Determination of Bacterial Adhesion to Intestinal Mucus

Carlos Gusils, Vilma Morata, and Silvia González

1. Introduction

The epithelial cells in the small intestine are covered by a relatively thick layer of mucus, secreted by specialized cells, which consists of mucin, many small associated proteins, glycoproteins, lipids, and glycolipids. The mucus contains receptors that recognize specific adhesion proteins (1,2). Adhesion or close association of bacteria to the epithelial cells may further contribute to competitive exclusion. In addition, bacterial adhesion to intestinal mucus and epithelia seems to be important for individual stability of microbial flora (3). Although the mucus layer covering the gastrointestinal tract has been recognized for many years, it has only recently been of interest in the study of the adhesion between mucus and bacteria. In this chapter, we describe a method for the study of colonization of the gastrointestinal mucus by bacteria and determine the possible effects on adhesion of pathogenic organisms.

2. Materials

2.1. Growth Media

1. de Man-Rogosa-Sharpe (MRS) broth (4), instead of LAPTg broth, can be use for lactic acid bacteria growth. MRS broth: 10 g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 2 g/L ammonium citrate, 2 g/L K2HPO4, 0.2 g/L MgSO4-7H2O, 0.05 g/L MnSO4-4H2O, and 1.08 g/L Tween-80. Sterilize in the autoclave at 121 °C for 15 min (see Note 1).

2. Brain-heart infusion (BHI) broth is used for culturing enterobacteria. Composition: 200 g/L calf brains, 250 g/L infusion from beef heart, 10 g/L protease peptone, 2 g/L dextrose, 5 g/L sodium chloride, and 2.5 g/L disodium phosphate, pH 7.4 ± 0.2. Sterilize in autoclave at 121°C for 15 min.

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

2.2. Mucus Collection

1. Newborn animals.

2. Sterile Petri dishes with two microscope covers.

3. HEPES-Hanks' buffer (Sigma).

4. Sterile tubes of centrifuge.

5. Refrigerated centrifuge.

a. Solution A (0.2 M NaH2PO4): Weigh 27.6 g NaH2PO4-H2O and make to 100 mL with distilled water (dH2O).

b. Solution B (0.2 MNa2HPO4): Weigh 53.05 g of Na2HPO4-7H2O and make to 100 mL with dH2O.

c. To prepare 1 L of 0.1 M phosphate buffer, pH 7.0, mix 195 mL of solution A and 305 mL of solution B and bring to 1000 mL with dH2O.

2.3. Growth of Lactobacilli in Chicken Small Intestinal Mucus

1. An active culture (16 h at 37°C) grown in MRS broth.

2. HEPES-Hanks' buffer (Sigma).

4. Physiological solution (PS): 8.5 g/L sodium chloride.

5. Rogosa SL agar (Merck): 10 g/L peptone from caseine, 5 g/L yeast extract, 20 g/L D(+) glucose, 6 g/L potassium dihydrogen phosphate, 2 g/L ammonium citrate, 1 g/L Tween-80, 15 g/L sodium acetate, 0.575 g/L magnesium sulfate, 0.034 g/L iron (II) sulfate, 0.12 g/L manganese sulfate, 15 g/L agar-agar, pH 5.5 ± 0.2. Suspend 74.5 g in 1 L of demineralized water by heating in a boiling water bath or in a current of steam; adjust the pH to 5.5 with acetic acid 96% (see Note 2).

6. Spectrophotometer.

2.4. In Vitro Adhesion Assay

1. An active culture grown in MRS broth for about 16 h at 37°C.

2. [Methyl, 1,2-3H] thymidine (specific activity 117 Ci/mM; Sigma).

3. Sterile mucus.

4. HEPES-Hanks' buffer.

5. 50 mg/mL Bovine serum albumin (BSA; Sigma).

6. Sterile polystyrene tissue culture wells (Corning).

7. 5% Sodium dodecyl sulphate (SDS): weigh 5 g and make up to 100 mL in dH2O with gentle stirring. Store at room temperature (see Note 3).

8. Scintillation liquid.

2.5. Characterization of Bacterial Adhesion to Mucus

1. An active bacterial culture grown in MRS broth for about 16 h at 37°C.

2. Sterile mucus.

2.5.1. Proteolytic Digestion

1. 1 mg/mL Trypsin (activity: 10,600 U/mg protein; Sigma).

2. 1 mg/mL Protease K (activity: 10-20 U/mg protein, Sigma).

3. Phosphate buffered saline (PBS), pH 7.0.

2.5.2. Characterization of Adhesion

1. Water bath at different temperatures (30, 37, and 42°C).

2. 0.1 M PBS at different pH values (6.0, 7.0, and 8.0).

3. 0.2 mMof different carbohydrate (Sigma) solutions (fucose, fructose, galactose, glucose, lactose, mannose, ^-acetyl galactosamine, ^-acetyl glucosamine, sialic acid, and sucrose).

4. 1 mM CaCl2 (Anedra, Argentina) in PBS.

2.6. Inhibition of Adhesion Assay

1. Pathogenic bacteria (for example, enterobacteria): Active cultures grown in BHI broth for about 16 h at 37°C.

2. An active bacterial culture grown in MRS broth for about 16 h at 37°C.

3. Sterile polystyrene tissue culture wells.

5. HEPES-Hanks' buffer.

6. Bacterial spent culture supernatants (SCS).

3. Methods

3.1. Mucus Collection

1. Obtain 5-cm samples from different sections of intestinal tract in asceptic conditions and put on a sterile microscope cover slip.

2. Isolate mucus from the sample walls by gently scraping with a sterile microscope cover slip, and put in sterile tubes of the centrifuge.

3. Dilute mucus twice with 10 M HEPES-Hank's buffer, pH 7.4, and shaken vigorously for 5 min.

4. Centrifuge once at 11,000g (10 min at 4°C) and once at 26,000g (15 min at 4°C) (see Note 4).

5. Store aliquots of the supernatant at -20°C.

3.2. Growth of Lactobacilli in Chicken Small Intestinal Mucus

1. Grow the strains in MRS broth at 37°C for 16 h.

2. Harvest the cells by centrifugation at 4000g for 5 min.

3. Wash the pellet obtained twice with PS (see Note 5).

4. Resuspend the cells in 5 mL PBS (0.1 M, pH 7.0).

5. Dilute the mucus in HEPES-Hanks' buffer (1:4).

6. Sterilize 3-mL aliquots of diluted mucus using UV light for 10 min.

7. Inoculate into 3 mL of UV-treated mucus an aliquot of 30 ^L (10-3 dilution) of overnight bacterial culture.

8. Incubate at 37°C in a water bath.

9. Determine the optical density at 560 nm against blank (non-inoculated UV-treated mucus) and plated dilutions of inoculated and non-inoculated mucus on Rogosa SL agar, every hour for the first 8 h and after 12, 16, and 24 h of incubation.

10. Plot CFU/mL and OD values against incubation time.

3.3. In Vitro Adhesion Assay

1. Grow the strains of lactic acid bacteria in LAPTg broth at 37°C for 16 h.

2. Harvest the cells by centrifugation at 4000g for 5 min.

3. Wash the pellet obtained with PS twice (see Note 5).

4. Resuspend the cells in 5 mL PS.

5. Inoculate 50 ^L in LAPTG broth with 1% [methyl, 1,2-3H] thymidine (specific activity 117 Ci/mM, Sigma).

7. Collect cells by centrifugation at 4000g for 5 min.

8. Wash twice with HEPES-Hanks' buffer.

9. Resuspend the cells in HEPES-Hanks' buffer.

10. Add 300 ^L of mucus and BSA in polystyrene tissue culture wells.

12. Wash twice with HEPES-Hanks' buffer to remove mucus or BSA excess.

13. Add 200 ^L of bacterial suspension to the immobilized mucus and BSA coating.

14. Incubate tissue culture plates for 1 h at 42°C.

15. Wash twice with HEPES-Hanks' buffer to remove unbound bacteria.

16. Add 5 mL of 5% SDS to each well and incubate the plates overnight at 37°C to lyse the bacteria.

17. Add the lysed cells from each well to 2 mL of scintillation liquid.

18. Calculate the adhesion ratio (%) comparing the radioactivity of the original bacterial suspension with the final radioactivity from the lysed cells.

3.4. Characterization of Bacterial Adhesion to Mucus

1. Prepare the culture inoculum as described in steps 1-9 from Subheading 3.3.

3.4.1. Proteolytic Digestion

1. Incubate mucus and mucus fractions with 1 mg/mL trypsin and 1 mg/mL protease K for 1 h at 37°C (see Note 8).

3. Determine adhesion capacity by method described in steps 13-18 from Subheading 3.3.

3.4.2. Characterization of Adhesion

1. Prepare the culture inoculum and immobilized mucus as described in Subheading 3.3.

2. Incubate the plates at different temperatures (30, 37, and 42°C) and pH values (6.0, 7.0, and 8.0), and with 0.2 mM of different carbohydrate solutions, 1 mM CaCl2, 0.15 mM EDTA.

4. Determine adhesion capacity by method described in steps 13-18 from Subheading 3.3.

3.5. Inhibition of Adhesion Assay

1. Prepare the culture inoculum (pathogenic bacteria) and immobilized mucus as described in Subheading 3.3.

2. Incubate beneficial bacteria or spent culture supernatants (SCS; see Note 9) with mucus in polystyrene tissue culture wells for 1 h at 42°C.

4. Challenge labeled pathogenic bacteria for 1 h at 42°C.

5. Wash twice with HEPES-Hanks' buffer.

6. Determine adhesion capacity by the method described in steps 13-18 from Subheading 3.3.

4. Notes

1. LAPTg broth (5), instead of MRS broth, can be use to lactic acid bacteria growth. LAPTg broth: 15 g/L peptone, 10 g/L yeast extract, 10 g/L glucose, 10 g/L triptone, and 1% Tween-80, pH 6.4 ± 0.2. Sterilize in autoclave at 121°C for 15 min.

2. This media doesn't have to be autoclaved.

3. Solution may become cloudy at temperatures below 20°C, but warming to 30°C and mixing may restore clarity.

4. The centrifugation removes epithelial cells and large cellular debris, to eliminate contaminants.

5. Cell washes are important to minimize carryover of products from culture.

6. The incubation time can be longer (8 or 16 h).

7. This stage is necessary to immobilize the mucus in the plates.

8. Use equal volumes of mucus or fractions (without proteolytic enzymes) as control.

9. SCS are obtained from overnight cultures in LAPTg broth by centrifugation (3000g) and sterilization by filtration; they are then used in the inhibition assays.

References

1. Kim, Y. S., Morita, A., Miura, S., and Siddiqui, B. (1984). Structure of glycoconjugates of intestinal mucosal membranes. Role of bacterial adherence. In: Attachment of organisms to the gut mucosa (Boedecker, E. C., ed.). CRC, Boca Raton, FL, pp. 99-109.

2 Metcalfe, J. E., Krogfelt, H. C., Krivan, C., Cohen, P. S., and Laux, D. C. (1991) Characterization and identification of a porcine small intestine mucus receptor for K88ab fim-brial adhesion. Infect. Immun. 59, 91-96.

3. Mikelsaar, M., Mandar, R., and Sepp, E. (1998). Lactic acid microflora in the human microbial ecosystem and its development. In: Lactic Acid Bacteria, Microbiology and Functional Aspects, Food Science and Technology (Salminen, S. and von Wright, A., eds.). Marcel Dekker, New York, pp. 279-342.

4. de Man, J. C., Rogosa, M., and Sharpe, E. (1960) A medium for the cultivation of lactoba-cilli. J. Appl. Bacteriol. 23, 130-155.

5. Raibaud, P., Caulet, M., Galpin, J. V., and Mocquot, G. (1961) Studies on the bacterial flora of the alimentary tract of pigs II. Streptococci: selective enumeration and differentiation of the dominant group. J. Appl. Bacteriol. 24, 285-291.

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