Production of Antimicrobial Substances by Lactic Acid Bacteria I

Determination of Hydrogen Peroxide

María Silvina Juárez Tomás, María Claudia Otero, Virginia Ocaña, and María Elena Nader-Macías

1. Introduction

Restoration of the balance of different ecological niches has been proposed as a way to control the income of pathogenic microorganisms (1). The genus Lactobacillus has been used in different human and animal tracts as probiotic microorganisms with this objective in mind (2). The characteristics of the strains proposed as probiotics have been published (3) or patented under the process of elaboration of different types of products (4-6). One of the mechanisms suggested to control the vaginal ecosystem is the production of antagonistic substances (lactic acid, bacteriocins, or H2O2) (7-9). The H2O2-producing microorganisms present in the vagina of healthy women have been suggested as some of the bacteria responsible for maintenance of ecological balance, mainly in pregnant women (10,11). The absence of these microorganisms is related to a higher risk of: bacterial vaginosis (12), recurrent urinary tract infections by Escherichia coli (13), and acquisition of human immunodeficiency virus type 1 (HIV-1) (14). Bauer (15) has proposed that H2O2-producing lactobacilli also might exert control over vaginal cancer through specific interactions of reactive oxygen species, such as superoxide anion, hydroxyl radicals, and hypochlorous acid. The conversion of H2O2 into more toxic compounds during the oxidative process is potentiated by peroxidase and halures. This enzyme and some halures, such as chloride and bromide, are present in vaginal washes in sufficient amounts (16) to allow an optimal environment for successful inhibition of pathogens.

In vitro tests provide an approach for determining the ability of lactobacilli to produce H2O2. The H2O2 amounts produced in such systems are probably not a direct reflection of what happens in the vaginal tract of women or animals, which is not yet know. However, there is a registered patent (4) with an H2O2-generating L. crispatus strain, also supporting the use of H2O2-producing lactobacilli to restore the vaginal ecosystem

The detection of H2O2 can be performed with different methods, including a number of plate and spectrophotometric tests (17). As the results reported in the references are quite different, we optimized two methods to perform the screening and study of the kinetics of production of H2O2 by Lactobacillus strains isolated from human and bovine vagina. The qualitative method on plate (18) employs horseradish peroxidase incorporated in agar medium, which oxidizes the chromogenic substrate (tetramethyl-benzidine) to a purple-blue pigment in those colonies that produce H2O2. The quantitative spectrophotometric method uses horseradish peroxidase and involves oxidation of o-dianisidine in the presence of the H2O2 produced during the growth of lactobacilli in broth media (19). In previous papers, the isolation and identification of human and bovine vaginal lactobacilli were reported (20,21), and the microorganisms with potential probiotic use were selected by applying the methodology described in the present chapter (22-24). The techniques had the following objectives:

1. Screening for the production of antimicrobial substances among human and bovine vaginal Lactobacillus strains, by using a plate diffusion technique (8).

2. Detection of H2O2-producing lactobacilli by a qualitative plate method (18) and a modified quantitative spectrophotometric method using o-dianisidine, horseradish peroxidase (19)

3. Study of the kinetics of production of hydrogen peroxide by selected Lactobacillus strains.

2. Materials

2.1. Screening of Antimicrobial Substances Generated by Lactic Acid Bacteria

2.1.1. Microorganisms

1. Lactobacillus strains isolated from vaginal swabs from humans and cows. The methods applied for the isolation and identification of bovine and human vaginal lactobacilli were previously reported (20,21).

2. Human vaginal pathogens: Escherichia coli, Klebsiella sp., group B Streptococcus sp., Enterococcus faecalis, Staphylococcus aureus, and Candida sp. (from the Culture Collection of the Instituto de Microbiología "Luis C. Verna" of the Universidad Nacional de Tucumán, Argentina). Streptococcus agalactieae ATCC 1020 (from the American Type Culture Collection) was also employed.

3. Bovine pathogens (isolated from clinical material of metritis): Escherichia coli 99/14 and Corynebacterium pyogenes 96/393 (from the Culture Collection of Estación Experimental Agropecuaria Balcarce, INTA).

2.1.2. Storage of Microorganisms

1. LAPTg broth: 1.5% peptone, 1% tryptone, 1% glucose, 1% yeast extract, 0.1% Tween-80 (pH 6.5). Autoclave at 121°C for 15 min and store at refrigeration temperature. The chemicals for LAPTg preparation were obtained from Britania Laboratories (Argentina).

2. Brain-heart infusion (BHI) agar: 20% brain infusion, 20% heart infusion, 1% peptone, 0.2% glucose, 2.5% Na2HPO4, 1.5% agar (pH 7.4). Autoclave at 121°C for 15 min and store at refrigeration temperature. Dehydrated BHI medium was obtained from Britania Laboratories (Argentina).

3. 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) and store at refrigeration temperature.

4. BHI broth with glycerol: the same as step 2 of this subheading without agar, added with 25% glycerol.

2.1.3. Plate Diffusion Technique

1. 2 N NaOH: Sterile distilled water, NaOH (anhydrous, Anedra, Argentina). Store at room temperature.

2. Catalase (EC 1.11.1.6) used at 1000 U/mL, (from bovine liver, Sigma, St. Louis, MO). Store at <0°C. Caution: Avoid contact and inhalation.

3. 0.22 ^m Membrane filters, 25-mm dia. (Millipore, Bedford, MA). Autoclave at 121°C for 15 min.

4. Peptone water (0.1% meat peptone). Autoclave at 121°C for 15 min and store at refrigeration temperature.

5. LAPTg agar (see Subheading 2.1.2.) with 1% agar.

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

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