The identification tests strongly suggest that this species was P. mirabilis, but the results aren’t 100%. Two tests on the API strip did not come out with the expected result. In the case of H2S production, the conditions may not have been strictly anaerobic. I cannot explain why the tryptophan deaminase test did not work. This strain of P. mirabilis was, uncharacteristically, not hemolytic. The urine samples were not fresh, therefore it is possible that this strain lost its hemolytic activity, or that it is simply not hemolytic.
b) P. mirabilis cell differentiation The cells seen in figure 2 show clearly the differences in the two main types of cell morphology, i) swarmer cells and ii) swimmer cells. i) The cells that grow on solid media are usually in the swarmer state. Swarmer cells can be any length between 10 and 80 ?m in length (ref. 68 in 3) and are covered in hundreds of long fimbrae. The cells are polyploid as a result of elongation and DNA replication with no cell division. ii) Cells that grow in liquid media are very similar to those of Enterobacteriaceae. They are highly motile rods of about 1.75 ?m in length (68 in 3) and have only a few fimbrae.
c) Colony patterns on NA One distinguishing feature of P. mirabilis is the pattern of its colony on NA, seen in figure 1. The ridges seen are the result of dense and sparse congregations of cells. When NA is inoculated with cells from liquid, the swimmer cells differentiate into swarmer cells by undergoing elongation and DNA replication without cell division. This can be thought of as a kind of lag phase. The swarmer cells then act as a group by slowly swarming, or migrating, out of the periphary of the inoculation point, as seen in figure 5.
After a point, the cells stop migrating and the cells undergo consolidation (seen as the ridges in figure 1). During consolidation the swarmer cells revert back to swimmer cells through cell division. The cycle of differentiation, migration and consolidation is repeated until nutrient levels deplete. Migration from the inoculation point is not the result of a global signal, as is seen in some species of myxobacteria, but merely a consequence of high cell density (4).
P. mirabilis projectile migrating outward from the inoculation point. Cell differentiation is thought to be important to enable P. mirabilis to migrate up the urinary tract. It has been hypothesised that P. mirabilis exists as a swimmer cell before it enters the urinary tract, then once it reaches the epithelial surface of the urinary tract it differentiates into a swarmer cell. This then enables the cell to swarm up the tract in a way that means it is not hindered by the villi (3).
d) Chemotactic response to amino acids Amino acids were not proved to be involved in any kind of chemotactic response, as is seen by the lack of pattern in figure 3. This was due to the fact that nutrient, as opposed to minimal, agar was used, and thus already contained amino acids. However, glutamine has been found to be a chemoattractant for swarming cells, and initiates the differentiation of P. mirabilis cells (3, 5).
e) Chemotactic response to chemicals Some chemicals affect the swarm of P. mirabilis, which contradicts one study by Williams et al (6). Figure 4 shows that the swarm is negatively affected by HCl, KOH, and by a lesser extent, FeSO4, as seen in figure 6. Between the HCl and KOH wells the pH is 7, and figure 6a shows that P. mirabilis is attracted to this pH. There also seems to be more growth towards KOH, which is to be expected as the urinary tract is relatively alkaline. It also shows that the cells are attracted to glucose. The water acts as a control, and demonstrates the distance that would have been swarmed over had other chemicals not been present. The results in figure 4 show that lactose, urea, KH2PO4 and NH4H2PO4 evoked no chemotactic response.
Swarming pattern of P. mirabilis as a result of the presence of a) HCl, KOH, water and glucose; b) FeSO4 Chemotactic responses are important for P. mirabilis when in the urinary tract. They determine the direction of the kidney, an environment to which P. mirabilis has adapted to. f) Attachment The vast number of fimbrae allows attachment of P. mirabilis cells to other cells. The effect of P. mirabilis cells on the McCoy cells can be seen in figure 7. The P. mirabilis cells can’t actually be seen, but a lot of cell debris can. This may not necessarily be due to the presence of P. mirabilis, it is probably a result of there not being any cell media.
McCoy cells after being incubated with P. mirabilis for 17 hours. If only tissue samples 2 and 3 are looked at from table 1, the decrease in OD shows that approximately 9% of P. mirabilis cells attached to the McCoy cells. Tissue sample 1 did not show any OD decrease, possibly because more McCoy cells detached than the control allowed for. Attachment is also vitally important for P. mirabilis because of the constant flushing of the urinary tract. The cells have to be able to withstand this host defence whilst migrating up the urinary tract, and to also remain attached to the kidney.
Further investigations This investigation was seriously limited by time. As a result, there are many aspects that I would have liked to investigated had there been more time, these being; i) Urease-induced crystal formation (as specified in 1) ii) Further investigation into chemotactic response to glutamine using minimal agar iii) Investigate why this strain shoed no hemolytic activity iv) Identify fimbrae types (as detailed in 1) v) Examine further differences between swarmer and swimmer cells, such as metabolic differences and cell wall and membrane differences (as detailed in 3).
REFERENCES
1.Mobley, H. L. T. 1996. Virulence of P. mirabilis. Urinary tract infections: pathogenesis and clinical management. 245-267. 2. Mims et al. 2000. Medical microbiology. 2nd edition: 221 3. Belas, R. 1996. P. mirabilis swarmer cell differentiation and urinary tract infection. Urinary tract infections: pathogenesis and clinical management. 271-295. 4. Itoh, H. et al. 1999. Periodic pattern formation of bacterial colonies. J. Phys. Soc. Japan. 68: 1436-1443. 5. Allison, C. et al. 1993. Cell differentiation of P. mirabilis is initiated by glutamine, a specific chemoattractant for swarming cells. Mol. Microbiol. 8: 53-60.