Hydroxylapatite Beads As an Experimental Model to Study the Adhesion of Lactic Acid Bacteria From the Oral Cavity to Hard Tissues

María del Carmen Ahumada Ostengo and María Elena Nader-Macías

1. Introduction

The oral environment contains many different types of microorganisms, including both Gram-negative and Gram-positive cocci, bacilli, and spirochetes. From the ecological point of view, the oral cavity is a perfect niche for certain bacteria such as lactobacilli (1) because they interact, forming different types of communities. Lacto-bacilli have been associated with the generation of caries in some reports, secondary to the cariogenic Streptococcus (2).

In previous papers, the isolation and identification of 145 strains from healthy subjects and from subjects with active caries (3-5) were performed. Strains were characterized by their surface properties and also by the production of inhibitory substances (3-5). From all the strains, one isolated from the teeth of healthy patients and another from a patient with caries, sharing some surface properties, were selected for further study of their adhesion properties in an experimental model by using hydroxylapatite beads.

Adhesion is the first step in the association of microorganisms with surfaces or mucous membranes. The first approach is a nonspecific interaction of both surfaces; later some other types of interactions can occur, involving more specific mediators of adhesions (6,7).

Many different assays are available to study the adhesion phenomenon; some of them use predictive characteristics, and others use experimental models resembling the in vivo situation (1). Two model systems predominate. The most widely used has been saliva-coated hydroxylapatite or hydroxylapatite coated with buffers, proteins, and other substances (1).

In an attempt to increase knowledge of the mechanisms of adhesion of oral lactoba-cilli with hard surfaces, this chapter describes an experimental model for studing the adhesion between lactobacilli and hard tissues represented by hydroxylapatite, the component most abundant in the teeth. The following steps were performed:

1. Obtaining the microorganisms.

2. Preparation of the hydroxylapatite beads.

3. Adhesion assay.

2. Materials

2.1. Microorganisms and Culture Media

1. Probiotic microorganisms: The bacteria used in this study were Lactobacillus salivarius CRL (Centro de Referencia para Lactobacilos Culture Collection) 1414 and L. plantarum CRL 1356. They were isolated from the mouths of different groups of patients and characterized as described previously (2-4).

2. Storage and culture.

a. Milk-yeast extract: 10% skim milk, 1% glucose, 0.5% yeast extract, pH 6.0.

b. Sterilize by autoclaving.

c. Store at refrigerated temperature.

d. All the culture media and/or components were obtained from Britania-Argentina.

3. Lactobacilli subcultivation.

a. LAPTg broth: 15 g/L peptone, 10 g/L tryptone, 10 g/L glucose, 10 g/L yeast extract, 1 mL Tween-80, pH 6.5).

b. Sterilize by autoclaving.

c. Store at refrigerated temperature.

4. Peptone water (diluent): 0.1% meat peptone.

5. Adhesion buffer: 50 mM KCl, 1 mM KH2PO4, 1 mMCaCl2, 0.1 mMMgCl2, pH 6.0. Store at refrigerated temperature. The prepared buffer can be stored under refrigeration for up to 6 mo, to control contamination.

2.2. Preparation of Hydroxylapatite Beads

1. Hydroxylapatite powder: fast flow (flow rate 115 mL/h/cm2), mol wt = 1004 (ICN Biomedicals). Store at room temperature.

2. Powder and liquid glass ionomer filling material with type II dose dispenser (Klepp Universal Shade, Germany). Store at room temperature.

3. Adhesion buffer: as described in Subheading 2.1., item 5, pH 6.0. Store at refrigerated temperature.

4. Syringe restorative dispenser (3M Dental Products), with spatula and support glass.

5. Peptone water (diluent): 0.1% meat peptone distributed in glass tubes and stored at refrigeration temperature.

6. Other materials:

a. Automatic pipets.

b. Petri dishes.

c. Tissue homogenizer with Teflon pestle (MSE, speed continuously variable).

d. Glass tubes.

e. Glass slides.

f. Sterile tips for pipets.

g. Tube centrifuge (Rolco SRL model 2036, Argentina).

3. Methods

3.1. Preparation of Probiotic Bacteria Cell Suspension (see Note 1)

1. Lactobacilli were subcultured in LAPTg broth no more than three times, by using a 2% inoculum.

2. Each subculture was incubated for 12 h at 37°C.

3. The bacterial cells were collected by centrifugation at 2000g for 15 min and washed with the adhesion buffer.

4. Three different concentrations of microorganisms were established for each assay.

5. The Optical density was determined previously according to a calibration curve by which the number of colony-forming units (CFU)/mL was determined (by the successive dilution method and plating in agar media).

6. The three optical densities at 540 nm used in each assay were 0.1, 0.3, and 1.0, corresponding to 1 x 106 CFU/mL, 1.2 x 108 CFU/mL, and 1.3 x 109 CFU/mL for L. platarum CRL 1356 and 1 x 106 CFU/mL, 2 x 107 CFU/mL, and 2 x 108 CFU/mL for L. salivarus CRL 1414.

3.2. Preparation of Beads With Hydroxylapatite

1. The beads were prepared by mixing (with a dose dispenser) two parts of hydroxylapatite with two parts of powdered glass ionomer and 400 ^L of liquid in a glass support.

2. The syringe was loaded with the mixture, and the material was spilled out, forming the beads on the same glass support.

3. The beads were allowed to rest at room temperature for 24 h.

4. Then the weight of the beads was determined, and the beads were distributed in Eppendorf tubes (approx 0.22 g of beads) and autoclaved.

5. The beads were stored at refrigeration temperatures and stabilized for 30 min at room temperature immersed in 1mL adhesion buffer before the experiment.

3.3. Adhesion Assay

1. Microorganisms.

a. Before beginning the adhesion experiment, the number of initial microorganisms was determined by serial dilution in peptone water (dilutions 1:10 to 1:1,000,000).

b. Aliquots were plated in LAPTg agar.

c. The plates were incubated under microaerophilic conditions (Forma Scientific CO2 incubator model 3185, water-jacketed) for 48 h at 37°C.

d. This step was performed after the washing step described before.

a. Beads were stabilized at room temperature.

b. After sterilization in autoclaving, they can be stored at refrigeration temperature, before being stabilized in the buffer at room temperature.

c. Beads were added to 1 mL of adhesion buffer.

d. The beads were always used at a similar weight, approx 0.22 g and stored in an Eppendorf tube.

3. Adhesion assay.

a. Aliquots of 2.5 mL of microorganisms were placed in contact with the beads 1 mL of adhesion buffer.

b. The mixture was taken to the shaker in a thermostatic water bath and incubated for 1 h at 37°C, 50 opm (see Note 2).

c. After incubation, the beads were separated from the supernatant with a spatula in aseptic conditions.

d. They were washed twice with adhesion buffer by gently moving the tubes for 5 min at room temperature.

e. Disintegration of the beads was performed by using a Teflon pesttle connected to a tissue homogenizor (resuspended in 1 mL of water peptone).

f. Aliquots of the supernatant were serially diluted in peptone water (as described before in step 1a.) and plated in LAPTg agar for determining the number of nonadherent microorganisms.

g. The number of bacteria adhering to the beads was determined in the bead homogenates by serial dilution in peptone water and by plating aliquots in LAPTg agar.

h. The plates were incubated in microaerophilic conditions (Forma Scientific CO2 incubator, model 3185, water-jacketed) for 48 h at 37°C.

i. Controls were represented by bacterial suspensions without beads.

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