Production of Antimicrobial Substances by Lactic Acid Bacteria II

Screening Bacteriocin-Producing Strains With Probiotic Purposes and Characterization of a Lactobacillus Bacteriocin

Virginia S. Ocaña and María Elena Nader-Macías 1. Introduction

Bacteriocins have been defined as proteinaceous, bactericidal substances synthesized by bacteria, which usually have a narrow spectrum of activity, only inhibiting strains of the same or closely related species (1). The term bacteriocin-like substance is applied to antagonistic substances that are not completely defined or do not fit the typical criteria of bacteriocins. They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria as well as fungi (2).

Lactobacillus species are the dominant microorganisms isolated from the vagina of healthy premenopausal women (3). In this environment, they exert a protective effect against pathogenic microorganisms by different mechanisms such as production of antimicrobial agents, which include organic acids, hydrogen peroxide, and probably bacteriocins (2,4-6). The production of bacteriocins by vaginal lactobacilli has been demonstrated in vitro; however, it is not yet well established whether they are produced in vivo as another antagonistic mechanism exerted by the normal microflora.

Bacteriocin-producing bacteria as well as bacteriocins per se are of growing interest as biological controls in the manufacture of beverages and fermented products, mainly in the area of dairy products (7). These bacteria have also been proposed as probiotic candidates for human or animal use.

The objectives of the present chapter are to describe the methods employed for:

1. Detection of production of bacteriocins among vaginal Lactobacillus strains.

2. Characterization of the bacteriocin or bacteriocin-like substances.

3. Study of the kinetics of production and mode of action of bacteriocins.

4. Determination of the inhibition of pathogenic microorganisms by bacteriocin-producing strains in mixed cultures.

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. Materials

2.1. Microorganisms

1. Lactobacillus strains. The microorganisms employed for this study were 134 Lactobacillus strains isolated from human vaginal swabs. The methods applied for the isolation and identification of lactobacilli have been previously reported (8).

2. Pathogenic microorganisms. These were kindly provided by the Culture Collection of the Instituto de Microbiología "Luis C. Verna" (Universidad Nacional de Tucumán, Argentina). They had been isolated from vaginal swabs and identified by standard techniques. Streptococcus agalactiae ATCC 27956, Enterococcusfaecium ATCC 19434, Enterococ-cus faecalis ATCC 19433 (all from American Type Culture Collection), Enterococcus faecium CRL 318 and E. faecalis CRL 341 (both from Cerela Culture Collection) were also used.

2.2. Culture, Dilution, and Conservation Media

1. LAPTg broth: 1.5% peptone, 1% tryptone, 1% glucose, 1% yeast extract, 0.1% Tween-80, pH 6.5. Sterilize at 121°C for 15 min (autoclaved), and store at refrigerated temperature.

2. LAPTg agar: 1.5% peptone, 1% tryptone, 1% glucose, 1% yeast extract, 0.1% Tween-80, 1.5% agar-agar, pH 6.5. Sterilize at 121°C for 15 min and store at refrigerated temperature.

3. LEL: 10% low fat milk, 0.5% yeast extract, 1% glucose. Sterilize at 121°C for 15 min and store at refrigerated temperature.

4. LBS agar: 1% peptone from casein, 0.5% yeast extract, 2% glucose, 0.6% potassium dihydrogen phosphate, 0.2% ammonium citrate, 0.1% Tween-80, 1.5% sodium acetate, 0.0575% magnesium sulfate, 0.0034% iron (II) sulfate, 0.012% manganese sulfate, 1.5% agar-agar, pH 5.5. Sterilize at 121°C for 15 min and store at refrigerated temperature.

5. Blood agar: Brain-heart infusion agar supplemented with 2% human blood. Add the blood to the 45°C melted agar and pour into Petri dishes. Store the plates at 4°C.

6. Chocolate agar: Brain-heart infusion supplemented with 2% human blood. Add the blood to the melted agar, heat at 70°C, and pour into Petri dishes. Store the plates at refrigerated temperature.

7. Streptococcus faecalis (SF) agar: Streptococcus Selective Agar (Merck, Germany), prepared according to the manufacturer's indications, final pH 7.4. Sterilize by autoclaving and store at refrigerated temperature.

8. Peptone water: 1% meat peptone in distilled water. Store at room temperature.

2.3. Proteolytic Enzymes and Other Reagents Employed for the Characterization of Bacteriocins

All of these were obtained from Sigma (St Louis, MO).

1. 10 mg/mL Proteinase K, trypsin, type II, and type XV protease in phosphate buffer, pH 7. Filter-sterilize the solutions (0.22-^m pore size membranes; Millipore) and store at -20°C.

2. 10mg/mL Chymotrypsin in Tris-HCl buffer, pH 8.0, with 0.01 M CaCl2. Filter-sterilize the solution (0.22-^m pore size membranes; Millipore) and store at -20°C.

3. 10mg/mL Pepsin solution in citrate buffer, pH 3.0. Filter-sterilize the solution (0.22-^m pore size membranes; Millipore) and store at -20°C.

4. 1 M a-Amylase in phosphate buffer, pH 6.9. Filter-sterilize the solution (0.22-^m pore size membranes; Millipore) and store at -20°C.

5. 0.1 M Sodium m-periodate in acetate buffer, pH 4.5. Filter-sterilize the solution (0.22-^m pore size membranes; Millipore) and store at -20°C.

6. 0.1 g/mL Lipase solution in PBS, pH 7.7, with 0.01% CaCl2. Filter-sterilize the solution (0.22-^m pore size membranes; Millipore) and store at -20°C.

7. Chloroform (Merck, Germany).

2.4. Accessories

1. Plastic straws, cut in 7-8-cm pieces and sterilized in glass flasks.

2. Sterile cotton swabs, used to take vaginal samples and to spread Neisseria and Gardnerella on agar plates.

3. 0.22-^m Membrane filters (Millipore).

3. Methods

3.1. Microorganisms

1. Lactobacillus storage: Isolated lactobacilli are grown in 5 mL of LAPTg for 12 h at 37°C, subcultured twice in the same media under identical conditions, and then separated by centrifugation at 2000g for 15 min. The pellets are resuspended in 5 mL of LEL and stored at -20 °C.

2. Lactobacillus growth: Frozen lactobacilli are subcultured tree times in LAPTg broth and incubated for 12-14 h at 37°C prior to performing the studies. The inocula for the cultures were 2%. Overnight cultures grown in LAPTg broth are centrifuged at 2000g for 30 min, and the supernatants were separated for detection of bacteriocins.

3. Pathogen growth and storage: Enterococcus, Staphylococcus, Listeria, Candida and Streptococcus species are grown in LAPTg broth and stored in milk-yeast extract at -20°C. They are subcultured three times in LAPTg for 12-14 h at 37°C prior to perform the inhibition studies. The inocula for the cultures are of 2%. Neisseria gonorrhoeae strains are grown in chocolate agar and Gardnerella vaginalis in blood agar, both incubated microaerophilically at 37°C. They are not stored but immediately employed for the inhibition assays.

3.2. Detection of Antimicrobial Activity

To perform screening for bacteriocin production, the following steps were followed:

1. The Lactobacillus supernatants (obtained as described in Subheading 3.1., step 2) were neutralized with 2 N NaOH, treated with catalase (1000 U/mL) for 1 h at 25°C, and filter-sterilized (Millipore, 0.22-^m pore size). The treated supernatants were stored at 4°C while the plates with the indicator strains were prepared.

2. Preparation of plates with indicator strains: The pathogens (grown as described in Subheading 3.1., step 3) were diluted in peptone H2O (1:10 or 1:100 dilutions to obtain concentrations of 107-108 CFU/mL, mixed with 10 mL of 45°C melted LAPTg agar, and poured onto Petri dishes. Colonies of microorganisms isolated in blood or chocolate agar are transferred to the same media by using cotton swabs to test the antimicrobial activity of lactobacilli supernatant in pure cultures.

3. Effect of Lactobacillus supernatants on pathogen growth: 25 ^L of lactobacilli supernatant aliquots (obtained as described in Subheading 3.2., step 1) were assayed on pathogen plates into which 4-mm holes had been punched with sterilized straws and that were filled with the supernatant. The plates are incubated for 5 h at room temperature (see Note 1) and then for 24 h at 37°C. The diameters of the inhibition halos are measured after the incubation.

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