Bacterial Strains
Lactobacillus strains were isolated from fermented cucumbers, which are the most popular fermented foods consumed in Poland. Juice of fermented cucumbers was seeded on MRS agar (De Man, Rogosa and Sharpe agar, BTL, Warsaw, Poland) and incubated in aerobic condition for 24 h at 37 °C. Bacteria were isolated and identified from selected colonies. Isolates which were Gram-positive and catalase-negative rods were further identified using API 50CHL (bio-Merieux, Marcy l’Etoile, France) and MALDI-TOF MS analysis (Bruker, Billerica, Massachusetts, USA). Of 26 isolates, 4 strains of Lactobacillus plantarum and 4 strains of Lactobacillus brevis were selected for further study. One of the selection criteria was the growth capacity of these strains in the presence of bile salts and gastric juice which was checked by methods described by Li et al. [22].
P. mirabilis (strain C7) was derived from encrusted biofilm formed on urinary catheters of long-term catheterized patients and was deposited in the bacterial strain collection at the Department of Biology of Bacteria, the University of Lodz. The method of isolation of this strain and its characteristics had been described in our previous study [31].
Adhesion Assay
The ability of the Lactobacillus strains to adhere to both intestinal (Caco-2) and urinary (Hu609) epithelial cells was determined. In this assay, the cells were grown in a suitable medium line, in the case of Caco-2, it was Dulbecco’s medium; for Hu 609, RPMI 1640 medium was used. Media were supplemented with 10% heat-inactivated fetal calf serum (FCS, Lonza, Walkersville, MS, USA), 2 mM ultraglutamine (Lonza), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Polfa Tarchomin, Warsaw, Poland). For the adhesion assay, all types of epithelial cells (1 × 105 cells per well) were seeded into 24-well plates and grown for 24 h in a humidified incubator with 5% CO2 at 37 °C in culture medium supplemented only with 10% fetal bovine serum (FCS, Lonza) and 2 mM ultraglutamine (Lonza). Afterwards, the cells were infected with 1-mL bacterial suspension (108 CFU/mL in culture medium with 10% FBS + 2 mM ultraglutamine) (Lonza). After 3-h incubation (37 °C, 5% CO2), the suspension was aspirated, and the cells were washed three times with PBS (pH 7.3) to remove free bacteria. To establish the number of adherent bacteria, a monolayer was lysed using 1% Triton X-100. After that, 1/10 dilution of the suspension was seeded on MRS plates and incubated for 24 h at 37 °C. The results are expressed as bacterial colony-forming units (CFU) recovered per milliliter.
Antibacterial Activity
The antimicrobial activity of Lactobacillus strains was checked by broth microdilution method. Cultures of Lactobacillus (MRS broth for 48 h at 37 °C) were centrifuged 8000×g for 20 min at 4 °C. The supernatant without the bacteria was recovered and separated into two aliquots. One of them was untreated; the second supernatant was neutralized to pH 6.5 with 1 N sodium hydroxide (NaOH). In the case of untreated supernatants, the pH ranged between 3.42 and 3.86. The supernatants were sterilized by filtration through Minisart® (Sartorius, Goettingen, Germany) syringe filters 0.2 μm. The antibacterial activity of Lactobacillus was checked against P. mirabilis C7. The strain was cultured in TSB medium (tryptic soy broth, BTL, Warsaw, Poland) for 24 h at 37 °C. In the experiment, the dilutions of supernatants were prepared in F-bottom 96-well plates. To each well, 100 μl of bacterial suspension (105 CFU/mL) in TSB to 100-μl tested supernatant was added. As controls, 200 μl TSB (negative control) and 200 μl bacterial suspension in TSB of the tested bacterial strains (positive control) were used. Inhibition of bacterial growth was detected by measuring turbidity at 600 nm using a microplate reader Multiskan Ex (Labsystems, Helsinki, Finland).
Analysis of the Influence of Lactobacillus on the Crystallization Caused by P. mirabilis
The effect of Lactobacillus on crystallization caused by P. mirabilis was analyzed in eight independent tests. The first probe (control) contained only P. mirabilis, in the remaining seven tests, P. mirabilis bacteria were incubated simultaneously with individually tested strains of Lactobacillus. Lactobacillus strains were cultured on MRS medium (De Man, Rogosa and Sharpe broth, BTL, Warsaw, Poland) for 48 h at 37 °C, while P. mirabilis was grown in a TSB medium (tryptic soy broth, Warsaw, BTL, Poland) for 18 h at 37 °C. Before each experiment, a suspension of both or only one species in 20 mL of synthetic urine was prepared. The number of bacteria per milliliter was determined spectrophotometrically at 600 nm (Ultrospec 2000, Pharmacia Biotech, Vienna, Austria), where the density of the suspension was adjusted to 1–5 × 105 bacteria per milliliter. Synthetic urine is a composition which corresponds to the mean concentration of the mineral components found during a 24-h period in normal human urine and was used in in vitro studies of uropathogens [19, 44]. The solution was prepared using the modified method previously described by McLean et al. [30] and consisted of the following components (g/L): urea (CH4N2O)—25.0, sodium chloride (NaCl)—4.6, potassium dihydrogen phosphate (KH2PO4)—2.8, sodium sulfate (Na2SO4)—2.3, potassium chloride (KCl)—1.6, ammonium chloride (NH4Cl)—1.0, creatine (C4H9N3O2)—1.1, calcium chloride dihydrate (CaCl2 × 2H2O)—0.651, magnesium chloride hexahydrate (MgCl2 × 6H2O)—0.651, sodium citrate (Na3C6H5O7)—0.65, sodium oxalate (Na2C2O4)—0.02 (Sigma, Poznan, Poland), and tryptic soy broth, 10.0 (BTL, Warsaw, Poland). After adjusting the pH to 5.8, the synthetic urine solution was sterilized by passing through a 0.2-μm pore-size filter (Minisart® Sartorius, Goettingen, Germany).
Bacterial suspension in synthetic urine was incubated without mixing for 24 h at 37 °C. At the beginning of the experiment (0 h) and after 5, 8, and 24 h, incubation pH, bacterial viability, and the degree of crystallization were evaluated in all cultures. A pure culture of P. mirabilis was used as a control system in all mentioned test. The numbers of bacteria in pure and mixed cultures, defined as CFU/mL (colony forming units per milliliter), were determined on McConkey medium (BTL, Warsaw, Poland) and MRS agar for P. mirabilis and Lactobacillus, respectively.
Crystallization was determined by spectrophotometric measurements, microscopic observation using direct phase-contrast microscopy (Nikon Eclipse 2000S, Tokyo, Japan), and its intensity was tested by chemical analysis. The turbidity of bacterial suspension in synthetic urine as an absorbance of light at 600 nm wavelength was measured (Ultrospec 2000, Pharmacia Biotech, Uppsala, Sweden). For chemical analyses, a sample (1 mL) of crystals with a bacterial suspension was centrifuged at 8000×g for 10 min. The obtained pellet was suspended in aqueous solution of nitric acid (30% HNO3, POCh, Gliwice, Poland) and incubated for 60 min at 100 °C. After mineralization, calcium and magnesium concentrations were determined by atomic absorption spectroscopy (AAS, SpectrAA-300 Varian, Palo Alto, USA). X-ray powder diffraction (XRD) was used for phase identification of a crystalline material. Analysis was performed on X’pert PRO MPD (PANalytical) diffractometer where CuKα radiation monochromatized by nickel filter was applied. Measurements were done in the range of 2θ angles from 3 to 90° with a continuous scan (step 0.0167°) where the measurement time of one step was 30 s. The X’Pert High Score Plus PANanalytical (Almelo, The Netherlands) software was used for indexing peaks in the XRD pattern.
For the evaluation of bacterial extracellular protein and polysaccharide content, 1 mL of each sample was taken at 0, 5, 8, and 24 h of incubation and centrifuged at 8000×g for 10 min in room temperature. Protein concentrations were determined by the method of Lowry et al. [24] using bovine serum albumin as a standard. The phenol-sulfuric acid method described by Masuko et al. [27] with glucose as a standard was selected for carbohydrate determination.
Statistics
The data are presented as mean ± standard deviation (SD) of three to five independent experiments. Statistical analyses were based on the Mann-Whitney U test performed using Statistica software version 13.3 pl (StatSoft, Krakow, Poland). The results were considered to be statistically significant at p < 0.05. Statistical differences between groups are indicated in the text.