Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Nature of the synergic combinationa
Growth condition Final OD600 Growth rate (μ; h−1) % Inhibitionc Control 2.6 0.48 ± 0.02 0 Nisin (150 IU/ml) 2.2 0.37 ± 0.01 22 S-layer (10 μg/ml) 2.6 0.49 ± 0.02 0 Nisin (150 IU/ml) + S-layer (10 μg/ml) 0.7 0.05 ± 0.01 89 S-layer (heat inactivated)b 2.8 0.53 ± 0.02 ≪0 S-layer (heat inactivated) + nisin (150 IU/ml) 2.5 0.42 ± 0.02 12 CCCP (50 μM) 0.9 0.11 ± 0.01 77 CCCP (50 μM) + S-layer (10 μg/ml) 0.4 0.04 ± 0.01 92 CCCP (25 μM) 1.2 0.21 ± 0.01 56 CCCP (25 μM) + S-layer (10 μg/ml) 0.6 0.11 ± 0.01 77 ↵ a Staphylococcus aureus cultures were assayed as described for Fig. 1, and the different compounds were added at the indicated concentrations. Growth curves were performed, and the growth rate, μ, was determined from the resulting plots. Final OD600s after 8 h of growth were also indicated.
↵ b Heat-inactivated S-layer (10 μg/ml) was obtained by heating at 60°C for 2 h.
↵ c Percentage of inhibition was calculated from the growth rates and related to the control.
