Inhibition of Bacillus anthracis spore outgrowth by nisin

Abstract

The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC(50)) and an IC(90) of 0.57 microM and 0.90 microM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC(90). These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.

FIG. 1.

Nisin does not alter germination initiation. (A) The data are expressed as the percentage of the OD₆₀₀ at time zero and 10 min relative to that of each culture at time zero. Shown is the mean of a single experiment conducted in triplicate as a representative of three independent experiments. Error bars indicate standard deviations. In all cases, the differences between spore refractility at 10 min relative to that at 0 min were statistically significant (P < 0.05). (B) At 0 and 5 min, samples were analyzed for heat resistance, as described under Materials and Methods. The data are expressed as the means of three experiments. Error bars indicate standard deviations. In all cases, the differences between the percentage of heat-resistant spores at 5 min relative to that at 0 min were statistically significant (P < 0.05).

FIG. 2.

Germination is required for the inhibitory action of nisin. The data are expressed as the mean of a single experiment conducted in triplicate and are representative of those from two independent experiments. Error bars indicate standard deviations. In all cases, the differences between the OD₆₀₀ at 18 h relative to that at 0 h were statistically significant (P < 0.05).

FIG. 3.

The inhibitory action of nisin is irreversible. The data are expressed as the mean of a single experiment conducted in triplicate and are representative of those from two independent experiments. Error bars indicate standard deviations. In each case in which the spores were exposed to nisin, the increase in the OD₆₀₀ at 18 h relative to that at 0 h was not statistically significant (P > 0.05).

FIG. 4.

B. anthracis spores do not develop into vegetative bacilli in the presence of nisin. (A) The data are expressed as the percentage of OD₆₀₀ at each time point relative to the OD₆₀₀ of each culture at time zero, which was the control in these experiments. The data are expressed as the means of a single experiment conducted in triplicate and are representative of those from three independent experiments. Error bars indicate standard deviations. (B) At time zero and 5 and 10 h, samples were removed and visualized by DIC microscopy. For each panel, a single spore is shown for clarity but is representative of all other B. anthracis spores within that sample. Bars, 6.5 μm. The data are representative of those from three independent experiments.

FIG. 5.

Nisin prevents B. anthracis spores from becoming metabolically active. (A) At 0, 7, and 10 h, culture supernatants were evaluated for the presence of LF and PA by immunoblot analysis. The samples in each lane were normalized for the volumes of the culture supernatants. The data are from a single experiment and are representative of data collected in three independent experiments. (B) At the indicated times, aliquots were removed from the cultures and were evaluated for oxidative metabolism by measuring spectrophotometrically the production of formazan at 570 nm, as described under Materials and Methods. (C) At time zero (i.e., prior to the addition of nisin) and 30 min, aliquots were removed from the cultures and evaluated for the membrane potential by measuring the DiOC₂-associated B. anthracis fluorescence by flow cytometry. The data are plotted as the MFI. (B and C) Means of the data from a single experiment conducted in triplicate. The data are representative of those from three independent experiments. Error bars indicate standard deviations. For each sample in panel C incubated in BHI medium, the difference between the membrane potential at 30 min in the presence and the absence of nisin was statistically significant (P < 0.05).

FIG. 6.

Effects of nisin on B. anthracis membrane integrity. At the indicated times, aliquots were removed from the cultures and evaluated for PI uptake, as described under Materials and Methods. The data were plotted as the geometric MFI. The means of the data from a single experiment conducted in triplicate are presented. The data are representative of those from three independent experiments. Error bars indicate standard deviations. In all cases, the differences in PI uptake in samples containing nisin at 30 and 60 min relative to that at 0 min were statistically significant (P < 0.05).

FIG. 7.

Effects of nisin on spore remodeling during germination. (A) At the indicated times, cultures were evaluated for the release of DPA, as described under Materials and Methods. The means of the data from a single experiment conducted in triplicate are presented. The data are representative of those from three independent experiments. Error bars indicate standard deviations. (B) After 90 min, the indicated samples were removed, fixed, and imaged by TEM, as described under Materials and Methods. RLU, relative light units.