Question

Scenario
Five-year-old Thomas is looking forward to starting kindergarten in the next few weeks. As a last summertime activity, his parents take him and his four older siblings to the county fair to enjoy the rides and see the animals. While at the fair, the family snacks on the fair food, drinks lemonade and soda, and wanders through the barns to visit with the horses, cows, sheep, goats, and chickens being kept there for the week.

Signs and Symptoms
Three days after the fair, Thomas and two of his sisters start to complain about feeling sick. Within a day, the entire family has severe stomach cramps, vomiting, and diarrhea. Over the next few days, the rest of family gradually starts to feel better, but not Thomas. His diarrhea continues and becomes tinged with blood.

His mother takes him to the family physician, who notes that Thomas is pale and anemic. He asks Thomas to try to "pee in the cup" for a urine sample, but Thomas says he cannot pee. The physician tells the family to go immediately to the hospital.

At the hospital, Thomas was diagnosed with acute renal failure and catheterized for a urine specimen. Samples of blood and stool were also collected and sent to the clinical lab.

Testing
The fecal specimen was plated on several types of differential and selective media, including MacConkey Agar and ChromAgar 0157, a selective and differential medium specifically for Escherichia coli 0157:H7, a pathogenic strain of E. coli.

Question 2: MacConkey Agar contains lactose as the differential agent. Lactose-fermenting bacteria (like E. coli) produce pink-colored colonies and non–lactose fermenting bacteria (S. dysenteriae) do not. What does the MacConkey Agar culture result shown here indicate about the bacteria isolated from Thomas’s stool?

"Mauve"-colored colonies were observed on the surface of the ChromAgar 0157 agar plate, which is a positive test result for E. coli 0157:H7. This strain and others like it are also known as enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli (STEC).

Question 4: E. coli is a human commensal that normally does not cause disease. Why is the lab reporting E. coli as the cause of Thomas’s disease?

E. coli 0157:H7 is different from commensal strains because it produces a toxin, called a verotoxin or Shiga toxin, that destroys small blood vessels, such as those found in the glomeruli of kidneys.

This toxin is the product of the stx gene. There are two variants, stx-1 and stx-2. The Stx-2 toxin is 400 times more toxic than Stx-1. Serological or DNA tests for the toxin are confirmatory for STEC.

PCR was done using primers specific for stx DNA. The primers amplify a DNA fragment approximately 900 base pairs long. PCR products were separated by gel electrophoresis, along with a 100 bp DNA ladder for size comparison. On the gel shown here, the largest fragment in the ladder is 1,000 bp, and the smallest is 500 bp.



Question 5: What do the results of the PCR analysis show?

To determine which variant of the toxin the bacteria were producing, restriction fragment length polymorphism (RFLP) analysis was also done, using two restriction endonucleases, AccI and HincII. Using these enzymes, the expected restriction fragment lengths for the two gene variants are:
Sxt-1: 705 bp, 158 bp, and 32 bp
Sxt-2: 555 bp, 262 bp, and 62 bp



Question 6: What do the results of the RFLP analysis show?

Diagnosis
Based on the lab report, Thomas’s diagnosis was changed to hemolytic uremic syndrome (HUS) caused by an infection with E. coli 0157:H7, which is the most common strain of STEC.

Question 7: The "A" subunit of the Stx toxin inhibits protein synthesis and induces apoptosis. How could this lead to the massive tissue damage seen in HUS?

Answer 1

Question 2:

Mac-Conkey's agar is a selective and differential media. It is a selective medium because it does not allow the growth of most species of the Gram-positive organisms. It is a differential media because it allows the differentiation between the lactose-fermenting and non-lactose fermenting bacteria based on the difference in the color of the colonies formed.

The composition of Mac-Conkey's agar is:

a. Peptone (pancreatic digest of gelatin)

b. Protease Petone (meat and casein)

c. Lactose monohydrate

d. Bile salts

e. Neutral red

f. Sodium chloride

g. Crystal violet

h. Agar

i. Distilled water

The crystal violet and bile salts prevent the growth of the Gram-positive organisms, while the lactose serves as the differentiation agent. Lactose is the carbohydrate source that can be used only by the lactose-fermenting bacteria. When the lactose-fermenting bacteria ferment lactose, then the acidic environment produced will lower the pH of the medium to less than 6.8. The indicator neutral red turns red at a pH below 6.8, causing the colonies of lactose-fermenting bacteria to be red or pink.

The non-lactose fermenting bacteria use the peptone present in the medium instead of lactose. They do not change the acidity of the medium, hence they grow as colorless or transparent colonies. This allows for the differentiation between the lactose-fermenting and non-lactose fermenting bacteria using Mac-Conkey's agar.

The colonies produced by the bacteria isolated from Thomas's stool in Mac-Conkey's agar would be red or pink. This is because E.coli can ferment lactose, i.e., it is a lactose fermenting bacteria. thus, it would lower the acidity of the medium to below 6.8 pH, which would cause the indicator to turn red. hence, the colonies growing would also be seen as pink or red colonies.

Thus, the Mac-Conkey agar culture result would indicate that the bacteria isolated from Thomas's stool is a lactose-fermenting type of bacteria.

The ChromAgarO157 is a selective medium which allows the identification of E.coli O157: H7 based on the development of a particular color. The chromogenic substrate present in the medium changes color in the presence of E.coli O157: H7 thus helping to identify the particular strain of E.coli.E.coli O157: H7 has been implicated in mild non-bloody diarrhea to severe bloody diarrhea (hemolytic colitis), hemolytic uremic syndrome (HUS) and death.

Mauve colored colonies observed on the surface of ChromAgar O157: H7 indicates the presence of E.coli O157: H7. This means that the bacteria which was isolated from Thomas's stool was E.coli O157: H7.

Question 4: E.coli is a human commensal. Commensalism can be defined as the long-term biological interaction between two organisms where one benefits but the other derives neither benefit nor harm. E.coli is normally found in the gut of humans well as animals, but there are certain types of E.coli which are capable of causing an infection.

The E.coli O157: H7 also known as the Shiga toxin-producing E.coli (STEC) is one such type of E.coli that can cause illnesses ranging from mild non-bloody diarrhea to severe bloody diarrhea (hemolytic colitis), hemolytic uremic syndrome (HUS) and death.

The STEC can cause HUS which is the most common cause of kidney failure in children. The damage to the blood vessels by the Shiga toxin or the verotoxin is the main cause of kidney failure. The toxins Stx 1 and Stx 2 are produced by the STEC. The HUS is more often associated with strains of e.coli that produce Stx-2 rather than Stx-1.

The toxins consist of two subunits - A and B. The B subunit is a pentamer of five identical subunits that are bound covalently to the A subunit. The A subunit is responsible for binding to the ribosome of the eukaryotic cell and halting the protein synthesis, while the B subunit binds to the Gb3 receptor found in the endothelial cells. This mechanism of action is responsible for the HUS and the associated kidney failure.