Case A

Case B

Case C

Background: Bacteria belonging to the Vibrio group are commonly found in brackish and marine environments worldwide. Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus are the most important human pathogens within this family. Some of the members of this group can cause diseases like gastroenteritis, open wound infections and septicemia. These diseases are related to the ingestion of contaminated water and seafood as well as to the exposure to contaminated water.

There are several factors linked to severity of the symptoms. People with diabetes, liver diseases and with impaired immune system are more likely to suffer a more severe disease. There are several clinical cases of Vibrio vulnificus reporting gastroenteritis, open wound infections and septicemia. Infections with this bacterium may lead to death. Vibrio parahaemolyticus is involved in most of the gastroenteritis is related to the ingestion of raw oysters and seafood. Nevertheless, Vibrio cholerae has a greater pandemic potential and is responsible for several pandemics throughout the entire world. As of today, thousands of cases and deaths were reported every year.

This bacterium is the causative agent of cholera. The disease is characterized by severe watery diarrhea, vomiting and prostration. Severe dehydration may occur due to the fact that the patient will experience 20 -30 bowel movements within a day. The diarrhea will be watery and profuse and may lead to death within a few hours if patient does not receive appropriate rehydration.

There are many serogroups of V. cholera, of which 01 and 0139 were related to pandemic episodes. Other serogroups such as non-01 and non-0139 were related to less virulent outbreaks. V. cholerae 01, Vibrio vulnificus and Vibrio parahaemolyticus harbor many genes that encode different virulent factors and the expression of these genes accounts for the severity of the disease caused by this strain. The genes coding for toxins as well as other several virulence factors within Vibrio cholerae 01, Vibrio vulnificus and Vibrio parahaemolyticus can be targeted using molecular tests such as PCR which is critical to differentiate pathogenic from other variants.

Procedure: For each case, DNA is isolated from cultured bacteria, and multiplex PCR using primers specific for the three Vibrio species is performed to see which organism is causing the infection. The individual primers as well as control DNA samples for the three species are provided. The following PCR fragments sizes are obtained: V. cholerae, 162 bp; V. parahaemolyticus, 367 bp; V. vulnificus, 205 bp.

Bioinformatics: BLAST the sequence of the PCR product to determine which toxin gene is being detected and which strain is implicated in the infection.

Case A. A hurricane struck the coastal area of the island. The residents were evacuated ahead of time, but many of them came back trying to rescue their belongings right after the hurricane passed. The houses were still flooded with one foot of water. Despite the efforts of the authorities, some of the residents managed to walk inside the houses and spent hours trying to recover what is left. One day later, two elderly ladies, neighbors Emily and Luisa, reported to the emergency room with fever, severe vomiting and abdominal pain. Their pulse was weak and their blood pressure was low. The skin was dry and lacked turgor. Both women presented painful leg cramps. The profuse diarrhea had dehydrated both of them in such a severe way that they were experiencing cardiac and respiratory problems. Although it was obvious to the doctors that they were dealing with a gastrointestinal disease, the absence of blood, based on the color of the diarrhea, was the key for the treatment as well as for the laboratory tests that had to be done to confirm the cause of the disease. Later that day two other males and one female from the same neighborhood arrived at the hospital with similar but less severe symptoms.

Stool samples from all patients were collected and sent to the laboratory for further microbiological, serological and molecular analysis of the suspected microorganism. Vibrio was indicated by selective cultures. DNA was extracted from these cultures to perform PCR analysis of the potential toxin genes.

1. Why is the color of the stool important in the presumptive diagnosis?
2. How do you think the dehydration may affect the laboratory analysis such as CBC or urinalysis?
3. How are the leg cramps are related to the cholera disease?
4. Which culture methods should be used in order to isolate the bacteria from the stool sample?
5. What kinds of information do the cultural methods provide?
6. What did you need to know in order to determine the Vibrio serogroup?
7. Using the information that is available with cultural and serogroup methods, were you are able to discriminate between the strains infecting all of the patients?
8. Did the patients get infected by same strain? How do you know?
9. What are the advantages and disadvantages of the molecular methods and the bioinformatics analysis?
10. What are the advantages and disadvantages of using cultural methods?

Case B. It is midsummer, and the Ortiz family is enjoying an extended vacation. However, the family became concerned because of the sudden appearance of abdominal pain, nausea, vomiting and diarrhea in two of their family members, brothers Marcos and Vincent. They were wondering about the possible cause, as they were all eating the same foods during their vacations. Suddenly, one of them remembered that the day before they bought raw oysters from a street vendor. Almost all of them consumed the oysters, but, why were only two sick? After several hours, the brothers were becoming more ill so they decided that was time to visit the emergency room. At the hospital they were interviewed about the general health status of the sick men. Both are diabetic, but in general they do not have any other health problems. Both were treated with antibiotics and rehydrated. Blood samples and stool samples were sent to laboratory for further analysis. After treatment, both men recovered and were questioned further about the food that they consumed within the last few days. The doctors paid attention to the fact that they had consumed raw oysters and order an analysis for the detection of suspected bacteria. Selective culture methods indicated possible infection with Vibrio. DNA was extracted from these cultures to perform PCR analysis of the toxin genes.

1. Which is the toxin that pathogenic strains of Vibrio parahaemolyticus harbor?
2. Compare this toxin with the cholera toxin. Consider clinical, molecular and physiological factors.
3. Why were only two of the family members were ill if everyone ate raw oysters?
4. Does the fact that this occurred in summer have something to do with this outbreak?
5. What are the advantages and disadvantages of the molecular methods and the bioinformatics analysis?
6. What are the advantages and disadvantages of using cultural methods?

Case C. The annual family picnic gathered a lot of family members at the beach. Many generations enjoyed the beautiful day. However, the grandmother did not feel well that day. She is diabetic and she had an open wound in the leg that had not improved. Finally, she decided that maybe the salty water will helped her in the healing process so she entered into the water for a while. Two days passed and she became even more ill. The wound was worse and she could not stand. The family doctor hospitalized her and ordered cytological and microbiological examination of the wound. The Doctor discussed the results with the family and they were startled by the fact that the cause appeared to be a marine bacterium that must have been acquired while she enjoyed the beach. DNA was isolated from the cultured bacteria to identify which species of bacteria was the culprit.

1. If all the family were at the beach, why did only the grandmother become ill?
2. Why do she and many people think that salty water will improve the condition of the wound?
3. Which are the differences between the different biotypes within Vibrio vulnificus?
4. Do other Vibrios have different biotypes? If so, what are the differences between them?