Clara Silva, Rosario Rey, and María Elena Nader-Macías
The use of probiotic microorganisms has been widely promoted in the last 20 yr
(1). They have been used in the gastrointestinal tract as capsules or as fermented milks
(2). The characteristics of the strains proposed as probiotics have been published (3) or patented under an elaboration process (4-6). The first step in designing a probiotic product is to isolate and characterize strains with some beneficial properties. The second step is to determine the optimal conditions to obtain the highest amount of viable microorganisms, together with the study of the best conditions to produce antagonistic substances (7).
Urinary tract infections (UTIs) constitute a common cause of illness in pre- and postmenopausal women. It was estimated that 40-50% of adult women suffer a cystitis during their life. Ninety percent of acquired ambulatory UTIs and 30% of nosocomial infections are produced by Escherichia coli (8). The healthy human urinary tract is free of microorganisms, except for the anterior urethra, which is colonized by indigenous microbiota. The vaginal environment is a dynamic and complex ecological system with a highly heterogeneous microflora; thus favorable conditions exist for the colonization process, which is also affected by factors external to the tissues. The distal urethra and periurethral areas are separated ecological niches, both covered by the vaginal secretions that contain approx 109 microorganisms/mL (9). In these secretions, members of the genus Lactobacillus are predominant. Bacterial colonization does not increase because of the urinary flux, which clears the bacterial cells from the outer surfaces, as well as other factors such as pH, osmolarity, and urea concentration (10,11).
From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ
The work of Redondo-Lopez et al. (12) and Hillier et al. (13) supports the fact that the ecological stability of the microflora, its integrity in equilibrium with the different host tissues, and the effects exerted by various mechanisms and factors all protect against incoming pathogenic microorganisms. In the same way, loss or disruption of the normal genital microbiota, particularly the lactobacilli species, implies that an increase in urogenital infections will follow. The composition of the vaginal microbiota is influenced by estrogen levels, as reported by some researchers who have shown that this hormone stimulates glycogen deposition in the epithelial tissue (14). Not much is known about the microbial implications of the steroid-hormonal effect on the urogenital tract. Different characteristics can be influenced by hormones involved in the maintenance of the microecosystem (15). The reproductive hormones produce a number of physiological changes in the host, including increased phagocytic activity, which can influence the response to an infectious process (16). However, modifications in carbohydrate and protein metabolism by these hormones can result in changes of the indigenous microflora of the host, owing to alteration of the colonized tissues (17).
One of the biggest challenges for UTI therapy is recurrence in adult women: even though one episode can be rapidly eliminated, it is soon followed by another, sometimes caused by a different microorganism (18). Different methodologies have been adopted for the treatment of recurrences: local hygiene with disinfectants, long- or short-term prophylaxis with oral antibiotics using low doses, oral or topical therapy with estrogens, daily consumption of cranberry juice, and others (19). Ingestion or application of products containing viable lactobacilli is currently accepted to maintain or modify the normal microbiota. In some cases partially effective results were achieved, even though the optimal strategy was not applied (20).
In previous papers, an experimental model was set up to study the effect of lactoba-cilli in the urogenital tract of mice. Autochthonous lactobacilli were isolated from the vagina of normal BALB/C mice. The strains were identified by their morphology and by biochemical and physiological testing and later classified according to Bergey's Manual. In vitro adhesion was tested by their ability to adhere to uroepithelial cells, according to Reid et al. (12). One strain, L. fermentum CRL 1058, was selected by the adherence test and also by other surface characteristics and because it was largely prevalent in our isolates. To study the colonization capability of this L. fermentum strain in the urinary tract, adult female BALB/c mice were used as the experimental model. To prevent the microorganisms from being cleared by the urine flux, they were prepared in agarose beads and inoculated intraurethrally. The optimal dose and the physical forms were studied to determine the colonization capability of this probiotic strain. Effective colonization was obtained by the intraurethral inoculation of agarose beads in three doses containing 107 colony-forming units (CFU)/12 h (22,23). In other studies, in vivo experiments were performed to assess the effect of antibiotics and hormones on colonization of both lactobacilli and uropathogenic E. coli (24,25).
The aim of the present work was to study the effect of estradiol on the kinetics of colonization of L. fermentum CRL 1058 and a uropathogenic E. coli strain in the uri nary tract and the histological modifications produced by them. The following areas were investigated:
1. Determination of microorganisms, the methods to be applied for their use, and preparation for inoculation.
2. Determination of the animals to be used.
3. The amount of hormones in sera.
4. The number of microorganisms in the tissue homogenates.
5. Cytological, histological, and ultrastructural studies performed to determine the effect of the hormones on the colonization capability of L. fermentum and E. coli.
1. L. fermentum CRL 1058: isolated from the vagina of 2-mo-old Balb/c mice, by using the procedure described previously (20).
2. Uropathogenic E. coli: isolated from an adult female patient with a diagnostis of pyelonephritis (from the Collection of Culture Strains, Bacteriology Department, Instituto de Microbiología; courtesy of Dr. L.C. Verna, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán.) The strain had the following characteristics related to pathogenicity: mannose-resistant hemagglutination (MRHA), hemolysin producer, pyelonephritogenic capability (assayed by intraurethral inoculation in 2-mo-old female BALB/c mice and later isolated in different organs of the urinary tract; see Note 1).
2.2. Preparation of Beads for Inoculation
1. Phosphate-buffered saline (PBS), pH 7.0.
3. Peptone water: 0.1% meat peptone in distilled water.
4. LAPTg broth: 15 g/L peptone, 10 g/L tryptone, 10 g/L glucose, 10 g/L yeast extract, 1 mL/L Tween-80, pH 6.5. Autoclave at 121°C for 15 min. Store at refrigeration temperature. The chemicals for LAPTg preparation were obtained from Britania Laboratories (Argentina).
2.3. Culture Media
1. Milk-yeast extract: 10% skim milk, 1% glucose, 0.5% yeast extract, pH 6.0. Autoclave for 20 min at 115°C (3/4 atm).
2. LAPTg broth.
3. LAPTg agar: The same as LAPTg broth with 15 g/L agar.
4. MRS broth: Dehydrated MRS was obtained from Biokar Diagnostics (Beauvais, France). Composition: 10 g/L peptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 1.08 g/L Tween-80, 2 g/L dipotassium phosphate, 5 g/L sodium acetate, 2 g/L ammonium citrate, 0.2 g/L magnesium sulfate, 0.05 g/L manganese sulfate, pH 6.5. Autoclave at 121°C for 15 min. Store at refrigeration temperature.
5. MRS agar: The same as MRS broth with 15 g/L agar.
6. McConkey's agar (Merck): 17.0 g/L casein peptone, 3.0 g/L meat peptone, 5.0 g/L sodium chloride, 10.0 g/L lactose, 1.5 g/L oxgall salt, 0.03 g/L neutral red, 0.001 violet crystal, 13.5 g/L agar, pH 7.1 ± 0.2.
7. Cystine lactose-deficient electrolyte Britania: 4.0 g/L peptone, 3.0 g/L meat extract, 0.128 g/L L-cystine, 4.0 g/L tryptamine, 0.02 bromothymol blue, 10.0 lactose, 15.0 agar, pH 7.3 ± 0.2.
9. Brain-heart infusion (BHI; Britania): 200 g/L goal brain infusion, 250 g/L bovine heart, 10 g/L peptone, 5g/L sodium cloride, 2.0g/L glucose, 2.5 g/L disodium phosphate. pH 7.4 ± 0.2.
10. Other materials.
a. Sterile tubes.
c. Petri dishes.
d. Incubator for 37°C (Forma Scientific, model 3185.
1. Mice: 2-mo-old female BALB/c mice from the inbred colony of the Instituto de Microbiologia at the Universidad Nacional de Tucumán (weighing approx 30 g). Animals were housed in plastic cages and fed ad libitum; environmental conditions were kept constant. Each experiment was carried out with groups of 20-25 mice. The Centro de Referencia para Lactobacilos (CERELA) Committee of Ethics approved the protocol used for animal studies (see Note 3).
2. Mice Sacrifice.
b. Bonnet string.
d. Pasteur pipets for blood extraction.
h. Petri dishes.
i. Sterile tubes containing 2 mL 0.1% peptone water for tissue homogenization. j. Homogenizer.
1. Estradiol valerate (Progynon depot, Schering): 10 mg in 1-mL ampules. Hormones were inoculated at different doses, to determine which dose produced some type of effect. Evaluation of the hormonal effect was determined by serum estrogen level and by vaginal cytology, which permits observation of cell changes during the estral cycle. The protocol used is schematized in Fig. 1.
2.6. Cytological Studies
1. Saline solution (0.15 M NaCl) for vaginal washes. Sterilize by autoclaving and store at refrigeration temperature.
2. 76 x 26-mm Glass slides microscope.
3. 96° Ethanol (Biopack, Argentine). Store at room temperature.
4. Glass bottles for slides.
5. Graduated ethyl alcohols: 96°, 80°, 70°, 50°, 100°.
6. Harris hematoxylin (Biopack, Argentina). Store at room temperature.
7. Aqueous eosine (E.A.36; Biopur, Argentina). Store at room temperature.
1x108UFC 1x108UFC 1 x 10s UFC 1 x 107 UFC Days of mice sacrifice
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