Question

Bacteria are the most common cause of sepsis, with 62.2% of patients with positive blood cultures harboring Gram-negative bacteria and 46.8% infected with Gram-positive bacteria. This overlap can be explained by polymicrobial sepsis, which is frequently simulated in mouse models. While Escherichia coli can be found in approximately 1 in 6 culture-positive patients, Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) have made up an increasing percentage of sepsis with the advent of excessive antibiotic treatment. Now, you collected blood samples from a patient and have a blood culture with several bacterial colonies on the plate.

(1) Please describe the procedure to purify the DNA from the colonies. (17%)

(2) Please describe what gel electrophoretic technique you can use to identify MRSA and how to prove the isolated MRSA strain is mutated. (17%)

Answer 1

1. To purify the DNA from the colonies, the colony needs to be grown in appropriate broth and the cells needs to be pelleted after incubation. Once the cell pellet is obtained, lysis of the cell is done by using detergents and the cytosolic components come outside. This is centrifuged and the supernatant is only taken. The supernatant is added with cations that can precipitate the DNA such as magnesium containing compounds along with appropriate buffer and again centrifuged where the DNA is found in the pellet excluding all other lipid and protein moeity. This isolated DNA is then stored in low temperatures in appropriate buffers.

2. The mutant and the wild type gene of the microbe can be known by doing southern hybridisation where the DNA from both are run on the gel and then transferred onto a blot where appropriate probes are added to check for the mutations. In this way one can determine the mutated ones from the wild type ones.

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