Case Study Pathogenic Bacteriology 2009

1. Case StudyPathogenic Bacteriology2009 Case 26 Tsung-Han Hsieh Omar Ahmed Rochelle Songco

2. Case Summary • Patient: 80-year-old female had a cystocele repair performed. She was give a 7-day course of oral cephalexin but stopped the course after 3days. • Symptoms: 10 days postoperatively, she had diarrhea (multiple, watery, loose stool w/o blood) for 3days. She also had crampy abdominal pain, vomiting, and mild fever.

3. Case Summary • Blood cell count was normal, but 53% of immature polymorphonuclear cells were seen. • Urine culture revealed >100,000 CFU/ml of E. coli which was susceptible to all antimicrobial agents • Cultures for Salmonella, Shigella, Yerinia, and Campylobacterspp were all negative. • Enzyme immunoassay (EIA) showed positive for bacterial toxin in the stool sample. • Physical examination, electrolytes, liver enzymes, and lipase were within normal range.

4. Key Information Pointing to Diagnosis • >100,000 CFU/ml of E. coli in the urine culture -> CFU/ml greater than 100,000 is significant • Watery diarrhea without blood -> the organism is not invasive • EIA showed positive for bacterial toxin in the stool sample -> the organism produces enterotoxin

5. The Diagnosis for Case #26 • The causative organism was enterotoxigenic E. coli (ETEC) • Cystocele repair is predisposing factor. Since E. coli is a major cause of urinary tract infection, and the cystocele repair involves urinary tract, the organism could have the chance to get into the patient and cause diarrhea and other symptoms. • EIA was performed to specify which types of E. coli was causing the disease.

6. Classification,Gram Stain Results, and Microscopic Appearance of ETEC • Family: Enterobacteriaceae • Genus: Escherichia • Gram-negative bacilli • Facultative anaerobes Picture was taken from http://www.nirgal.net/graphics/e_coli.jpg

7. Diseases and Pathogenesis of Disease Caused by ETEC • Disease • E. coli is a major cause of urinary tract infection, gastroenteritis, neonatal meningitis, and septicemia. • Urinary tract infections can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection) • ETEC is a common cause of traveler’s diarrhea and diarrhea in children in developing countries. • Symptoms normally include watery diarrhea, nausea, abdominal cramps ,and low-grade fever for 1-5 days.

8. Diseases and Pathogenesis of Disease Caused by ETEC • Pathogenesis of Disease • Pili, capsule, outer membrane proteins, and O, H, and K antigens • Enterotoxins – it’s produced by ETEC and causes watery diarrhea. • LT (heat-labile) toxin- very similar to cholera toxin in both structure and mode of action. It binds binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP. Increased cAMP then causes ion imbalance that results in fluid transport into the bowel (watery diarrhea) • The ST (heat-stable) enterotoxin- a family of toxins which are peptides of molecular weight about 2,000 daltons. Their small size explains why they are not inactivated by heat. ST causes an increase in cyclic GMP in host cell cytoplasm. This also leads to secretion of fluid and electrolytes resulting in watery diarrhea.

9. Diseases and Pathogenesis of Disease Caused by ETEC • Pathogenesis of Disease • Particularly problematic as a nosocomial pathogen • It’s because E. coli is one of the M.O. that is abundant in the hospital, and it can be transmitted easily by fecal oral route.

10. Diagnosis/Isolation/Identification/ of ETEC • Diagnosis • The diagnosis is suggested by the clinical picture and confirmed by stool/urine culture. Serotyping and tests for virulence factors are occasionally performed for outbreaks. • Isolation • MacConkey agar (pink to red color with or without a zone of • precipitated bile) • ChromAgar ECC plates (light to dark blue) • Identification • Direct probe testing • EIA or ELISA

11. Therapy, Prevention and Prognosis of Patient Infected with ETEC • Therapy • Most infected persons will recover within a few days, without requiring any specific treatment • Electrolyte fluids are recommended for persons with diarrhea to prevent dehydration and loss of electrolytes. • Antibiotic course- only be give when the symptoms become severe. ETECis usually susceptible to a variety of chemotherapeutic agents, but is frequently resistant to common antibiotics such as trimethoprim-sulfamethoxazole and ampicillin. • It is essential to do susceptibility testing.

12. Therapy, Prevention and Prognosis of Patient Infected with ETEC • Prevention • Wash your hands thoroughly after going to the bathroom or changing diapers. • Eat only thoroughly cooked ground beef. • Cook ground beef products to an internal temperature of 160 degrees Fahrenheit.  • Avoid unpasteurized milk and juices. • Wash fresh fruits and vegetables thoroughly before eating. • Keep raw meat separate from ready-to-eat foods.

13. Therapy, Prevention and Prognosis of Patient Infected with ETEC • Prognosis • The disease is usually self-limiting. The infection will end on its own and is rarely life-threatening. • Treatment failures might due to incomplete antibiotic course. • Disease relapses frequently because E. coli is NF in the GI tract and it might turn into pathogen when the condition is right.

14. Primary Research Article Contributing to the Understanding of the Disease caused by ETEC • Seung Y. Koh, et al., Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli, Veterinary Microbiology, 2008. 130 : 191–197. • Experimental set up • Bacterial adherence assays • E. coli (different strains) were cultured overnight on 5% sheep blood agar at 32 ℃ . • Bacteria suspension, cell lines (IPEC-J2, IPEC-1, and INT-407), and D-Mannose were added into the plate wells. • Phase contrast microscopy and bright field microscopy under oil were used for observing the binding interaction. • Green fluorescence protein-tagged E. coli • E. coli was transformed with Green fluorescence protein (GFP). • Bacterial adherence assays was performed with GFP tagged E. coli. • Observation was made by confocal scanning laser microscopy

15. Primary Research Article Contributing to the Understanding of the Disease caused by ETEC • What did they find • Representative figure (A) Shows the degree of E. coli binding to porcine intestinal epithelial cells. (B) The confocal image showed GFP- transformed bacterial (ETEC strain B41) attachment to IPEC-J2 cells. (C) ETEC F18 strain 2134 intensively bound to IPEC-1cells. • Conclusion • Different strains of E. coli has different binding affinity to different cell lines. Fig. 1. Adherence of E. coli strains to IPEC-J2 and IPEC-1 cells • Stronger binding affinity could result in releasing of more enterotoxin. • Porcine intestinal epithelial cell lines can be used as in vitro study of pathogenesis of ETEC. • How does this article relate to and support the case? • This article relates and supports the case by discussing the relationship between the binding of ETEC to the host cell and the releasing of enterotoxin.

16. Take Home Message • Disease involves • Traveler’s diarrhea and diarrhea in children in developing countries • Typical symptoms are • Water diarrhea, nausea, abdominal cramps ,and low-grade fever • Pathogen is • Enterotoxin: LS and ST • Diagnostics include test • Stool/urine culture, EIA and Serotyping for virulence factor • Therapy is based on • Premixed oral rehydration solutions, sometimes antibiotic course

17. Take Home Message • Prognosis • The disease is usually self-limiting and rarely life threatening • Prevention is • Keep hands clean, don’t eat raw meat, and wash raw vegetable thoroughly before eating • Transmission is • Via fecal-oral route or direct contact of contaminants. • Threat is • Major endemic health threat in developing countries

18. References • Connie R. Mahon, Donald C. Lehman, George Manuselis. Diagnostic Microbiology; 3rd ed.;Saunders, an imprint of Elsevier Inc.:2006; Chapter 22. • Seung Y. Koh, Sajan George, Volker Bro¨zel, et al, Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli.Veterinary Microbiology, 2008. 130 : 191–197. • Robin McKenziea, Michael Darsleyc, Nicola Thomasc, et al. A double-blind, placebo-controlled trial to evaluate the efficacy of PTL-003, an attenuated enterotoxigenic E. coli (ETEC) vaccine train, in protecting against challenge with virulent ETEC. Vaccine, 2008, 26: 4731–4739 • McQueen, Nancy. Winter 2009. Lec 5 Enterobacteriaceae

19. Acknowledgments • Dr. Nancy McQueen • Omar Ahmed • Rochelle Songco