1. IV: Enterobacteriaceae: Lactose Fermenters
UNIVERSITY OF COLORADO
SCHOOL OF MEDICINE
DEPARTMENT OF PATHOLOGY
RESIDENCY TRAINING PROGRAM
MICROBIOLOGY
IV: Enterobacteriaceae:
Lactose Fermenters
Case Study: Hemolytic Uremic Syndrome

2. Hemolytic Uremic Syndrome
CASE HISTORY
An 11-year-old male experienced stomach cramps followed by bloody diarrhea a few hours later. The patient was hospitalized. Blood cultures were drawn on admission.
The blood platelets were very low. X-ray revealed significant intestinal edema. Kidney output dropped to zero.
The child received 3 units of fresh frozen plasma, the first of 11 plasma exchanges administered over the next three weeks. In the second week, pericarditis was diagnosed and treated with pericardiocentesis, followed by surgery to excise the pericardium.
The patient became hypotensive and convulsive. Intestinal perforation occurred and surgery was performed to correct this complication.
After 5 additional surgeries, the child gradually recovered.
Adapted from case originally presented by Dr. James Beebe, Director Microbiology Laboratory, Colorado Dept. Public Health. CACMLE Teleconference, July 25, 2001.

3. Agar Plate Appearance of Blood Culture Isolate
Appearance of the isolate from the admission blood culture as seen on the surface of sheep blood agar (left upper), Standard MacConkey agar (left lower) and sorbitol MacConkey agar (right) after 48 hours incubation at 35o C.
The colonies are entire, convex, and smooth. The colonies on standard MAC are pink-red, and on sorbitol MacConkey are non-pigmented.
What presumptive species identification might be made? What spot test reaction might be helpful?
What serogroup does the reaction on sorbitol-MacConkey agar suggest? Does this provide a definitive identification?
What types of stool specimens should be screened with sorbitol/MAC?
Agar Plate Appearance of Blood Culture Isolate
ANSWERS

4. Colony Appearance on Rainbow Agar ANSWER
Appearance of this isolate on Rainbow agar (a product of Biolog, Inc., Hayward, California).
Observed are commensal E. coli strains in the background (black pigmentation) and a few scattered Shiga-toxin producing strains (red pigmentation).
Biochemical identification is necessary as not all colonies having these appearances are E. coli. Also, not all colonies with a red pigmentation are serogroup 0157:H7
What additional tests might be performed to establish a definitive species identification of this isolate?
ANSWER
Colony Appearance
on Rainbow Agar

5. Identification using the Vitek GNI Card
The biocode number on the Vitek GNI card codes out to Escherichia coli, 98%; Hafnia alvei, 2%. An astute observer noted that the sorbitol reaction was negative. Serotype 0157:H7 was suspected.

6. DIAGNOSIS: Hemolytic Uremic Syndrome Etiologic Agent: Escherichia coli
Serogroup 0157
Recapitulation on next page

7. Escherichia coli Serogroup 0157: Recapitulation
Outbreaks of foodborne illness have been associated with consumption of ground beef, roast beef, raw milk, yogurt, and unpasteurized apple juice. Person-to-person transmission occurs, as well as water-borne and swimming-associated infections.
Cases peak in the warm months. Primary reservoir is cattle, but deer and elk may carry the agent as well.
As few as 10 viable cells can initiate infection in humans. Illness is characterized by bloody diarrhea, typically after 3-8 days incubation.
After infection, humans shed E coli 0157 for as long as 71 days. Close observation is needed when treating with antibiotics as illness may be exacerbated, promoting the emergence of hemolytic uremic syndrome. Hemolytic uremic syndrome is defined by a traid of features (acute renal failure, thrombocytopenia, and microantiopathic hemolytic anemia) and is the leading cause of acute renal failure in children.
As presented here, a large portion of EHEC isolates belong to serogroup 0157:H7, which may be suspected as this serogroup does not produce acid from sorbitol (appears “lactose-negative” on sorbitol MacConkey agar). Suspected isolates should be confirmed with specific 0157:H7 antisera.
Review of pathogenic E. coli on next page

8. Escherichia coli may be categorized into major groups according to serotypes and virulence mechanisms. ETEC are the most common cause of travelers’ diarrhea and have caused several food-borne outbreaks in the United States. A subset of the ETEC, serogroup 0157: H7, includes strains that produce bloody diarrhea and often hemolytic uremic syndrome. There are an estimated 78,420 cases of ETEC in the United States each year.
Review on next page

9. Pathogenic E. coli: Review
Enterotoxigenic E. coli (ETEC): for heat-labile and/or heat-stable enterotoxins that produce a secretory diarrhea (“traveler’s diarrhea”) similar to that of Vibrio cholerae. Surface attachment of the bacterial cells to intestinal epithelial cells is prerequisite to toxin production.
Enteroinvasive E. coli (EIEC): are capable of penetrating the intestinal epithelial cells and producing an inflammatory diarrhea similar to that caused by Shigella species. This strain can be suspected when observing blood, mucus, and segmented neutrophils in fecal smears. Both ETEC and EIEC strains are recovered primarily from patients who have traveled to foreign countries.
Enteropathogenic E. coli (EPEC): cause non-bloody diarrheal syndromes primarily in infants. The pathogenesis is unclear; however, the inflammatory reactions and epithelial degenerative changes that are observed in tissue sections may be secondary to adhesive properties of the bacterium, believed to be plasmid related.
BACK

10. BACK What presumptive species identification might be made?
Observed on the blood agar and MAC plates are colonies representative of a lactose-fermenting gram negative bacillus. The absence of colony pigmentation on Sorbitol-MacConkey agar suggests a 0157 serogroup.
What spot test reaction might be helpful?
A positive spot indole reaction would help confirm the identification of E. coli.
As mentioned before, the lack of acid production of the colonies growing on Sorbitol MacConkey agar serves as a screen for serogroup 0157, one important agent of hemolytic uremic syndrome.
Does this provide a definitive identification?
Approximately 80% of E. coli stains are sorbitol negative. However, most sorbitol negative glucose fermenters are E. coli. In either case, serogrouping studies must be performed for confirmation.
What types of stool specimens should be screened with sorbitol/MAC?
Obviously sorbitol/MacConkey agar plates will not be set up on all stool specimens. However, those that appear bloody, or those obtained f rom a patient with a history of passing bloody specimens should be screened. Cefixime and tellurite are added in some sorbitol/Mac formulas to make the medium even more selective for recovery of E.coli 0157.
BACK

11. E. coli Identification: Recapitulation
In summary, above are key biochemical characteristsics by which E. coli can be identified, and by which 0157 serogroups can be differentiated from non-0157 and commensal strains. Note that acid production from sorbitol and rhamnose, and a negative reaction for glucuronidase are key presumptive biochemical clues, and resistance to cefoxime and tellurite are more definitive.
BACK