Case A

Background:  Salmonella enterica is a species of bacterium that can cause serious digestive illness in humans. Salmonella bacteria pass from the feces of people or animals to other people or other animals. Many different serovars of S. enterica exist, for example Enteritidis, Typhimurium, and Paratyphi. The first two are the most common in the United States, although serovar Paratyphi and others are present as well. Salmonella bacteria were discovered over 100 years ago by the assistant of an American scientist named Salmon, for whom they are named.

Salmonellosis is an infection with Salmonella bacteria. Most persons infected with Salmonella develop diarrhea, fever, and abdominal cramps 12 to 72 hours after infection. The illness usually lasts 4 to 7 days, and most persons recover without treatment. However, in some persons the diarrhea may be so severe that the patient needs to be hospitalized. In these patients, the Salmonella infection may spread from the intestines to the blood stream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics. The elderly, infants, and those with impaired immune systems are more likely to have a severe illness.

Case A.  Sarah, a 6-year-old girl, was admitted to the hospital with a 4-day history of fever, vomiting, abdominal pain, and diarrhea.  Lab tests indicated that her white blood cell count is elevated and that she has mild liver dysfunction.  A stool culture taken on admission yielded a Gram negative rod that appeared to be Salmonella.  Blood, nasopharyngeal, and urine cultures were negative.  Sarah had no history of overseas travel, and there was no indication that she had ingested any suspect foods.  No other family members were ill.  Sarah has a pet turtle, a red-eared slider, for which she is the sole caregiver.  A water specimen from the turtle’s tank also yielded a culture of Salmonella, as turtles are natural carriers of this disease-causing organism. To determine whether the bacteria isolated from the turtle’s tank and from Sarah were the same, genomic DNA was isolated from each of the bacteria samples.

Procedure: To analyze this case, digest each of the DNA samples with XbaI.  Then digest each of the original DNA samples (not the XbaI-digested samples) with BlnI.  Run the digested samples on a gel, using a 0.5% agarose gel.  (Note that this would normally be done using pulse-field gel electrophoresis, due to the large sizes of the DNA fragments, but here the fragments will run correctly using the standard agarose gel procedure). Do the band patterns for the DNA isolated from Sarah and from the turtle match?

DNA samples (the first two are serovars of Salmonella enterica)

• Salmonella Paratyphi
• Salmonella Typhimurium
• Sarah
• turtle

Multiplex PCR can be used to determine whichs serovar of Salmonella Sarah has.  To perform this procedure, use the Salmonella primers to run PCR on the DNA isolated from the turtle’s tank, the DNA isolated from Sarah, and from control DNA samples isolated from the Paratyphi and Typhimurium serovars.  The primers file contains two sets of primers, one which identifies all serovars of Salmonella and amplifies a 204 bp DNA fragment; the other is specific for the Typhimurium serovar and amplifies a 402 bp fragment.  Use a 1.0% agarose gel to run the PCR products.

Bioinformatics: What gene is amplified by the primers? Use BLAST to identify the gene associated with the gel fragments. Click on the gel fragment from one of the samples. The sequence of that DNA should be visible in the lower window (if not, check the Sequence box above the window). The entire sequence, just a portion containing the repeat region, can be sent for BLAST analysis (the latter will work better if using fragments from the Southern blot). See the BLAST tutorial for more detailed instructions.

Additional sequences of similar genes from other species of Salmonella and also from E. coli in the Bioinformatics folder included with this case. Open these sequences and build a tree from the Opened & Processed window. How similar are the  Paratyphi and Typhimurium serovars, compared to the other bacterial types included?

1. Which serovar of Salmonella is Sarah infected with? Has that particular serovar been associated with Salmonellosis in the U.S?
2. Do the results prove that Sara contracted the Salmonella infection by handling the turtle?
3. How should Sarah be treated so that she can recover from the infection?
4. What is the level of risk associated with pet turtles, and is it legal to sell them?
5. What other pets are associated with possible bacterial or viral infections?
6. What techniques are currently used to test for Salmonella infection?