Adv. microbiology case study

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1. Case study created by : Robert Patrick Fredal II I C. Diff- iculty Ahead

2. Patient History A 67 year old female presented to the hospital because of ongoing watery diarrhea that occurred up to 7 times a day for the past five days. Signs of dehydration were noted and she has also been complaining of moderate to severe abdominal pain, fever, nausea, and loss of appetite. Upon further investigation this patient was treated with antibiotics a week prior due to a nosocomial urinary tract infection acquired after her lengthy hospital stay for a broken hip.

3. Lab results Because of the patients prior antibiotic history an anaerobic culture was added to the routine stool culture The anaerobically incubated media grew gram positive spore producing bacilli Occult blood was detected in the specimen along with a small amount of pus Upon microscopic examination many white blood cells were noted A colonoscopy was performed and revealed areas of inflammation and pus along with necrosis of the colon wall www.gihealth.com

4. What is the possible diagnosis of this patient? What organism could be causing this? She was diagnosed with pseudomembranous colitis , confirmatory tests were done and came up positive for Clostridium difficile Other pathogenic species of the Clostridium genus include: C. botulinium (botulism) this organism produces an active neurotoxin and can have CNS involvement such as blurred vision C. perfringens ( gas gangrene) cause of amputations in diabetics Clostridium tetani (tetanus) which causes painful muscle spasms that can often lead to respiratory complications

5. Clostridium difficile: the basics It is One of the most serious nosocomial infections worldwide A ssociated with antibiotic associated diarrhea (20-30% of all cases) can range from self limiting diarrhea and flu-like symptoms to life-threatening colitis It can be a small part of an adults normal gut flora More than half a million people become ill a year because of this organism In recent years C. difficile has because more frequent than ever before, more severe, and even more difficult to treat The most virulent strain (BI/NAP1/027) has increased toxin production and drug resistance http://www.vancocin-us.com/healthcareprofessionals/aboutcdifficile

6. Cultural characteristics Gram positive bacilli They can look club shaped with a bulge at each end Forms endospores Strict anaerobe Grows well on SBA at 37 degrees celcius Colony morphology; Glossy, gray/white , circular colonies with a rough edge, fluoresce green-yellow under UV light, non-hemolytic, and a characteristic farmyard smell peritrichous flagella http://lancastria.net/blog/new-drug-to-treat-c-diff.html http:// depts.washington.edu/molmicdx/mdx/tests/cdiff.shtml

7. Culture media Cycloserine C efoxitine Fructose A gar (CCFA) Selective medium used for C. difficile D- cycloserine and cefoxitine antibiotics are added to inhibit most other organisms Colonies appear large, flat, yellow , ground glass look , and a filamentous edge can be observed Spores are absent when grown on artificial media along with reduced motility Has a characteristic “Farmyard” smell http://www.cdc.gov/media/dpk/2014/dpk-hai.html

8. Biochemical reactions Non hemolytic Sachharolytic Indole negative Lecithinase negative Hydrolyzes Aesculin http://haveyroo.blogspot.com/2009/07/clostridium-difficile-or-c-diff.html

9. Identification Cell cytotoxicity assays are considered the gold standard test for detection of C.  difficile toxin by observing the cytopathic effects of the toxin in cell culture Enzyme ImmunoAssays (EIA) and Enzyme-Linked I mmunosorbent A ssay (ELISA) tests are available for use and can detect toxins A and B PCR is also now starting to be used

10. Clinical Significance

11. Virulence factors Enterotoxin (Toxin A) Stimulates chemotaxis and induces cytokine production that leads to hyper secretion of fluids from the bowel Cytotoxin ( Toxin B) Causes depolymerization of actin accompanied by loss of the cellular cytoskeleton (cytopathic effect) Binary toxin It is unclear the role of this toxin but it may synergistically increase the virulence of toxins A & B Adhesion factor Aids in binding to cells in the colon Hyaluronidase ( produces hydrolytic activity) Spore formation Allows for the organism to remain viable under harsh conditions and for long periods of time

12. Pathogenic Mechanisms Antibiotics reduce normal flora which allows for C. diff to start to multiply and begin to overtake the normal gut bacteria Toxins are produced and cause degradation of the cell cytoskeleton This causes loss of cell shape and decreased adherence to eachother Fluid leaks occur which leads to the watery diarrhea The toxins can also erode away the colon membrane and in severe cases can cause bowel perforation

13. http://www.cdiff-support.co.uk/about.htm

14. Susceptibility testing  susceptible to metronidazole and vancomycin  resistant to clindamycin , fusidic acid, and   fluoroquinolone http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19000

15. Treatment First you should discontinue taking antibiotics that likely caused the infection Keep thoroughly hydrated Normally oral metronidazole is given(DOC), but in more complicated infections oral vancomycin is administered Human micro biota transplants (poo transplant) Repopulates the gut and suppresses the growth of C. diff Probiotics Surgery may be needed depending on the damage to the bowel ( colectomy)

16. Prevention and disease control WASH YOUR HANDS with soap and water Alcohol-based hand cleansers do not kill C.diff spores Patient is isolated in a room that includes an attached bathroom Contact precautions Cleansing with bleach to disinfect any potentially contaminated surfaces http://www.nanobugs.com/shop/sposters.html