Synergistic effects of the Lactobacillus acidophilus surface layer and nisin on bacterial growth

Abstract

We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.

FIG. 1.

Effect of S-layer, nisin, or both on the growth of S. enterica, S. aureus, and B. cereus. To determine the effect of nisin and S-layer protein separately or in combination, bacterial cells were first cultured at 37°C for 16 h with agitation. These cultures were diluted in fresh medium (0.5 ml into 10 ml) and distributed in aliquots, each containing either the S-layer, nisin, or both, and OD₆₀₀ was monitored every hour. Three or more independent experiments were performed for each bacterium. Nisin was added at the following concentrations: 2,500 IU/ml for Salmonella enterica, 250 IU/ml for Bacillus cereus, and 150 IU/ml for Staphylococcus aureus; the concentration of S-layer was 10 μg/ml.

FIG. 2.

Type of effect of the combination of nisin and S-layer. Exponentially growing bacteria were incubated in the presence of either nisin or nisin and S-layer. (A) Effect on Bacillus cereus with nisin (250 IU/ml) or nisin and S-layer (250 IU/ml and 10 μg/ml, respectively). (B) Effect on Staphylococcus aureus with nisin (150 IU/ml) or nisin and S-layer (150 IU/ml and 10 μg/ml, respectively). Dotted line, cfu/ml; unbroken line, OD at 600 nm.

FIG. 3.

S-layer integrity during incubation with Salmonella. Viable cells from Salmonella enterica were washed once with phosphate-buffered saline buffer, resuspended at an optical density at 600 nm of 1 mixed with S-layer protein (10 μg/ml), and incubated at 37°C with constant rotation to keep the contents in suspension at the indicated times. At different times S-layer was analyzed by Western blotting. Electrotransfer to parablot polyvinylidene difluoride was according to the manufacturer's instructions (Macherey-Nagel, Germany). Polyclonal antibody anti-S-layer was used at 1:1,000.

FIG. 4.

S-layer treatments. S-layer preparations were submitted to different treatments and analyzed by PAGE, zymography, and Western immunoblotting as previously described (16). Lane 1, S-layer; lane 2, pretreatment with nisin of L. acidophilus cultures before S-layer preparation; lane 3, heat-inactivated S-layer (2 h at 60°C); lane 4, S-layer preparation incubated with nisin.