16US MLSC4061Advanced Clinical Microbiology
Name: ______
Module 3 Assignment
Instructions: Answer the following questions. Follow theguidelines in the course syllabus for proper submission of your document.All references, including course materials, must be cited using APA guidelines at the end of the document. If the answer was found in course materials, a citation is required. If a direct quote is used, the reference should be cited using APA style. Failure to follow the citation directions will result in a deduction of 10% of the possible assignment points.
Grading: A total of40 points will be converted to 20 points for this assignment.
Due Date: Thursday, June 23rdat 11:55 PM (EST)
Part I: Answer the following questions about Anaerobic bacteria.
1. What 2 enzymes may anaerobes lack to protect them free radicals? / Dismutase, Catalase2. List two predisposing factors for anaerobic infections. / Trauma to skin/mucous membrane, Vascular stasis, Tissue necrosis, etc.
3. Describe the benefit of using BBE agar in an anaerobic culture. / It inhibits most facultative anaerobes and allows presumptive identification of BacteroidesfragilisGroup
4. Name one method of treatment, besides antibiotics, that may be used to treat gangrene. / Amputation or Hyperbaric oxygen
5. Which genera of anaerobes produce spores? / Clostridium
6. Which foodborne disease is characterized by flaccid paralysis? Name the organism that causes this disease? / Botulism, Clostridium botulinum
7. Which disease is characterized by convulsive muscular spasms, difficulty breathing, and lockjaw? Name the organism that causes this disease. / Tetanus, Clostridium tetani
8. What are the 4 main Genera of anaerobic GNB? / Prevotella, Porphyromonas, Bacteroides, Fusobacterium
9. If an anaerobic GNB is resistant to Vancomycin, Kanamycin, Colistin, and Bile, what group or organisms does it belong to? / Bacteroidesfragilis Group
10. Lipase and Lecithinase are enzymes that are useful in speciating what genus of anaerobes? / Clostridium
11. Large, boxcar GPB with a double zone of hemolysis are characteristic what organism? / Clostridium perfringens
12. f a sample is positive on the Glutamate dehydrogenase, what organism is present in the sample? Does this mean the organism is causing disease? Why or why not? / Clostridium difficile
No
Must demonstrate toxic production
13. Which specialty media is used to isolate the organism from the prior question? What is the characteristic appearance of colonies grown on this media? / Cycloserine-Cefoxitin-Fructose Agar (CCFA). Selective and differential for C. difficile. Other organisms will grow on CCFA butC. difficilehas characteristic colonies: Yellow with aground-glass appearance. Fermentation of fructose causes color of agar to change to yellow
14. Describe the gram stain morphology of Fusobacterium. / Longer, slender, fusiform, tapered, GNB
15. Name 3 specialty strength antibiotic sensitivity disks used to characterize anaerobic GNB. / Vancomycin, Kanamycin, Colistin
PART II: ANTIBIOTIC MATCHING REVIEW – use each once.
Antimicrobial Review: Match the antibiotic with the mode of ACTION.Antimicrobial Group / Effect on bacteria
E__ Fluoroquinolones, Rifampin
B__ Aminoglycosides, Tetracycline
A__ β‐Lactams
D__ Polymixins
C__ Sulfonamides, Trimethoprim / A. Inhibit cell wall synthesis
B. Inhibit protein synthesis
C. Disrupts metabolic processes
D. Disruption of cell membrane
E. Inhibit DNA synthesis
Antimicrobial Review: Match the antibiotic with the mode of RESISTANCE.
Antimicrobial Group / Mechanisms of Resistance
E__ Aminoglycosides
A__ Fluoroquinolones
C__ β‐Lactams
B__ Vancomycin
D__ Macrolides / A. Decreased uptake, altered target
B. Altered target, target overproduction
C. Enzymatic destruction, altered target, decreased uptake
D. Efflux, altered target
E. Enzymatic modification, decreased uptake, altered target
Part III: CASE STUDIES
Case 1
A microdilution tray with dilutions of four antibiotics is set up and incubated at 35°C for 24 hours (Image 1, patient X). A Quality Control strain of E. coli is set up and run at the same time (Image 2). This QC strain has a known MIC range of values for each of the four drugs being tested. Image 3 is a patient (Y). Review the data below and proceed to answer the following questions.
Antibiotic Sensitivity Reference TableRow / Drug / Susceptible BP
ug / ml / Resistant BP
ug / ml / E. coli QC MIC range ug/ml
A / Ciprofloxacin / ≤1 / ≥4 / 0.5‐2
B / Ampicillin / ≤8 / ≥ 32 / 1‐4
C / Gentamicin / ≤4 / ≥ 16 / 0.5 ‐2
D / Cephazolin / ≤8 / ≥ 32 / 2‐8
a.Using the provided reference chart, interpret the AST for patient X (image 1).To complete the table below, list the MIC of each drug (A through D) for the patient X isolateand the interpretation (S, I or R) for the report.
Drug / MIC ug / ml / InterpretationCiprofloxacin / 2 / I (Intermediate)
Ampicillin / > 256 / R (Resistant)
Gentamicin / 0.5 / S (Susceptible)
Cephazolin / 64 / R (Resistant)
b. Based on the MIC data for the QC isolate (Image 2), state your interpretation on the validity of your tests and explain your subsequent action. /
Based on the QC run, Ampicillin has an MIC of 16 against the QC E.coliwhich is above the range expected. Ampicillin should not be reported. The QC test should be repeated. If it is OK, the Ampicillin report can be released. If it is still out of range, a cause must be investigated.
c. Patient Y appears to have endocarditis, an infection of the heart valves. Multiple blood cultures have grown what appears to be a small pleomorphic gram negative bacillus.
Using the provided reference chart, describe your interpretation of this MIC data for Patient Y (shown, Image 3). / The key observation here is that no growth occurred in the wells that did not contain any antibiotic, therefore, the test is not valid as the media failed to support the growth of the organism, or the culture was not viable when inoculated. It is not possible to determine the MIC from this data.
d. Differentiate between bacteriostatic and bacteriocidal agents. / bacteriostatic = suppress growth so that immune system can clear the infection
bacteriocidal = kills the organism completely
e. Define MIC / Minimum inhibitory concentration = the lowest concentration of an antibiotic that inhibits growth
CASE 2
A construction worker was injured at a work site, cutting a deep gash in the top if his foot. He immediately bandaged the wound. The following day he experienced pain and tenderness in the wound site, noting visible swelling and inflammation (Image 1). Upon in exam in the Emergency room several hours later, he presented with a fever. A foul odor was noted when the bandage was removed, and the patient reported that the wound appeared much worse this afternoon than it had in the morning. The wound appeared very swollen, with red and blackish streaks ascending from the wound upward toward the top of his foot.
Case 2, Image 1Case 2, Image 2
Case 2, Image 3Case 2, Image 4
Case 2- Fluid was aspirated from the large lesion shown in Image 1 using a needle and syringe and sent to the lab for Gram-stain and culture. What consideration is important in the transport of this specimen and how would you suggest this sample be transported to the laboratory?
Inject the specimen into an anaerobic transport vial/tube which is the preferred transport system for fluids.
- Based on the information in Image 2, name several media that would be useful to you for initial plating.
Anaerobic SBA
Egg Yolk Agar
Cooked meat
Thioglycollate broth
- Describe the growth seen the next day shown in Image 3, what is your observation that helps in the identification of the species of this organism?
- Describe what is seen on the Egg Yolk agar plate in Image 4.
- What is the most likely identification of this organism? (genus and species)
CASE 3
A 70 year old female patient is recovering from surgery to treat a severe diverticulitis. Three days post-op, she exhibits a fever. Ultra-sound indicates that an abscess has formed, requiring additional surgery to drain the abscess. The surgeon suspects the presence of anaerobic and aerobic organisms in the abscess. She is particularly concerned about the presence of Bacteroidesfragilis .The abscess material was sent to the lab for processing. Image 1 shows the gram smear of the abscess material.
Case 3, Image 1Case 3, Image 2
Case 3, Image 3
Case 3- What are two important characteristics of B. fragilisthat make its presence a particular concern?
Antibiotic resistance
- Based on the Gram-stained smear of the material aspirated from the abscess (shown in Image 1), should the surgeon be concerned about the presence of B. fragilis?
- A selective differential agar is set up with the abscess material and incubated under anaerobic conditions. Two days later, the plate is examined and is shown in Image 2. Describe the significance of this finding.
- Describe and interpret the test shown in Image 3. Do these findings support theidentifiation of B. fragilis?
- The primary culture of this sample grew predominantly the Gram-negative bacillus noted in the images along with lesser amounts of mixed bacterial flora. What other types of organisms are likely to be in this specimen besides B. fragilis?