Published Ahead of Print 22 September 2008. 10.1128/AAC.00625-08.

2008, 52(12):4281. DOI:Antimicrob. Agents Chemother. van der Donk and Steven R. BlankeIan M. Gut, Angela M. Prouty, Jimmy D. Ballard, Wilfred A. Outgrowth by Nisin

SporeBacillus anthracisInhibition of

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2008, p. 4281–4288 Vol. 52, No. 120066-4804/08/$08.00�0 doi:10.1128/AAC.00625-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inhibition of Bacillus anthracis Spore Outgrowth by Nisin�

Ian M. Gut,1 Angela M. Prouty,1 Jimmy D. Ballard,4 Wilfred A. van der Donk,2,3*and Steven R. Blanke1,3*

Department of Microbiology, University of Illinois, Urbana, Illinois 618011; Department of Chemistry, University of Illinois, Urbana,Illinois 618012; Institute for Genomic Biology, University of Illinois, Urbana, Illinois 618013; and Department of Microbiology and

Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731044

Received 13 May 2008/Returned for modification 17 July 2008/Accepted 10 September 2008

The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillusspecies. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. Byusing B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC50)and an IC90 of 0.57 �M and 0.90 �M, respectively. Viable B. anthracis organisms were not recoverable fromcultures containing concentrations of nisin greater than the IC90. These studies demonstrated that spores loseheat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism ofinhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for theaction of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing theestablishment 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 sporestructures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncouplingof two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism andthe shedding of the external spore structures.

Lantibiotics are methyllanthionine-containing cationic anti-microbial peptides produced by several gram-positive bacteria(11). Nisin is a 34-amino-acid peptide produced by Lactococ-cus lactis subsp. lactis (ATCC 11454), which has emerged as animportant prototype for the study of the novel antibacterialproperties and structure-activity relationships characteristic ofthe lantibiotics (5, 33). Like all lantibiotics, nisin is ribosomallytranslated and is then posttranslationally modified to generatethree noncyclic nonproteogenic amino acids, dehydroalanine,and dehydrobutyrine and five lanthionine or methyllanthioninethioether rings (11).

The utility of nisin derives from its capacity to act upongram-positive bacteria by two entirely different mechanisms(15, 46). Nisin forms pores in lipid membranes (46), but it alsofunctions as a transglycosylase inhibitor that disrupts cell wallbiosynthesis via lipid II binding and mislocalization (21, 55).Because it functions as a “two-edged sword,” microbes havebeen relatively refractory to the emergence of resistance tonisin, despite its widespread and persistent use as a preserva-tive in the food industry (15, 46).

An additional and poorly understood activity of nisin is itscapacity to prevent the outgrowth of spores from several gram-positive bacteria, including several Bacillus species (9, 10, 40,42). To date, nisin inhibition of Bacillus spore outgrowth hasbeen documented by various methods, including the spectro-photometric measurement of liquid culture turbidity (3), theenumeration of CFU (4, 14, 32, 35, 43), well diffusion assays on

solid agar (14, 39), and microscopic observations (41). Al-though these approaches are useful, they have provided fewdetails about nisin’s mode of action against Bacillus spores.Currently, it has not been experimentally established whethernisin inhibits spore outgrowth by preventing germination ini-tiation or, alternatively, preventing a step downstream of ger-mination initiation. Additionally, the requirement for germi-nation for the action of nisin has not been addressed. Finally,it is not clear whether or not the action of nisin requires activelygrowing organisms, analogous to many other antibiotics.

To address these issues, the effects of nisin on Bacillus sporesand their development into replicating bacilli were evaluatedby using spores from Bacillus anthracis Sterne 7702 as a model.The results from these studies indicate that nisin does notinhibit germination initiation; instead, germination is requiredfor irreversible inhibition. Nisin acted rapidly upon germinat-ing spores to prevent the establishment of oxidative metabo-lism or the membrane potential, possibly by a mechanism in-volving the disruption of membrane integrity. Nisin did notinhibit the removal of the outer spore structures (e.g., theexosporium, cortex, and spore coat). Collectively, these datasuggest that nisin acts upon spores immediately after the ini-tiation of germination and effectively blocks the capacity of B.anthracis to proliferate and produce virulence factors.

MATERIALS AND METHODS

Spore preparations. Spores were prepared from B. anthracis Sterne 7702, asdescribed previously (49). The enumeration of spores or bacilli was performedwith a Petroff-Hauser hemocytometer under a light microscope at �400 magni-fication (Nikon Alphaphot YS, Mellville, NY). A typical spore preparationyielded 10 ml of spores at a concentration of 2.0 � 109 spores/ml.

Nisin purification. A sample of 500 mg nisaplin (50% denatured milk proteins,2.5% nisin, 47.5% sodium chloride) was suspended in 30% acetonitrile (Sigma,St. Louis, MO) with 0.1% trifluoroacetic acid (10 ml; Sigma). The suspension wassonicated for 20 min, followed by centrifugation at 1,500 � g for 10 min to

* Corresponding author. Mailing address for Wilfred A. van derDonk: Department of Chemistry, University of Illinois, Urbana, IL61801. Phone: (217) 244-5360. Fax: (217) 244-8533. E-mail: vddo.nk@illinois.edu. Mailing address for Steven R. Blanke: Department ofMicrobiology, University of Illinois, Urbana, IL 61801. Phone (217)244-2412. Fax: (217) 244-6697. E-mail: sblanke@life.uiuc.edu.

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remove all insoluble material. Reverse-phase high performance chromatography(Waters, Milford, MA) was performed with a PrePack C4 semipreparative col-umn (diameter, 25 mm; length, 100 mm; Waters) with a gradient of 0 to 100%acetonitrile. Under these conditions, nisin had a retention time of 28 min.Acetonitrile and trifluoroacetic acid were removed from fractions containingnisin by rotary evaporation, followed by lyophilization to remove the water. Priorto use, lyophilized nisin was weighed on an analytical balance and was dissolvedin 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS; pH 6.8) to yield thedesired concentration. The identity of purified nisin was confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (GeneralElectric, NY). As an additional quality control measure, purified nisin wasevaluated for its inhibitory activity against Lactococcus lactis 117 (ATTC 15577)cells grown in GM17 broth (3.7% M17 medium, 0.5% dextrose; BD Biosciences)at 30°C. Purified nisin inhibited L. lactis 117 with a 50% inhibitory concentration(IC50) of 0.0021 �M, in excellent agreement with the findings of previous studies(6, 27, 28), indicating that the purification protocol yielded nisin with the ex-pected biological activity.

Culture of B. anthracis spores. B. anthracis Sterne 7702 spores at a concen-tration of 4.0 � 106 spores/ml, unless indicated otherwise, were incubated inbrain heart infusion (BHI) medium supplemented with nisin (0.1, 1, 10, and 100�M), ciprofloxacin (0.01, 0.1, 1, and 10 �M), or 0.1 M MOPS (pH 6.8; Sigma) asa mock control. In these studies, the changes in germinating spores caused bynisin were compared to those induced by ciprofloxacin, an antibiotic recom-mended for use for the treatment of anthrax. The published MIC of ciprofloxacinagainst B. anthracis is 0.193 �M (34), which was consistent with the resultsobtained in preliminary experiments (data not shown), thus providing a basis forthe range of ciprofloxacin concentrations used in these studies. Each of theciprofloxacin studies was repeated twice with independent preparations ofspores. For nongerminating conditions, 0.1 M MOPS (pH 6.8) was substitutedfor BHI medium. All incubations were performed at 37°C under aeration (180rpm on a rotary shaker [Thermo Fisher Scientific Inc., Waltham, MA] or asindicated otherwise) and ambient CO2 (e.g., 0.03% CO2). In pilot experiments,spores were incubated in alternative media, which were Luria-Bertani (LB; 10g/liter Bacto tryptone, 5 g/liter NaCl, 5 g/liter Bacto yeast extract; BD Diagnos-tics), RPMI 1640 medium (ATCC) containing fetal bovine serum (FBS; 10%;JRH Biosciences, Lenexa, KA), minimal essential medium (MEM; JRH Bio-sciences) containing FBS (10%), or Dulbecco’s MEM (DMEM; JRH Bio-sciences) containing FBS (10%).

Determination of IC50s and IC90s of nisin against endospores. B. anthracisendospores at a final concentration of 4.4 � 104, 4.4 � 105, 4.4 � 106, or 4.4 �107 spores/ml were incubated in BHI medium supplemented with various con-centrations of nisin (0.05 �M to 100 �M) or 0.1 M MOPS pH 6.8 (as a negativecontrol). The IC50s and IC90s were derived from plots of the optical density at600 nm (OD600) at 16 h versus the nisin concentration and are the concentrationsof nisin that inhibited B. anthracis growth in BHI medium by 50% and 90%,respectively.

CFU quantification. Spores were serially diluted and plated on agar platescontaining LB medium (10 g/liter Bacto tryptone, 5 g/liter NaCl, 5 g/liter Bactoyeast extract, 15 g/liter Bacto agar; BD Biosciences). After 12 to 18 h at 37°C, theB. anthracis colonies were counted, and the numbers of CFU/ml were calculatedfrom those counts.

Spore hydration. The hydration of spores was determined by measuring theloss of spore refractility at 600 nm by using a Synergy 2 plate reader (BioTekInstruments, Inc., Winooski, VT). B. anthracis spores were incubated, as de-scribed under “Culture of B. anthracis spores,” except that a 96-well plate wasused and the plate was shaken for 15 s prior to each read. The data are presentedas a percentage of the OD600 at each time point relative to the OD600 of thespore suspensions at the beginning of the experiment (time zero).

Heat resistance. Spores were diluted into 0.1 M MOPS (pH 6.8) containingD-alanine and D-histidine (both at 10 mM; Sigma), to prevent the further ger-mination initiation of dormant spores, and identical aliquots were incubated ateither 65°C or on ice for 30 min. Viable B. anthracis organisms were quantifiedby plating serial dilutions and enumerating the CFU. The percentage of heat-resistant spores was calculated by dividing the numbers of CFU recovered fromthe samples heated at 65°C by the numbers of CFU recovered from the samplesincubated on ice.

DIC microscopy. At the indicated times, samples were removed from the B.anthracis cultures and fixed by incubation in 3% formaldehyde (Sigma) for 30min at 37°C, followed by the mounting of samples on glass slides in 20% glycerol(Sigma). Differential interference contrast (DIC) microscopy images were col-lected with an Applied Precision assembled DeltaVision epifluorescence micro-scope containing an Olympus Plan Apo �100 oil objective with a numerical

aperture of 1.42 and a working distance of 0.15 mm, and the images wereprocessed with the SoftWoRX (Issaquah, WA) Explorer Suite program.

Immunoblot analysis. At the indicated times, samples removed from B. an-thracis cultures grown in the presence of 0.2% (wt/vol) bicarbonate at 37°C under5% CO2 were centrifuged for 10 min at 21,000 � g. The culture supernatantswere denatured by the addition of an equal volume of 2� sodium dodecyl sulfate(SDS) sample buffer (4% SDS, 100 mM Tris, 0.4 mg bromophenol blue/ml, 0.2M dithiothreitol, 20% glycerol). The samples were boiled for 5 min and wereresolved by SDS-polyacrylamide gel electrophoresis (10% acrylamide). The con-tents of the gels were electrotransferred to nitrocellulose membranes (Pierce,Rockford, IL). The membranes were probed for the presence of protectiveantigen (PA) and lethal factor (LF) by utilizing anti-PA (QED Bioscience Inc.,San Diego, CA) and anti-LF (QED Bioscience Inc.) mouse monoclonal anti-bodies, respectively. Goat horseradish peroxidase-conjugated anti-mouse immu-noglobulin G (Abcam Inc., Cambridge, MA) was used as the secondary antibody,and cross-reacting material was visualized after the blots were exposed to X-rayfilm (Denville Scientific Inc., Metuchen, NJ) in the presence of the enhancedchemiluminescence immunoblotting reagent (Pierce, Rockford, IL). For theexperiments for the investigation of an association between the presence of PAor LF with spores, spore homogenates were prepared by vortexing spore sus-pensions 10 times with 0.1-mm-diameter glass beads for 30 s.

Oxidative metabolism. Samples from each culture were diluted into 0.1 MMOPS (pH 6.8) containing D-alanine and D-histidine (both at 10 mM; Sigma), toprevent the further germination initiation of dormant spores. Each sample wasthen incubated with 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (tetrazolium; 5 mg/ml) for 30 min at 37°C. The conversion of tetrazoliumto formazan was measured at 570 nm with a Synergy 2 plate reader (12).

Membrane potential. The B. anthracis spores were incubated as describedunder “Culture of B. anthracis spores,” except for the presence of a fluorescentmembrane potential-sensitive dye, 3-3�diethyloxacarbocyanine iodide (DiOC2;300 nM; Invitrogen, Carlsbad, CA) (29). At the indicated times, the membranepotential was assessed by measuring the increase in B. anthracis-associatedDiOC2 fluorescence by flow cytometry (EPICS XL-MCL flow cytometer; Beck-man Coulter, Fullerton, CA), with excitation at 488 nm with an argon laser andmeasurement of the fluorescence emission through a band-pass filter at 525/20nm. At least 10,000 events were detected for each sample, and the data wereanalyzed by using the FCS Express 3.00.0311 V Lite Standalone software. Thedata were plotted as the geometric mean of the fluorescence intensity (MFI).

Membrane integrity. Membrane integrity was evaluated by measuring theuptake of propidium iodide (PI) (19, 50). Samples from each culture wereincubated with PI (60 �M; Molecular Probes Inc., Leiden, The Netherlands) inan ice bath for 10 min (1). B. anthracis-associated fluorescence was measured byflow cytometry as described above under “Membrane potential,” except that thefluorescence emission was measured with a band-pass filter at 675/20 nm.

Quantification of DPA. The release of dipicolinic acid (DPA; 2,6-pyridinedi-carboxylic acid) was monitored by measuring the fluorescence resonance energytransfer between DPA and terbium (25, 45). B. anthracis spores were incubatedin a manner similar to that described above under “Culture of B. anthracisspores,” except for the presence of TbCl3 (200 �M; Sigma). The DPA-terbiumcomplex was excited at 280 nm, and emission was monitored at 546 nm with aSynergy 2 plate reader.

TEM. The B. anthracis organisms from each culture were concentrated bycentrifugation (21,000 � g for 30 min), the pellets were resuspended in Kar-novsky’s fixative (26), and samples were prepared for transmission electronmicroscopy (TEM) analysis, as described previously (56). Images were collectedwith a CMI Hitachi H600 transmission electron microscope (Tokyo, Japan) inthe University of Illinois College of Veterinary Medicine Microscopy Facility.

Statistics. Error bars represent standard deviations. P values were calculatedby Student’s t test by using a paired, one-tailed distribution. A P value of �0.05indicates statistical significance.

RESULTS

Growth inhibition by nisin. Previous studies reported thatnisin prevented the growth of Bacillus spores derived fromseveral different species (4, 14, 32, 35, 43). To evaluate theaction of nisin against spores responsible for this inhibitoryactivity, spores were prepared from B. anthracis Sterne 7702,which is a strain commonly employed as a model for investi-gation of the early stages of anthrax disease (24, 47). To vali-date earlier results, B. anthracis spores were incubated in BHI

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medium, which has been used to induce the germination of B.anthracis spores (17), supplemented with either nisin (0.05 �Mto 100 �M) or buffer control (0.1 M MOPS, pH 6.8). Pilotexperiments confirmed that 0.1 M MOPS (pH 6.8) alone doesnot induce spore germination (data not shown). In BHI me-dium inoculated with 4.4 � 106 spores/ml, nisin inhibited B.anthracis growth, with IC50s and IC90s of 0.57 �M and 0.90�M, respectively (Table 1). The inhibitory activity of nisinagainst spores was not strictly dependent on BHI medium, asnisin inhibited B. anthracis growth to the same degree in LBmedium or in MEM, DMEM, or RPMI 1640, each supple-mented with 10% FBS (data not shown). Relative to the num-bers of CFU in cultures at the initial time point, approximately10,000-fold more CFU was recovered at 10 h from culturessupplemented with either 0.1 �M nisin or 0.1 M MOPS (pH6.8). In contrast, no detectable CFU was recovered from 10-hcultures that had been supplemented with 1, 10, or 100 �Mnisin (data not shown).

On the basis of the results of these experiments, subsequentstudies were conducted with 4.0 � 106 spores/ml because thisconcentration of spores was sufficient to generate detectablereadouts for each assay and yielded IC50s and IC90s similar tothose calculated with a higher spore concentration (4.4 � 107

spores/ml; Table 1) and at the same time allowed several fullsets of experiments to be conducted from each spore prepara-tion.

Nisin does not inhibit germination initiation. The inabilityto recover CFU from cultures of B. anthracis spores supple-mented with nisin could be due to the irreversible inhibition ofgermination initiation. To evaluate this possibility, germinationinitiation was first monitored by measuring the characteristicloss of spore refractility that accompanies hydration of thespore structure, as indicated by a decrease in the OD600 (30,53). These experiments revealed a loss in refractility of �65%by 10 min in both the presence and the absence of nisin (Fig.1A), indicating that nisin did not detectably alter hydration ofthe spores following germination initiation. Similarly, the lossof spore refractility was not inhibited in the presence of eventhe highest tested concentration of ciprofloxacin (10 �M), aDNA gyrase inhibitor that acts upon bacteria by a mechanismfundamentally different from that of nisin. Incubation ofspores with nisin or ciprofloxacin alone (in the absence ofknown germinants) did not result in a loss of spore refractility(data not shown). These results are consistent with the notion

that neither nisin nor ciprofloxacin inhibits B. anthracis growthby blocking germination initiation.

A second hallmark of germination initiation is the rapid lossof spore resistance to heat (30, 53). In both the presence andthe absence of nisin, spores demonstrated a �80% loss in heatresistance by 5 min (Fig. 1B), providing additional evidencethat germination initiation was not altered in the presence ofnisin. When spores were incubated in 0.1 M MOPS (pH 6.8)supplemented with nisin, no loss of heat resistance was ob-served (data not shown), again confirming that nisin does notinduce germination. Taken together, these results indicate thatthe loss of recoverable CFU from cultures of spores supple-mented with nisin was not due to the inhibition of germinationinitiation, thereby ruling out the possibility that such a mech-anism underlies the inhibitory action of nisin against spores.Instead, these data would seem to indicate that the loss ofrecoverable CFU from cultures of spores supplemented withnisin may be due to nisin-mediated killing of the germinatedspores.

Germination initiation is required for the inhibitory actionof nisin. Whether germination initiation is necessary for nisinto act against spores was investigated next. Spores were incu-bated in BHI medium, in BHI medium supplemented with 10�M nisin, and in 0.1 M MOPS supplemented with 10 �M nisin.

FIG. 1. Nisin does not alter germination initiation. (A) The dataare expressed as the percentage of the OD600 at time zero and 10 minrelative to that of each culture at time zero. Shown is the mean of asingle experiment conducted in triplicate as a representative of threeindependent experiments. Error bars indicate standard deviations. Inall cases, the differences between spore refractility at 10 min relative tothat at 0 min were statistically significant (P � 0.05). (B) At 0 and 5min, samples were analyzed for heat resistance, as described underMaterials and Methods. The data are expressed as the means of threeexperiments. Error bars indicate standard deviations. In all cases, thedifferences between the percentage of heat-resistant spores at 5 minrelative to that at 0 min were statistically significant (P � 0.05).

TABLE 1. IC50 and IC90 of nisin against B. anthracis sporesa

No. of spores/mlb IC50 (�M)c IC90 (�M)d

4.4 � 104 0.17 � 0.01 0.41 � 0.024.4 � 105 0.19 � 0.01 0.44 � 0.014.4 � 106 0.57 � 0.03 0.90 � 0.014.4 � 107 0.63 � 0.06 0.98 � 0.01

a Three independent experiments were performed in triplicate with differentspore preparations and nisin purifications. The values are reported as the aver-ages of three experiments.

b Spores were freshly prepared from B. anthracis Sterne 7702.c Defined as the nisin concentration that inhibits the growth of cultures of

B. anthracis spores by 50% at 16 h.d Defined as the nisin concentration that inhibits the growth of cultures of

B. anthracis spores by 90% at 16 h.

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After 1 h, the spores were washed to lower the concentrationof nisin in solution to approximately 1 nM, which is well belowthe IC50. After the spores were washed, they were introducedinto fresh BHI medium. As expected, spores that had beenpreincubated with nisin under germinating conditions did notgrow when they were introduced into fresh BHI medium (Fig.2). In contrast, spores preincubated with nisin in the absence ofgerminant demonstrated robust growth in fresh BHI medium.These data indicate that germination initiation is requisite forthe inhibitory activity of nisin against spores.

To establish the point during the germination process atwhich nisin-mediated inhibition becomes irreversible, sporeswere preincubated in BHI medium (to induce germination)supplemented with either nisin (10 �M) or 0.1 M MOPS. Atvarious times, samples from each culture were washed exten-sively to lower the concentration of nisin in the spore suspen-sions to levels well below the IC90 and were introduced intofresh BHI medium. These experiments revealed that the ex-posure of B. anthracis spores to nisin for as few as 5 min undergerminating conditions completely blocked the growth of thegerminated spores in fresh medium lacking nisin (Fig. 3).

These results suggest that the inhibitory action of nisin againstspores becomes irreversible soon (�5 min) after germination isinitiated.

Nisin prevents spore development into vegetative bacilli. Toobtain additional insights into the stage of germination atwhich nisin arrests the development of spores into replicating,vegetative bacilli, the extended growth of cultures was moni-tored in the presence or the absence of nisin. As expected, theOD600 of each of the samples decreased initially, reflecting therapid hydration of spores that characteristically follows germi-nation initiation (Fig. 4A). After approximately 50 min, cul-tures supplemented with 0.1 �M nisin demonstrated clear bac-terial growth, albeit at a lower rate than cultures lacking nisin(Fig. 4A). In contrast, there was no evidence of growth incultures supplemented with higher concentrations of nisin ei-ther at 180 min (Fig. 4A) or at extended time points (12 h; datanot shown). Examination of samples removed from these cul-tures at 5 and 10 h by DIC microscopy revealed characteristicchains of vegetative bacilli from cultures supplemented witheither 0.1 �M nisin or the buffer control (Fig. 4B), whereas nobacilli were present within cultures supplemented with 1, 10, or100 �M nisin (Fig. 4B). These results indicate that at concen-trations greater than the IC90, nisin prevents the developmentof germinated spores into vegetative bacilli. By comparison,

FIG. 2. Germination is required for the inhibitory action of nisin.The data are expressed as the mean of a single experiment conductedin triplicate and are representative of those from two independentexperiments. Error bars indicate standard deviations. In all cases, thedifferences between the OD600 at 18 h relative to that at 0 h werestatistically significant (P � 0.05).

FIG. 3. The inhibitory action of nisin is irreversible. The data areexpressed as the mean of a single experiment conducted in triplicateand are representative of those from two independent experiments.Error bars indicate standard deviations. In each case in which thespores were exposed to nisin, the increase in the OD600 at 18 h relativeto that at 0 h was not statistically significant (P � 0.05).

FIG. 4. B. anthracis spores do not develop into vegetative bacilli inthe presence of nisin. (A) The data are expressed as the percentage ofOD600 at each time point relative to the OD600 of each culture at timezero, which was the control in these experiments. The data are ex-pressed as the means of a single experiment conducted in triplicate andare representative of those from three independent experiments. Errorbars indicate standard deviations. (B) At time zero and 5 and 10 h,samples were removed and visualized by DIC microscopy. For eachpanel, a single spore is shown for clarity but is representative of allother B. anthracis spores within that sample. Bars, 6.5 �m. The dataare representative of those from three independent experiments.

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germinated spores developed into vegetative bacilli by 5 h inthe presence of 1 �M ciprofloxacin (data not shown), which isa concentration sufficient to completely inhibit the prolifera-tion of vegetative bacilli.

Spores incubated with nisin do not produce lethal toxin.Although nisin prevents the development of spores into vege-tative bacilli, the extent to which the action of nisin impairsadditional events associated with spore germination was notclear. For B. anthracis, an important consideration is whetheror not virulence factors, such as lethal toxin (LT) (51), arereleased prior to nisin-mediated killing of germinated spores.Spores were incubated under conditions that are known toinduce LT production (23). In the absence of nisin, the twocomponents of the bipartite LT, LF and PA, were both readilydetected by immunoblot analysis (Fig. 5A). In contrast, neitherLF nor PA was detected within culture supernatants preparedfrom B. anthracis cultures supplemented with 10 �M nisin. Bycomparing the intensity of the cross-reacting material in two-fold serially diluted cultured supernatants, the amounts of PAand LF were determined to be reduced at least 32- and 16-fold,respectively, in cultures supplemented with nisin compared tothe amounts in cultures supplemented with the buffer control(0.1 M MOPS, pH 6.8; data not shown). Additional experi-ments showed that neither LF nor PA was detected withinhomogenates of B. anthracis spores incubated for 1 h in BHImedium supplemented with nisin (data not shown), ruling outthe possibility that LF or PA was present but spore associated.Finally, pilot experiments indicated that nisin did not causeeither LF or PA to precipitate out of solution (data notshown), ruling out another possible explanation for the ab-sence of these proteins in B. anthracis cultures supplementedwith nisin.

The action of nisin prevents spores from becoming meta-bolically active. Dormant B. anthracis spores are metabolicallyinactive (48). One potential explanation for the lack of detect-able LF or PA in B. anthracis cultures supplemented with nisinis the inability of germinating spores to establish an activemetabolism. To evaluate this hypothesis, the cellular produc-tion of NAD(P)H was monitored as a measure of oxidativemetabolism by determining the reduction of tetrazolium toformazan in an NAD(P)H-dependent manner (12). In the ab-sence of nisin, the robust production of formazan was detected,beginning at 5 to 10 min after the initiation of germination, andthe levels of formazan generated continued to increase duringthe course of the experiment (3 h) (Fig. 5B). In the presence of1, 10, or 100 �M nisin, small but detectable levels of formazanproduction were detected within 5 to 10 min after the initiationof germination, but formazan production did not continue toincrease after this time. Formazan production was detected inthe presence of 0.1 �M nisin, albeit at a lower rate than in theabsence of nisin. Formazan production was inhibited in thepresence of 1 and 10 �M ciprofloxacin (data not shown) butoccurred in the presence of 0.01 and 0.1 �M ciprofloxacin,albeit to a lesser extent (approximately 65% and 40%, respec-tively, compared to the amount of formazan produced in theabsence of antibiotic).

Because oxidative metabolism is linked to the establishmentof an electrochemical gradient across the cytoplasmic mem-brane, the effects of nisin on the establishment of a membranepotential within germinating spores were evaluated. In the

presence of germinant, B. anthracis demonstrated significantlystronger staining with the membrane potential-sensitive dyeDiOC2 (29) than in the absence of germinant, indicating theestablishment of a membrane potential by 30 min subsequentto germination initiation (Fig. 5C). In contrast, spores incu-bated in the presence of nisin demonstrated significantly lessDiOC2 staining at 30 min (Fig. 5C), which indicated that at thisearly time point, nisin interfered with the establishment of amembrane potential in germinating spores. By 5 and 10 h after

FIG. 5. Nisin prevents B. anthracis spores from becoming metabol-ically active. (A) At 0, 7, and 10 h, culture supernatants were evaluatedfor the presence of LF and PA by immunoblot analysis. The samples ineach lane were normalized for the volumes of the culture supernatants.The data are from a single experiment and are representative of datacollected in three independent experiments. (B) At the indicatedtimes, aliquots were removed from the cultures and were evaluated foroxidative metabolism by measuring spectrophotometrically the pro-duction of formazan at 570 nm, as described under Materials andMethods. (C) At time zero (i.e., prior to the addition of nisin) and 30min, aliquots were removed from the cultures and evaluated for themembrane potential by measuring the DiOC2-associated B. anthracisfluorescence by flow cytometry. The data are plotted as the MFI. (Band C) Means of the data from a single experiment conducted intriplicate. The data are representative of those from three independentexperiments. Error bars indicate standard deviations. For each samplein panel C incubated in BHI medium, the difference between themembrane potential at 30 min in the presence and the absence of nisinwas statistically significant (P � 0.05).

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germination initiation, spores incubated in the presence of 0.1�M nisin demonstrated DiOC2 staining similar to that ofspores in the absence of nisin (94.9% MFI of spores in theabsence of nisin; data not shown), indicating that these sporesrecovered and ultimately developed a membrane potential,albeit at a lower rate. Spores incubated in the presence ofhigher concentrations of nisin (1, 10, and 100 �M) did notdemonstrate increased DiOC2 staining at later time points (5or 10 h; data not shown). Notably, in the presence of cipro-floxacin (0.01, 0.1, 1, and 10 �M), germinating spores displayedDiOC2 staining comparable to that in the absence of antibiotic(approximately 80 to 100%; data not shown), indicating that, incontrast to nisin, ciprofloxacin did not prevent the establish-ment of a membrane potential. Taken together, these studiessuggest that nisin acts upon spores immediately after the ini-tiation of germination and that at concentrations nonpermis-sive for spore outgrowth (as demonstrated in Fig. 4) nisinprevents B. anthracis from becoming metabolically active.

Effects of nisin action on membrane integrity. The absenceof oxidative metabolism in germinating spores in the presenceof nisin could be due to a loss of membrane integrity (19, 50).To explore this possibility, germinating spores were evaluatedfor increases in membrane permeability by measuring the up-take of PI by flow cytometry. These experiments revealed thatby 30 min, nisin induced 2-, 6-, 13-, and 56-fold increases in theamount of PI taken up by spores incubated with 0.1, 1, 10, and100 �M nisin, respectively, relative to the amount taken up byspores incubated in the absence of nisin (Fig. 6). The results ofthse experiments suggest that within germinating B. anthracisspores, nisin induces a dose-dependent disruption of mem-brane integrity. In contrast, germinating spores exhibited onlya modest increase in PI uptake (less than twofold; data notshown) in the presence of ciprofloxacin (0.01, 0.1, 1, or 10 �M),further supporting the idea that nisin and ciprofloxacin inhibitthe outgrowth of B. anthracis spores by fundamentally differentmechanisms.

Nisin does not prevent spore remodeling during germina-tion. The inhibition of spore outgrowth could be due to dis-ruption of the extensive remodeling of the spore structure that

accompanies germination. To evaluate this possibility, thecharacteristic release of DPA from the spore structure, whichoccurs shortly after germination initiation (25, 54), was moni-tored. Both the magnitude and the rate of DPA release weresimilar in the presence or the absence of nisin (Fig. 7A).

To evaluate if nisin inhibits downstream remodeling of thespore structure, which includes hydrolysis of the cortex andrelease from the spore coat and exosporium. In the absence ofnisin, TEM analysis revealed that the core, cortex, spore coat,and exosporium were readily evident in dormant spores (e.g.,spores incubated in 0.1 M MOPS, pH 6.8) but that only thecore remained in germinated spores (Fig. 7B). Nisin did notinhibit the loss of the cortex, spore coat, or exosporium inspores that had been incubated in BHI medium (Fig. 7B).Taken together, these results indicate that the nisin-mediatedaction against spores likely does not involve the inhibition ofthe extensive remodeling of the spore structure that accompa-nies germination.

DISCUSSION

The inhibitory action of nisin against spores of Bacillus andClostridium pathogens has been recognized for well over 50 years(7, 36). However, the mode of action responsible for preventingspore outgrowth had not previously been characterized in detail.By using B. anthracis Sterne 7702 as a model, the data presentedhere demonstrate that spores lose their heat resistance and be-come hydrated in the presence of nisin, thereby ruling out apossible mechanism of inhibition in which nisin blocks germina-tion initiation. Rather, germination initiation is requisite for theaction of nisin. These observations are consistent with the findings

FIG. 6. Effects of nisin on B. anthracis membrane integrity. At theindicated times, aliquots were removed from the cultures and evalu-ated for PI uptake, as described under Materials and Methods. Thedata were plotted as the geometric MFI. The means of the data froma single experiment conducted in triplicate are presented. The data arerepresentative of those from three independent experiments. Errorbars indicate standard deviations. In all cases, the differences in PIuptake in samples containing nisin at 30 and 60 min relative to that at0 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 ofDPA, as described under Materials and Methods. The means of thedata from a single experiment conducted in triplicate are presented.The data are representative of those from three independent experi-ments. Error bars indicate standard deviations. (B) After 90 min, theindicated samples were removed, fixed, and imaged by TEM, as de-scribed under Materials and Methods. RLU, relative light units.

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of a previous report on subtilin, a close analog of nisin that alsoinhibits spore outgrowth without disrupting spore hydration (31).The current study revealed for the first time that nisin rapidly andirreversibly inhibits growth by preventing the establishment ofoxidative metabolism and the membrane potential in germinatingspores, possibly revealing an underlying explanation for the ab-sence of B. anthracis proliferation. On the other hand, nisin hadno detectable effects on the typical changes associated with thedissolution of the outer spore structures (e.g., the spore coats,cortex, and exosporium). Thus, the action of nisin reveals insightsinto germination by uncoupling two critical sequences of eventsnecessary for the outgrowth of spores: the establishment of me-tabolism and the shedding of the external spore structures.

The capacity of nisin to prevent germinating B. anthracisspores from establishing a full membrane potential or oxida-tive metabolism was likely linked to the disruption of mem-brane integrity. Although nisin at 1 �M induced only a 6-foldincrease in the amount of PI uptake above background,whereas at 100 �M nisin induced a 56-fold increase (Fig. 6),spore outgrowth and metabolic activity were still inhibited andspores were unable to establish a full membrane potentialthrough 10 h. In contrast, ciprofloxacin, a DNA gyrase inhib-itor which is recommended for use for the treatment of B.anthracis infections (8), did not prevent the establishment of amembrane potential in germinating spores and had an almostnegligible effect on membrane integrity, consistent with thenotion that these two antibiotics (i.e., ciprofloxacin and nisin)inhibit the cellular proliferation of B. anthracis in fundamen-tally different ways.

Two distinct mechanisms, membrane pore formation andthe prevention of cell wall biosynthesis, contribute to the bac-tericidal activity of nisin against vegetative gram-positive bac-teria (6, 21, 46). Our studies do not directly reveal which, ifeither, of these two mechanisms is primarily responsible forpreventing the outgrowth of B. anthracis spores. However,nisin’s capacity to disrupt the integrity of the membrane ofgerminating spores suggests that the membrane pore-formingactivity may be important for the inhibition of spore out-growth. In black lipid systems, nisin-induced pores allow theefflux of ATP (46), and it is conceivable that in germinatingspores, the efflux of ATP through nisin-induced pores coulddeprive B. anthracis of the energy required for macromolecularsynthesis and oxidative metabolism. Moreover, the formationof nisin-induced pores (20) can counteract the proton effluxrequired for membrane potential establishment and ATP for-mation (38, 48). Because the nisin-mediated inhibition of out-growth requires germination initiation, its target of actionlikely becomes accessible only subsequent to germination ini-tiation. It cannot currently be ruled out that nisin inhibition ofcell wall biogenesis, especially at lower nisin concentrations, atwhich the disruption of membrane integrity is more modest,may also contribute to the prevention of spore outgrowth.Moreover, considering the structural differences betweenspores and vegetative bacilli, one also cannot dismiss the pos-sibility that nisin may act upon germinating spores by a mech-anism fundamentally different from that which it uses againstbacilli. One study with Bacillus cereus implicated accessiblethiol groups within B. cereus spores as potential targets fornisin, with the result being outgrowth inhibition (41), althougha specific molecular target was not identified in that report.

Prior structure-activity studies suggested that the dehydroala-nine in position 5 of nisin is important for the inhibition ofBacillus spore outgrowth (9, 41), but this dehydrated residue isnot essential for bioactivity in vegetative cells. In contrast, amore recent study reported that this dehydroalanine was notessential for nisin’s inhibitory activity against Bacillus subtilisspores (44). In L. lactis, truncated nisin A mutants lacking ringsD and E were unable to permeate the membranes or cause adisruption of the membrane potential, but these mutants re-tained the capacity to inhibit the outgrowth of B. subtilis spores(44). These results point to an activity other than pore forma-tion, possibly inhibition of cell wall biogenesis, for the inhibi-tion of spore outgrowth by nisin. Thus, structure-activity rela-tionships for the identification of residues important for thevarious consequences of nisin against spores remain an impor-tant focus of future work.

Nisin is an FDA-approved natural product that has beenused for 40 years for food preservation, due in part to theselective toxicity of this lantibiotic toward gram-positive bac-teria (13, 15, 52). In this study, B. anthracis was used as amodel, but previous work indicated that nisin is also inhibitoryagainst spores from other Bacillus species (3, 7, 30, 32, 35, 43),as well as from Clostridium species (36), suggesting that thenew information on the inhibitory activity of nisin obtained inthis study will be applicable to determination of the mechanismof action of nisin against spores from these other organisms aswell. Here, nisin was demonstrated to act upon and kill ger-minated spores of B. anthracis prior to development into elon-gated and dividing bacilli and before LT was generated. No-tably, this mode of activity is in contrast to the modes of activityof several other widely used classes of antibiotics, includingciprofloxacin, whose mechanisms of action require ongoing cellactivity and/or proliferation (2, 16, 18, 22) and which are thusnot as likely to be effective against germinating spores. Collec-tively, these properties potentially make nisin an attractivechemotherapeutic agent for prophylaxis or postexposure treat-ment of spore-forming Bacillus or Clostridium pathogens.

ACKNOWLEDGMENTS

We thank Barbara Pilas and Ben Montez from the R. J. CarverBiotechnology Center at the University of Illinois—Urbana/Cham-paign (UIUC) for assistance with flow cytometry. In addition, theauthors acknowledge the assistance of Lou Ann Miller from the Cen-ter for Microscopic Imaging, within the College of Veterinary Medi-cine at UIUC, for assistance with TEM.

This work was supported by NIH-NIAID award U54-AI057156 tothe Western Regional Center for Excellence for Biodefense andEmerging Infectious Diseases Research (to S.R.B., J.D.B., T. M.Koehler, and P. I. D. Walker), a Chemical Biology Interface TrainingGrant from the National Institutes of Health (5 T32GM070421 toI.M.G.), and grant RO1-GM58822 from NIGMS (to W.A.V.D.D.).

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