Three pathogenic traits of Proteus mirabilis were looked at. P. mirabilis differentiates from a swimmer state to a swarmer state when in the urinary tract to enable it to migrate up the urinary tract. Chemotaxis enables the cells to determine which direction the kidney is. This investigation found that amino acids gave no chemotactic response, but that certain chemicals did. HCl, KOH and FeSO4 acted as chemorepellants, with glucose and pH7 being chemoattractants.
INTRODUCTION
Urinary tract infections are common and diverse. Most infections are not serious and are short-lived. Proteus mirabilis is a common soil organism that can cause urinary stones, acute pyelonephritis or cystitis when it infects the urinary tract (1, 2). P. mirabilis has many unique pathogenic traits, but I shall only investigate a few of them. Traits investigated include cell morphology (because it is unique), chemotactic response to amino acids and chemicals (to see how motility is affected), and attachment to eukaryotic cells (to mimic how it might attach in the urinary tract).
1. Proteus mirabilis identification Urine samples were spread onto CLED agar plates (prevents swarming of Proteus) and blood agar plates (identifies hemolytic strains) and incubated overnight at 37oC. Characteristic Proteus colonies were identified on CLED agar (these being blue, smooth convex colonies), and a swarming colony covered the blood agar. An API test was set up according to the kit instructions and a BLAST sequencing method was also set up to determine whether the species was P. mirabilis.
2. Morphology Cell morphology was investigated by inoculating the centre of a nutrient agar (NA) plate, and some nutrient broth (NB) with a colony from the CLED plate and examining cells from the plates under the electron microscope. 3. Chemotaxis a) Chemotaxis chamber: One well and half of the channel was filled with 10% nutrient agar. The opposite well and the rest of the channel were filled with 1M hydrochloric acid. This was repeated in other chambers substituting 1M HCl with either 1M potassium hydroxide, 1M glucose or sterilised distilled water (SDW).
These chemicals were chosen because they are chemicals found in the body, with SDW acting as the control. The well containing NA was inoculated with 5??l of Proteus grown in nutrient broth. The chamber was enclosed in a petri dish containing a layer of SDW to prevent evaporation when incubated at 37oC. To compare colony numbers from each well, 10 ?l were removed from the wells that didn’t contain NA, diluted to 10-3, spread onto CLED agar and incubated at 37oC.
b) Amino acids: Chemotactic responses to amino acids were investigated. Amino acids used were serene, lysine, glycine and glutamine, each chosen because of their different properties. 1M solutions of these amino acids were diluted to 10-1, 10-2 and 10-3. Filter discs were soaked in each of these solutions and were then set up as shown in figure 1. The centre was inoculated by one colony from the CLED plate. NA plate set up for chemotactic response to amino acids.
c) Chemicals: Another method of investigating chemotactic responses to various chemicals found in the body was investigated. NA plates were set up as in figure 1, but 3 mm wells were cut out from the agar instead of using filter discs. The chemicals used were 1M solutions of HCl, KOH, glucose, potassium dihydrogen orthophosphate (KH2PO4), ammonium dihydrogen orthophosphate (NH4H2PO4), ferrous sulphate (FeSO4), urea and lactose. Plates were inoculated and incubated as the amino acid plates were.
4. Attachment The overlaying media was removed from a fresh culture of McCoy cells. A large loopful of cells was taken from an NA plate to inoculate 1 ml SDW. The optical density of this cell suspension was measured and then poured on to the McCoy cells. A control was also set up to allow for any McCoy cells detaching from the surface. For this, 1 ml of SDW was poured on to the McCoy cells. These were then incubated for 17 hours at 37oC. After this time the solutions were poured off and the OD measured. The final OD was calculated by subtracting the control OD from the OD of the cells after incubation.
RESULTS
1. Proteus mirabilis identification The API test confirmed that the swarming bacteria were indeed Proteus mirabilis. However, the results from two of the tests (hydrogen sulphide production and the presence of tryptophan deaminase) were not as expected. The BLAST results showed a 96% match (382/395 base pair match) to P. mirabilis. P. mirabilis grown on blood agar showed no sign of hemolytic activity.