Characterization of the Bacteriocin

Among 134 vaginal lactobacilli, Lactobacillus salivarius subsp. salivarius CRL 1328 (CERELA Culture Collection) was selected for further study because it was able to inhibit the growth of Enterococcus, Listeria, Neisseria, and Gardnerella strains in the plaque assay for a substance different from acid or hydrogen peroxide.

3.3.1. Effect of Temperature

1. Heat resistance of the antimicrobial substance is studied by heating aliquots of the filter-sterilized supernatant to 60°C, 80°C, and 100°C for 10 min and also after autoclave treatment (121°C at 15 min).

2. The heated supernatant aliquots were cooled down and tested for activity as described before (see Subheading 3.2., step 3).

1. The pH of supernatant aliquots was adjusted to 2.0-8.0 with 2 NNaOH or 10% HCl prior to the filtration process.

2. Different pH supernatants were filter-sterilized and tested for activity as described before (see Subheading 3.2., step 3).

3.3.3. Activity of the Antimicrobial Substance After Treatment

With Proteolytic Enzymes, Sodium m-Periodate, and Organic Solvents

3.3.3.1. Preparation of Reagent Solutions

1. Proteinase K, trypsin, type II, and type XV protease solutions.

a. 10 mg of each enzyme was dissolved in 1 mL of phosphate buffer, pH 7, filter-sterilized (0.22-^m pore size membranes; Millipore) and stored at -20°C.

b. To treat the spent lactobacilli supernatants, the pH was adjusted to 7, and 10 ^L of the enzyme solution were mixed with 90 ^L of supernatants, to obtain a final enzyme concentration of 1 mg/mL.

2. Chymotrypsin solution.

a. 10 mg of enzyme were dissolved in 1 mL of Tris-HCl buffer, pH 8.0, with 0.1 M CaCl2. The CaCl2 must be dissolved in Tris-HCl buffer and then the enzyme can be added.

b. The enzyme solution was filter-sterilized (0.22-^m pore size membranes; Millipore) and stored at -20°C.

c. To treat the lactobacilli supernatants, the pH was adjusted to 8, and 10 ^L of the enzyme solution were mixed with 90 ^L of supernatants, to obtain a final enzyme concentration of 1 mg/mL (with a final CaCl2 concentration of 0.01 M).

3. Pepsin solution.

a. 10 mg of the enzyme were dissolved in 1 mL of citrate buffer, pH 3.0, filter-sterilized (0.22-^m pore size membranes; Millipore), and stored at -20°C.

b. To treat the lactobacilli supernatants, the pH 3.0 was adjusted with 10% HCl, and 10 ^L of the enzyme solution were mixed with 90 ^L of supernatants, to obtain a final enzyme concentration of 1 mg/mL.

4. a-Amylase solution.

a. The enzyme was resuspended in phosphate buffer, pH 6.9, to obtain a solution of a 1 M concentration.

b. The enzyme solution was filter-sterilized (0.22-^m pore size membranes; Millipore) and stored at -20°C.

c. To treat the lactobacilli supernatants, the pH was adjusted to 6.9 with 1 N NaOH, and 10 ^L of the enzyme solution were mixed with 90 ^L of supernatants, to obtain a final enzyme concentration of 0.1 M.

5. Sodium rn-periodate solution.

a. Sodium rn-periodate was dissolved in acetate buffer, pH 4.5, to obtain a 0.1 M solution.

b. The enzyme solution was filter-sterilized (0.22-^m pore size membranes; Millipore) and stores at -20°C.

c. To treat the spent lactobacilli supernatant, the pH was adjusted to 4.5 with 10% HCl, and 10 ^L of the enzyme solution were mixed with 90 ^L of supernatants, to obtain a final enzyme concentration of 10 mM.

6. Lipase solution.

a. The enzyme was dissolved in PBS pH 7.7 with 0.01% CaCl2 to obtain a final concentration of 0.1 g/mL.

b. The solution was filter-sterilized (0.22-^m pore size membranes; Millipore) and stored at -20°C.

c. To treat the lactobacilli supernatants, the pH was adjusted to 7.7 with 1 N NaOH, and 10 ^L of the enzyme solution were mixed with 90 ^L of the supernatant, to obtain a final enzyme concentration of 10 mg/mL (containing 0.001% CaCk2).

3.3.3.2. Treatment of Lactobacilli Supernatant With Enzymes and Sodium m-Periodate and Study of the Remaining Antimicrobial Activity

1. The enzymes and sodium rn-periodate were diluted 1:10 in the spent lactobacilli supernatant (see Note 2) with a pH adjusted according to the maximal enzyme activity (as described in Subheading 3.3.3.1.) (see Note 3).

2. The mixtures are incubated at room temperature for 1 h and the remaining activity is tested as described in Subheading 3.2.

3. The effect of the enzyme solution diluted 1:10 in LAPTg (at the same final concentration as in spent supernatants) is also tested on the indicator plaques as controls (see Note 4).

3.3.3.3. Treatment of Lactobacilli Supernatant With Organic Solvents and Study of the Remaining Antimicrobial Activity

1. The supernatant is also treated with 10 and 25% chloroform and 25% ethanol. For chloroform treatment the steps are as follows

2. For chloroform treatment the steps were as follows:

a. The organic solvent was added to the spent supernatant containing the bacteriocin (10 ^L of chloroform and 90 ^L of spent supernatants, as well as 25 ^L of the organic solvent with 75 ^L of the supernatant).

b. The mix was vortex agitated for 1 min, and the phases were separated.

c. The organic phase was evaporated by heating at 70°C in a water bath (see Note 5).

d. The remaining dry residue was resuspended in LAPTg broth.

e. Both the residue pellet (resuspended in the same original volume of LAPTg broth) and the aqueous supernatant under extraction are assayed for bacteriocin activity as described in Subheading 3.2.

3. The effect of ethanol was study in a similar way.

a. 10 ^L of 96° ethanol was added to 90 ^L of spent supernatant.

b. After a 1-min vortex agitation, the ethanol is eliminated from the mixture by evaporation at 70°C.

c. The remaining pellet was dissolved in the original LAPTg volume and tested for activity as described in Subheading 3.2.

3.4. Kinetics of Bacteriocin Production

1. LAPTg broth was inoculated at 2% with the bacteriocin-producing lactobacilli (grown in LAPTg for 12 h, which is the early stationary phase).

2. The culture was incubated at 37°C and samples are taken every 3 h to determine the titer of the bacteriocin and the number of CFU/mL.

3. For titration, supernatants are separated from 2 mL of culture by centrifugation at 2000g for 30 min, filter-sterilized, and serially diluted in LAPTg broth. Samples of 25 ^L of each dilution were poured into the holes of LAPTg agar plates containing the chosen indicator strain (in this example, E. faecalis; see Note 6), and inhibition is measured as described in Subheading 3.2., step 2. The titer was expressed in arbitrary units (AU), defined as the inverse of the dilution (in mL) that produced inhibition.

4. The number of CFU/mL was determined by serial dilution of samples taken from the culture at different times using peptone H2O for the dilutions and LAPTg agar plates to determine the number of colonies grown.

3.5. Mixed Cultures of L. salivarius and E. faecalis

1. The effect of bacteriocin-producing L. salivarius on E. faecalis growth can be studied in mixed cultures of both microorganisms performed in LAPTg broth at 37°C.

2. The initial inocula is 106 CFU/mL for lactobacilli and 105 or 107 CFU/mL for enterococci.

3. The number of CFU is determined by the serial dilution method using peptone H2O for the dilutions and LBS or SF agar plates to count viable microorganisms.

4. The plates are incubated for 48 h at 37°C.

3.6. Mode of Action of the Bacteriocin

1. Supernatant fluid of a 12-h L. salivarius culture is obtained by centrifugation at 2000g for 30 min and filter-sterilized as described in Subheading 3.1., step 1.

2. Aliquots of 5 mL are lyophilized, resuspended in the same volume of LAPTg broth, inoculated with E. faecalis (103, 105, and 107 CFU/mL), and incubated at 37°C (see Note 7).

3. Samples are taken at different times, and the CFU/mL of Enterococcus were determined-by the serial dilution method using H2O peptone for the dilutions and SF agar plates to count the viable microorganisms. Enterococcus growth in LAPTg is used for controls.

4. Notes

1. Incubation at room temperature is an important step, which allows diffusion of the supernatant into the media prior to the incubation step, which in turn allows growth of the indicator microorganisms to begin.

2. The Lactobacillus supernatant was always obtained as described in Subheading 3.2., step 1.

3. The spent supernatant pH can be adjusted over a wide range after testing the pH resistance of the bacteriocin.

4. It was observed, for all cases, that the enzyme solutions (tested in controls) do not produce any kind of inhibition. In any case, control of their effect on the growth of indicator microorganisms in recommended.

5. Caution: Do not try to evaporate the solvents directly in the flame.

6. The indicator strain should grow in the same broth as the lactobacilli and should be chosen from sensitive species that are easily grown.

7. The purpose of lyophilizing and resuspending the spent lactobacilli supernatant in LAPTg broth instead of using the spent supernatant directly, is to ensure that inhibition is not caused by a deficiency of any metabolite. It is possible to work using this strategy because the bacteriocin is resistant to lyophilization.

Acknowledgments

This paper was supported by grants from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), PID-BID 395, and the Ministerio de Salud Pública de la República Argentina (Beca Carrillo Oñativia para investigadores formados).

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