Urinary Tract Infections: The Pathogenic Traits of Proteus mirabilis.

Urinary Tract Infections: The Pathogenic Traits of Proteus mirabilis

Sarah Padley

ABSTRACT

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).

METHOD

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 μl 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 μl 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.

Figure 1. 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 ...

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Figure 1. 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.

2. Morphology

The cells grown on NA were distinctly different to those grown in NB, as shown in figures 2.

Figure 2. a) P. mirabilis cell grown on NA, magnification x10. b) P. mirabilis cell grown in NB, mag. x20.

3. Chemotaxis

a) The chemotaxis chamber did not work as expected because there was no difference in the number of cells when plated onto CLED plates.

b) The results from the chemotactic responses to amino acids remain inconclusive. The results are shown in figure 3.

Figure 3. Graph to illustrate the distance between P. mirabilis growth and amino acid disc (positive values denote growth past the disc; negative values denote growth up to the disc; bars missing from the graph mean that growth stopped on the disc).

c) The chemical chemotactic responses are shown in figure 4.

Figure 4. Graph to illustrate the distance between growth of P. mirabilis and chemical well.

4. Attachment

The results are shown in table 1.

Table 1. Optical density of P. mirabilis cells before and after incubation with McCoy cells (final OD calculated by subtracting the control optical density, 0.119).

DISCUSSION

a) P. mirabilis identification

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 μm 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 μm 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).

Figure 5. 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.

a) b)

Figure 6. 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.

Figure 7. 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;

Urease-induced crystal formation (as specified in 1)
Further investigation into chemotactic response to glutamine using minimal agar
Investigate why this strain shoed no hemolytic activity
Identify fimbrae types (as detailed in 1)
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.

6. Williams, F. D. et al. 1976. Evidence against the involvement of chemotaxis in the swarming of P. mirabilis. J. Bacteriol. 127: 237-248.