide up work
separately often earn lower grades than students who collaborate together on the entire
assignment.

Please note that plagiarism will not be tolerated in this assignment. Your answers will be
submitted to TurnItIn.com and evaluated for similarity to source material and to material
submitted by other students. Suspected instances of plagiarism will be reported to Academic
Affairs and will result in severe grading penalties and loss of groupwork privileges.

IMPOPRTANT NOTE ON GRADING: Demonstration of group effort and peer Team Member
Performance Evaluations (administered via CATME) will be taken into consideration for
individual exam grades. In order to track individual effort, each group must complete the
Teamwork Contract on the next page and perform all work within the Google Doc. In order to
earn the privilege of the group exam score, each student must 1) fulfill all obligations as listed
on the Teamwork Contract and 2) demonstrate a level of effort expected for students in a
200-level course. Failure to contribute equally to the group effort will result in grading penalties
and loss of groupwork privileges.

Teamwork Contract (developed in part from Team Dynamics by Kolb and Francoeur)
Mission Statement:
Who is your point person to contact instructor, submit assignments, etc.?
We decided that Alex Barbera was going to be our group leader.

When, where, and how will your team meet?


We will meet up whenever everyone in our group is available. We will mostly come
together in Boyle, but the location did not matter as long as it is secluded. We will discuss the
time and place through text message, deciding what will work for our group.

Will there be a common agenda for meetings?


During our meetings we will work on our take home assignment as a group.

What is expected of each member?


Each member is expected to contribute to each question whether it be ideas for finding
articles, finding articles, or writing out the answers.

How will your team manage conflict?


If two members have conflicting ideas we will just explain each side until a conclusion is
formed.

List some key strengths/preferences of each member:


You might list things like temperament/preferences of each person, work experience, skills (e.g.
strong essay writing, research skills, or good public speaker), area of interest in retail, etc.
Alex: Strong leadership ability and organizational skills.
Bryce: Great at reading and interpreting scholarly articles, good public speaker/explainer.
Hannah: Strong at essay writing and wording, efficient in research and finding credible sources.

How will you ensure members are meeting their personal objectives?
Since we will all come together when we work we can monitor each other as we go.
The following questions pertain to the paper posted on Canvas:

Davis, L.E., Cook, G., and Costerton, J.W. 2002. Biofilm on ventriculoperitoneal shunt tubing as
a cause of treatment failure in coccidioidal meningitis. Emerging Infectious Diseases 8(4):
376-379.

1. (3 pts) Why are patients with indwelling catheters or medical implants so susceptible to
the development of fungal biofilms?
Patients with indwelling catheters or medical implants are susceptible to fungal biofilms
because they are able to adhere to broad variety of surfaces including catheters and medical
implants (1). C. albicans is an opportunistic pathogen so healthy individuals with this fungus
growing in their body, usually in their gastrointestinal tract, do not have any kind of disease
caused by this fungus (1). Patients with a medical implant are likely undergoing stress due to
surgery or another illness that is weakening their immune system so C .albicans, and other
fungal pathogens, have the opportunity to grow and infect the host. C . albicans is resistant to
antimicrobial agents, which in turn allows the biofilm to grow (1). Its resistance is attributed by
several properties, with upregulation of efflux pumps contributing to its resistance to antifungal
agents (1). Another agent is the biofilms extracellular matrix and its component beta-1,3-glucan,
which adds to the fungal resistance to drugs. In order for a pathogen to be successful, it must
be able to attach to its host, evade the hosts defenses, and grow (1). With that being said, this
pathogen is very successful in this scenario.

2. (5 pts) The patient was initially treated with the antifungal drug fluconazole (800 mg/day).
How does fluconazole work to treat a fungal infection? Would you expect fluconazole to have
negative effects on human cells? Fully explain your answer.
Fluconazole is an antifungal drug that works by attacking a cells membrane
functionality. It does this by stopping the fungal cells production of ergosterol, an important
molecule for the production of the cell membrane of the fungus (2). Without this molecule the
fungal cell membrane will hyperpolarize which leaves the cell unable to undergo many vital cell
processes (3). The fungus would then die and the infection would lessen. Human cells lack the
molecule ergosterol, a fungus-specific cholesterol, therefore the drug should only affect the
fungal cells and have no negative effects on humans(2).

3. (5 pts) Why did the healthcare team culture this infection using Sabouraud dextrose
agar? In your answer, be sure to fully explain the properties of this growth media (selective,
differential, enrichment, etc). What types of organisms could grow on this media, and how did
these results provide diagnostic information to the healthcare team? Do any special precautions
need to be taken when culturing Coccidiodes immitis in the laboratory setting?
Sabouraud dextrose agar is a selective agar used for the cultivation and growth of fungi
(2). It favors fungal growth like, Coccidiodes immitis, as compared to bacteria due to its acidic
pH, presence of antibiotics, and its ability to enhance the visibility of characteristic spores and
pigment in the fungi (2,4). The presence of growth on the plate allows healthcare providers to
draw a conclusion that the infection was fungal as opposed to a bacterial infection. C occidiodes
immitis must be cultured in biosafety level 3 laboratories (5). Laboratory personnel are expected
to be experienced in handling dangerous pathogens. The handling of C occidiodes immitis must
be done in biosafety cabinets, other physical containment devices, or wearing appropriate
protective clothing (6). It is also expected that facilities have airlock entries and directional
airflow that has the air that enters the lab exit to the outside of the facility (6). Because
sporulation is possible with Coccicioides immitis, unsafe laboratory procedures could result in
coccidiomycosis (7).

4. (4 pts) Diagnosis of Coccidiodes immitis was supported by staining with calcofluor white
and light miscroscopy. How does calcofluor white stain these types of cells? What would the
medical technician expect to see in a sample containing C. immitis stained with calcofluor
white?

Calcofluor white is a stain that binds to the chitin and cellulose found in fungi cell walls
(9). This stain works as a quick but nonspecific method to easily identify fungal infections in
tissues (8). When stained, the elements of the fungus were sharply defined upon use of a
fluorescence microscope (8). Because it is the chitin and cellulose in the fungal cell wall that is
targeted by the calcofluor white, when a sample of C.immitis that is stained with this particular
stain is observed under a fluorescence microscope, only the cell walls of the fungus and
endospores will be illuminated (10). The cells of C.immitis would look like large spheres with
several nuclei and some would contain endospores (15). The human cells would not be
illuminated because they do not have cellulose or chitin so a medical microbiologist would be
able to clearly pick out C.immitis amongst human cells.

5. (5 pts) Would it be possible to provide a definitive diagnosis of C. immitis using


immunofluorescence microscopy alone? Fully explain your answer, and describe one specific
example of how immunofluorescence microscopy can be used to diagnose an infection.

It would be possible to provide a definitive diagnosis as immunofluorescence microscopy


uses antibodies to specifically bind to one microbe (16). Adding the fluorescently treated
antibody that is specific for C.immitis into a sample of an unknown pathogen would definitively
determine whether or not C.immitis is present based on whether the antibodies attached and
illuminated the pathogen (16). An example of how immunofluorescence microscopy aids in the
diagnosis is that of autoimmune-vesiculo bullous disease of the skin (17). This type of
microscopy is a helpful tool to come to an exact diagnosis because it affords the opportunity to
detect the presence of antibodies that are specific to the disease (17).
6. (5 pts) Which portal(s) of entry was/were most likely utilized by C. immitis in this
particular patient? Propose a hypothetical mechanism of transmission explaining how this
patient became infected with this fungal pathogen.

The infection could come into contact with the patient by indirect and/or vehicle
transmission (2). This contact could be that of dirt, where spores can be present, or by
aerosolization(11). The portals of entry could potential be entry through skin and surgical
application (2).
It appears as though the skin lesion was the initial portal of entry for the mans
Coccidioidal meningitis (15). The mans bloodstream could have become infected with the
fungus or the endospore by the mans skin or open wound coming in contact with dirt containing
the fungus. This would have caused the lesion. The endospores could have adhered to the
shunt after the shunt was inserted into the patients ventricle (15). Since the CSF and serum
were tested positive for the presence of C. immitis antibodies, the spores could have adhered to
the shunt via the cerebrospinal fluid and initiated a biofilm before the medication was able to rid
the patient of the infection (15). This would explain why the disease would have seemed to have
gone away for four years, because during these four years the shunt could have harbored the
growth of an antifungal resistant biofilm that took time to grow and affect the man.

7. (8 pts) Provide a two paragraph summary (including scholarly sources) of what is


currently known about the role of biofilms in antifungal drug resistance of pathogenic fungi.

Biofilms are becoming increasingly resistant to common antifungal drugs such as


fluconazole. This increase stems from the bringing together of common human pathogens
together in one biofilm. Once together, these organisms are able to combine their drug resistant
ability, which is the case with C. albicans and C. dubliniensis. These two pathogens possess two
different efflux pumps allowing them to be resistant to more antifungal drugs together, more
specifically in this situation fluconazole (12). In fact, genes like MDR1, responsible for fluconazole
resistance, and CDR are upregulated when the biofilm is forming and developing(14).
The upregulation of genes that code for ergosterol synthesis, drug resistance transporters
and improved adherence, are present in fungal biofilms (13). Essentially, the presence of the biofilm
is improving the expression of the genes that improve membrane function, rid the fungal cells of
antifungal drugs, and improve the cells abilities to adhere to the host (13). Primary metabolism
genes, that synthesize amino acids, are upregulated in biofilms in order to form the biofilm (13). By
improving these components in all the individual cells within a biofilm, the fungal infection is more
successful at evading the effects of antifungal drugs.

8. (5 pts) Go to NCBI PubMed to research clinical investigations of the role of quorum


sensing and biofilm formation in other infections. Select a paper and provide the citation in CSE
format. Write a one paragraph summary of the paper for an audience of healthcare
professionals.

Quorum sensing has been found to be a factor in the formation of certain biofilms,
especially in the early stages of development. Another benefactor of quorum sensing is that it
allows a biofilm to move in a process called swarm motility, which is considered to be based off
of biofilm formation in its early stages (18). When a biofilm forms, it is able to send out a signal
to organisms that are passing by telling them that this is an area that has a lot of nutrients (18).
A fixed biomass with a high concentration of 3-OH-C12-HSL, an AHL chemical used in quorum
sensing, increases the formation of the biofilm, while a low concentration had no effect on the
formation (18). In addition, when the biofilm was also treated with 3-oxo-C8-HSL, another
quorum sensing chemical, the formation increased (18). These chemicals and their
corresponding formation rates supports the idea that multiple molecular signals can affect the
formation of the biofilm. N-acyl homoserine lactone or AHLs are a type of signaling molecule
utilized by many gram-negative bacteria, like the Rhizobium etli that is investigated in this article
(18). Quorum sensing is dependent on the presence of these signaling molecules and therefore
the regulation of biofilm formation is done by AHLs in the genus of Rhizobium (18). Overall,
quorum sensing plays an important role in the formation of biofilms and its related processes.

References
1. Nobile CJ, Johnson AD. Candida albicans Biofilms and Human Disease. Annual Review
Microbiology. 2015;69:7192. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930275/

2. Aliabadi Z, Foster J, Slonczewski. Microbiology The Human Experience. New York (NY):
W.W. Norton & Company; 2016.

3. Elicharova H, Sychrova H. Fluconazole treatment hyperpolarizes the plasma membrane of


Candida cells. Medical Mycology.
2013;51(8):785794.https://academic.oup.com/mmy/article-lookup/doi/10.3109/13693786.2013.
779038

4. Das S, Sharma S, Kar S, Sahu SK, Samal B, Mallick A. Is inclusion of Sabouraud dextrose
agar essential for the laboratory diagnosis of fungal keratitis? Indian J Ophthalmol.
2010;58(4):281285.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2907027/

5. Fisher MC, Koenig GL, White TJ, San-Blas G, Negroni R, Alvarez IG, Wanke B, Taylor JW.
Biogeographic range expansion into South America by Coccidioides immitis mirrors New World
patterns of human migration. Proceedings of the National Academy of Sciences.
2001;98(8):45584562. http://www.pnas.org.setonhill.idm.oclc.org/content/98/8/4558.full#ref-10

6. Biosafety Level 3 (BL3) NIH Guidelines for Research Involving Recombinant DNA Molecules
Appendix G-II-C. University of South Carolina.
https://www.sc.edu/ehs/Biosafety/Biosafety%20Level%203%20(BL3)%20-%20Appendix%20G-I
I-C.pdf

7. Section VIII-B: Fungal Agents - Centers for Disease ... Center For Disease Control. [accessed
2017 Sep 7]. https://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_viii_b.pdf

8. Monheit JE, Cowan DF, Moore DG. Rapid detection of fungi in tissues using calcofluor white
and fluorescense microscopy. Archives of Pathology & Laboratory Medicine.
1984;108(8):616618.http://europepmc.org/abstract/med/6204621
9. Saubolle MA, McKellar PP, Sussland D. Epidemiologic, Clinical, and Diagnostic Aspects of
Coccidioidomycosis. Journal of Clinical Microbiology. 2006;45(1):2630.
http://jcm.asm.org/content/45/1/26.short

10. Maza LMDL. Coccidioides immitis Eagle K, editor. New England Journal of Medicine.
1993;329(26):19351935. http://www.nejm.org/doi/full/10.1056/NEJM199312233292606#t=article

11. Fungal Diseases Valley Fever (Coccidioidomycosis). Centers for Disease Control and
Prevention. 2017 May 22 [accessed 2017 Sep 6].
https://www.cdc.gov/fungal/diseases/coccidioidomycosis/index.html

12. Fungal Biofilms and Drug Resistance - Volume 10, Number 1-January 2004 - Emerging
Infectious Disease journal - CDC. Centers for Disease Control and Prevention. 2010 Dec 21
[accessed 2017 Sep 6]. https://wwwnc.cdc.gov/eid/article/10/1/03-0119_article

13. Fanning S, Mitchell AP. Fungal Biofilms. PLoS Pathogens. 2012;8(4).


http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002585

14. Jabra-Rizk MA, Falkler WA, Meiller TF. Fungal Biofilms and Drug Resistance. Emerging
Infectious Diseases. 2004;10(1):1419.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031105/

15. Davis LE, Cook G, Costerton JW. Biofilm on Ventriculoperitoneal Shunt Tubing as a Cause
of Treatment Failure in Coccidioidal Meningitis. Emerging Infectious Diseases.
2002;8(4):376379.

16. Petris SD. Immunoelectron Microscopy and Immunofluorescence in Membrane Biology.


Methods in Membrane Biology. 1978:1201.
https://link.springer.com/chapter/10.1007%2F978-1-4613-4036-2_1

17. Babu RA, Chandra KLP, Chandrasekar P, Kumar KK, Reddy BVR, Reddy GS.
Immunofluorescence and its application in dermatopathology with oral manifestations:
Revisited. Journal of Orofacial Sciences. 2013;5(1):28.
http://www.jofs.in/temp/JOrofacSci512-5337121_144931.pdf

18. Dixit S, Dubey RC, Maheshwari DK, Seth PK, Bajpai VK. Roles of quorum sensing
molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation. Brazilian Journal
of Microbiology. 2017 Jul 8.
http://ac.els-cdn.com/S1517838217306147/1-s2.0-S1517838217306147-main.pdf?_tid=71c90a
0c-9337-11e7-b147-00000aacb362&acdnat=1504725380_1d9be2c0977eb4dac89d98755bfa0b
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