J I

12 h

12 h

12 h

Day 1 Day 2 Day 5 Day 7 Day IS

Fig. 1. Hormone inoculation scheme. H, hormone inoculation. The three bent arrows are the lactobacilli inoculation, and the single straight arrow is the E. coli inoculation. Days of the mice sacrifice were counted from the day of E. coli challenge.

8. Orange G (Biopur). Store at room temperature.

9. Xylene (Biopack). Store at room temperature.

10. Natural Canada balsam (Biopack). Store at room temperature.

11. 24 x 48-mm Glass slides.

12. Cover slides.

2.7. Histological Studies

1. 10% Formaldehyde (Biopack). Store at room temperature.

2. Increasing graduated ethyl alcohols (Biopack): 50°, 70°, 80°, 96°, 100°.

3. Xylene (Biopack). Store at room temperature.

5. Histoplast Fusion Point 56°-58°C (Biopack).

6. Sliding microtome (Zeiss).

7. Microtome blades.

8. 76 x 26-mm Glass microscope slides (Zeiss).

9. Decreasing graduated ethyl alcohols (Biopack): 100°, 96°, 80°, 70°, 50°.

10. Hematoxylin stain (Biopack). Store at room temperature.

11. Eosin yellow (Biopack).

12. Distilled water.

13. Natural Canadian balsam (Biopur).

14. 22 x 48-mm Glass cover slides.

15. Light microscope.

2.8. Electron Microscopy

1. Glutaraldehyde: 3.16% in 0.1 Mphosphate buffer, pH 7.4 (Pelco).

2. Washing buffer: 0.1 Mphosphate buffer, pH 7.4.

3. Osmium tetroxide.

4. Epoxy-type resin: Epon 812 (Spurr, Pelco).

5. Ultramicrotome.

6. Uranyl acetate.

7. Pb citrate.

8. Electron microscope (Zeiss 109).

3. Methods

3.1. Microorganisms

1. L. fermentum CRL 1058 stored in milk-yeast extract.

a. Screw-top glass tubes containing 5 mL LAPTg broth were inoculated with the isolated lactobacillus and incubated at 37°C for 24 h.

b. A 50-^L aliquot of these cultures was subcultured twice at 37°C for 12 h in 5 mL of the same medium.

c. The lactobacilli culture was centrifuged for 10 min at 2000g), and the spent supernatant was discarded.

d. The pellet was washed twice with 0.85% saline solution and suspended in 5 mL milk-yeast extract.

e. This bacterial suspension was distributed in 1-mL screw-top cryovials and stored at -20°C.

2. Uropathogenic E. coli: The microorganisms were identified by the methods described in Bergey's Manual of Determinative Bacteriology, standard techniques used in the laboratory, and API 50 CHL (Biomerieux-France).

a. Screw-top glass tubes containing 5 mL LAPTg broth were inoculated with the isolated E. coli and incubated at 37°C for 24 h.

b. The E. coli culture was centrifuged for 10 min at 2000g, and the spent supernatant was discarded.

c. The pellet was washed twice with saline solution (0.85% NaCl) and suspended in BHI + 25% glycerol.

d. This bacterial suspension was distributed in 0.3 mL screw-top cryovials and stored at -70°C.

e. They were subcultured in LAPTg broth for later use.

3.2. Bacterial Incorporation Into Beads

The agarose beads were prepared according to the method described by Cash et al. (2) with certain modifications, as follows:

1. L. fermentum was grown in LAPTg broth for 12 h at 37°C.

2. Organisms were harvested by centrifugation, washed twice with PBS, pH 7.0, and resus-pended in PBS.

3. A suspension of 108 colony-forming units (CFU)/mL was mixed with the same volume of 1% agarose in PBS and maintained at 37-40°C and three volumes of vaseline added at the same temperature.

4. The mixture was gentle vortexed for 3 min and after resting at room temperature for 2 min, cooled in an ice bath and maintained at 0°C for 7-10 min.

5. The beads were washed by centrifugation at 1000g with 0.1% peptone water to remove excess vaseline.

6. The supernatant was taken out with a Pasteur pipet.

7. The beads were observed under the microscope to check adequate size and shape.

8. Viability was determined by culturing the beads in MRS and LAPTg agar medium.

9. The beads were distributed in 3-mL volume tubes in and stored at -20°C.

3.3. E. coli Suspension

1. The uropathogenic strain was grown in either BHI or LAPTg broth.

2. Microorganisms were harvested by centrifugation, washed twice with PBS, pH 7.0, and suspended in 0.1% peptone water.

3.4. Estrogenic Stimulation

1. Estrogenic stimulation was performed by im inoculation of one dose (0.5 mg estradiol valerate for human use (Progynon depot). The dose was calculated according to the weight of the mouse (approx 30 g).

2. The hormone was inoculated for 48 h before inoculation with L. fermentum and E. coli.

3.5. Quantification of Serum Estriol

1. Mice were bled from the retro-orbital venous plexus before sacrifice at 24 h to 15 d after stimulation

2. Serum was separated by centrifugation and frozen at -20°C.

3. Quantitative determination of estriol was performed by using the microparticle enzyme inmunoassay (MEIA; Imx System, Abbott).

3.6. Microbiological and Histological Studies in Mice

3.6.1. Vaginal Samples

Vaginal washing was performed with 50 ^L of saline solution by using a syringe with a canule and aspirating the liquid. Samples were processed for the following studies:

1. Direct Gram staining.

2. Vaginal cytology.

3. Culture in selective media.

3.6.2. Bacterial Inoculation

The following procedure was used for microorganism inoculation:

1. Mice were anesthetized with sodium pentobarbital before intraurethral inoculation of L. fermentum in agarose beads (three doses of 1.5 x 108 CFU or 2.0 x 108 CFU 12 h between each dose, and 12 h later one dose of E. coli of 1.0 x 107 CFU or 1.0 x 108 CFU). A plastic catheter coupled to a syringe was used for this purpose.

2. After each inoculation, animals were returned to their cages.

3. During the experimental days indicated, the following samples were taken or studies performed:

a. Urine, for quantification of microorganisms.

b. Organ homogenates, to determine the degree of colonization, represented by the number of microorganisms in each organ.

c. Histological studies, to determine whether certain types of collateral effects or modifications could be seen, at the structural level.

d. Ultrastructural studies by transmission electron microscopy (TEM) to determine whether certain types of modifications had occurred at this level.

3.7. Urine Culture

Before sacrifice, urine was collected aseptically with a loop to (1) observe the presence of red blood cells, polymorphonuclear granulocytes, epithelial cells, and mucus and (2) quantify the numbers of microorganisms in CLED (see Note 4).

3.8. Bacterial Counts in Tissue Homogenates

1. Animals were sacrificed by cervical dislocation.

2. Their urinary tract organs (urethra, bladder, ureters, and kidneys) were removed aseptically, placed in 2 mL of 0.1% peptone water, and homogeneized with a Teflon pestle.

3. The urethra and ureters had been previously cut longitudinally with a scissors to allow release of microorganisms.

4. Sometimes homogenization of the bladder is difficult because of the fibrous nature of the tissue, and the bladder must be homogenized longer.

5. The samples were serially diluted (10-fold) in peptone water, from 1:10 to 1:1,000,000, using glass tubes with peptone water and automatic pipets; each dilution tube mixed vigorously in a vortex.

6. A 0.5-mL aliquot of each sample dilution was plated in duplicate in the following culture media: Lactobacillus selective agar (LBS), MRS, and McConkey's agar (differential for Gram-negative bacilli); 12-15 mL of melted agar (see Note 5).

7. LBS agar, MRS, and McConkey agar were added and mixed carefully while the plate was rotated, for even distribution of the bacteria. The agar media should be melted and maintained at a temperature of 45-50°C before use.

8. The plates were placed in the incubator at 37°C, for 48 h.

9. The number of CFUs was determined, selecting the data for plates that contained 30-300 isolated colonies (see Note 6).

10. Viable cells counts were made for each organ.

11. Results were expressed as means ± SD of the data obtained from three or four animals.

3.9. Cytological Studies

A syringe loaded with 0.9% physiological solution was introduced into the mouse vagina and aspiration was performed.

3.9.1. Fixation

The aspiration was spread over a glass slide and immediately fixed in 96° alcohol (see Note 7). The smears were left to dry in the open air for Papanicolaou smear.

3.9.2. Papanicolaou Smear Technique (25)

1. The smear should be hydrated by successive passages in alcohol with decreasing concentrations, as follows:

2. Distilled water for 1 min (see Note 9).

3. Harris's hematoxylin stain for 5 min.

4. Differentiation with acid water (water with the addition of 1 drop of HCl).

3.9.3. Dehydration (see Note 10)

Successive passages of alcohols of increasing concentration were made (see Note 8), as follows:

1. Ethyl alcohol 96°, two passages of 1 min.

2. Ethyl alcohol 100°, two passages of 1 min.

3. Xylene, two passages of 1 min.

3.9.4. Mounting and Observation (see Note 9)

1. The slides were mounted with Canadian balsam, and covered with a glass cover slide for microscopic observation.

2. The cytological slide was observed under light microscopy (ocular 10X and objective 40X), with different colors. The cells can be of three types: eosinophil surface cells, cyanophilic surface cells, cyanophilic intermediate cells, cyanophilic parabasal cells, leukocytes, or mucus. At least 200 cells are counted to determine the percentage of each of the cellular types observed.

3. An estrogenic state is characterized by the presence of eosinophils surface cells with or without a pycnotic nucleus. The background of the slide does not show inflammatory elements.

4. A progesteronic state is characterized by the presence of cyanophilic intermediate cells with vesicles nucleus. The background of the smear is dirty because of larger amounts of leukocytes and mucus.

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