International Journal of Food Microbiology 143 (2010) 125–135

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Morphology and physico-chemical properties of Bacillus spores surrounded or notwith an exosporiumConsequences on their ability to adhere to stainless steel

Christine Faille a,⁎, Yannick Lequette a, Annette Ronse a, Christian Slomianny b,Estelle Garénaux c,d, Yann Guerardel c,d

a INRA, UR638, Interfacial Processes and Hygienic Materials, 369 rue Jules Guesde, BP 20039, F-59651 Villeneuve d'Ascq cedex, Franceb INSERM-LPC, U1003, Université Lille1, Sciences et Technologies, Cité Scientifique, Bât. SN3, F-59655 Villeneuve d'Ascq cedex, Francec Univ. Lille1, Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, F-59650 Villeneuve d'Ascq cedex, Franced CNRS UMR 8576, F-59655, Villeneuve d'Ascq cedex, France

⁎ Corresponding author. Tel.: +33 320 435404; fax:E-mail address: faille@lille.inra.fr (C. Faille).

0168-1605/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2010.07.038

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 April 2010Received in revised form 9 July 2010Accepted 31 July 2010

Keywords:Bacillus sporeExosporiumMorphologyAppendagesSaccharidic composition

This studywas designed to elucidate the influence of spore properties such as the presence of an exosporium, ontheir ability to adhere tomaterials. This analysiswas performed on 17 strains belonging to the B. cereus group andto less related Bacillus species. We first demonstrated that spores of the B. cereus group, surrounded by anexosporium, differed in theirmorphological features such as exosporium size, number of appendages or hair-likenap length.Wealso found that the saccharidic composition of exosporiumdiffered among strains, e.g. concerninga newly identified rhamnose derivative: the 2,4-O-dimethyl-rhamnose. Conversely, spores of distant Bacillusspecies shared morphological and physico-chemical properties with B. cereus spores. Some external featureswere also observed on these spores, such as a thin loose-fitting layer, whose nature is still to be determined, or athick saccharidic layer (mainly composedof rhamnose andquinovose). The ability of spores to adhere to stainlesssteel varied among strains, those belonging to the B. cereus group generally being the most adherent. However,the presence of an exosporium is not sufficient to explain the ability of spores to adhere to inanimate surfaces.Indeed, when the 17 strains were compared, hydrophobicity and the number of appendages were the onlysignificant adhesion parameters. Furthermore, the differences in spore adhesion observed within the B. cereusgroupwere related to differences in the number of appendages, the exosporium length and to a lesser extent, thezeta potential.

+33 320 435426.

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1. Introduction

ManyBacillus species (B. cereus,B. subtilis,B. licheniformis,B. pumilus…)are of concern in the food industry, being readily isolated froma variety offoods (Banerjee and Sarkar, 2004; Salkinoja-Salonen et al., 1999; te Giffelet al., 1996; Valero et al., 2003), including cereal products, vegetables,spices, dried foods, dairy products and meat products. Many of themimpair quality and some can cause food-borne diseases, e.g. B. cereus,B. thuringiensis, B. licheniformis or B. subtilis (Brown, 2000). These bacteriaare able to produce spores resistant to heat-treatments or disinfectionprocedures (Dufrenne et al., 1994).

All Bacillus spores share a common architecture consisting in a set ofconcentric layers: a cortex and a coat surrounding the inner core.Conversely, anadditional loose balloon-like envelope called exosporium

is observed around spores of B. cereus strains and of the closely-relatedspecies forming the B. cereus group, e.g. B. thuringiensis, B. anthracis orB. mycoides. This structure has also been observed in other Bacillusspecies, such as B. alvei, B. brevis, or B. sphaericus, but it has not beenobserved in spores of B. subtilis or B. licheniformis (Hachisuka et al.,1984). The exosporium is made of a paracrystalline basal layersurrounded by an external hair-like nap, which has been observed onexosporia of B. anthracis (Sylvestre et al., 2003), B. thuringiensis (Plompet al., 2005a) and B. cereus (Gerhardt andRibi, 1964). The hair-like nap iscomposed of several collagen-like glycoproteins (Garcia-Patrone andTandecarz, 1995; Todd et al., 2003), which are composed of threedomains: anN-terminal domain involved in targeting andanchoring theprotein in the exosporium(Thompson and Stewart, 2008), the collagen-like region (CLR) and a proximal C-terminal domain, structurally similarto the C1qprotein of the complement in the immune system (Rety et al.,2005). Alongwith the presence of an exosporium, spores of the B. cereusgroup share common properties such as hydrophobicity and electro-negativity (Andersson et al., 1998). However, some differences in sporeproperties have been also reported within the B. cereus group, such as

126 C. Faille et al. / International Journal of Food Microbiology 143 (2010) 125–135

varying hydrophobicity (Andersson and Rönner, 1998; Tauveron et al.,2006), exosporium size (Tauveron et al., 2006), and the length of thehair-like nap (Sylvestre et al., 2003; Tauveron et al., 2006). BclAglycosylation also differs among Bacillus species as shown by thecomposition in saccharidic residues (Fox et al., 2003):while 3-O-methylrhamnose has been reported in the exosporia of B. anthracis, B. cereus,and B. thuringiensis (Fox et al., 2003; Garcia-Patrone and Tandecarz,1995), anthrose has so far only been identified in B. anthracis spores(Daubenspeck et al., 2004) and in few strains of B. thuringiensis orB. cereus species (Dong et al., 2008), but not in B. cereus ATCC 14579(Daubenspeck et al., 2004). In addition fucose and 2-O-methylrhamnose residues have been identified in B. cereus and B. thuringiensisspores but are absent from B. anthracis spores (Fox et al., 2003; Walleret al., 2004).

Variations in spore properties have also been observed betweenstrains of various species. Among these properties, the size of thedehydrated spore varies greatly among strains (Carrera et al., 2007). Insome species, such as B. cereus, B. thuringiensis, or B. pumilus (Hachisukaet al., 1984), spores are surroundedby appendages,whichvary in length(up to 2 μm) and in number among strains (Stalheim and Granum,2001; Tauveron et al., 2006). Physico-chemical properties also differamong spores of different species. Indeed, previous works have shownthat spores could be hydrophilic to hydrophobic and more or lesselectronegative (Ankolekar and Labbe, 2010; Buhr et al., 2008).

Elsewhere, B. cereus spores have been shown to firmly adhere to awide variety of materials typically encountered in food processingpremises, such as stainless steel, PEHD or Teflon® (Faille et al., 2002).Some authors put forward the hypothesis that a direct cause andeffect relationship would link the presence of the exosporium to theability of spores to adhere (Brahmbhatt et al., 2007). However, besidesthe presence of an exosporium, the various properties describedabove could also be suspected of playing a role in spore adhesion toinert surfaces and/or in their further resistance to flow. Therefore, anaccurate characterization of spore morphology and physico-chemicalproperties will provide important information concerning theirpossible behavior in adhesion processes.

The aim of this study was to investigate differences in the sporeproperties within the B. cereus group and among Bacillus species inorder to identify discriminating properties. We then investigatedwhether the presence of an exosporium was of a major importance inspore adhesion or if differences in the ability of spores to adhere couldbe rather attributed to other spore properties.

2. Material and methods

2.1. Strains and spore production

Seventeen Bacillus strains from various origins were used in thisstudy. Nine strains belong to the B. cereus species: B. cereus ATCC14579 is the type strain, B. cereus CUETM 98/4 was isolated from adairy processing line, B. cereus D1, D6, D17 and D31 were isolatedfrom patients with diarrheic symptoms following ingestion ofcontaminated foods, B. cereus 433, LM9 and CIP 5832 were isolatedfrom the environment. The latter seven strains have been provided bythe INRAUR 249 (LaMinière, France). Three other strains belonging tothe B. cereus group: B. thuringiensis 407 have been isolated from soil,B. thuringiensis 7138T is the type strain and the avirulent strainB. anthracis 9131 (Etiennetoumelin et al., 1995), lacking both pXO1and pXO2 plasmids,was provided by the Institut Pasteur (Paris, France).Lastly, five strains belonging to other Bacillus species were alsoanalyzed: B. subtilis CUETM 98/7 isolated from a dairy processing line,the type strain B. subtilis LMG 7135T, the type strain B. licheniformis LMG6933T, B. pumilus CUETM98/6, isolated from a dairy processing line andB. sporothermoduransMB581, provided by ILVO (Merelbeke, Belgium).

Spores were produced as previously described (Faille et al., 1999).When over 95% mature spores were observed, spores were scraped

from the agar surface, and resuspended in water. Spores were washedfive times in sterile water at 4 °C (15 min at 1200 g) and stored at 4 °Cin sterile water until use. Before each experiment, a further set oftwo washes was performed and spores were subjected to a 2.5-minultrasonication step in a ultrasonic cleaner (Bransonic 2510E-MT,42 kHz, 100 W, Branson Ultrasonics Corporation, USA) to limit thepresence of aggregates.

2.2. Transmission electron microscopy (TEM)

Whole spores were adsorbed onto Formvar-coated grids (15/95,Euromedex, EMS, Hatfield, USA) and examined after negative stainingwith 1% uranyl acetate (VWR, Fontenay-sous-Bois, France) on aHitachi H600 electron microscope at an accelerated voltage of 75 kV.Several TEM pictures were taken, and the spore and exosporium sizeswere obtained by measuring at least 30 individual spores. In order toallow the observation of nanometric features, spore sections wereobserved by TEM. The external part of exosporia being mainlycomposed of glycoproteins, a ruthenium red staining procedurepreviously described (Waller et al., 2004) and slightly modified in ourlaboratory, was performed. Briefly, cells were fixed in a solution of2.5% glutaraldehyde, 0.1 M cacodylate buffer pH 7, 500 ppm rutheni-um red (Sigma, St Louis, USA). After 15 min incubation at 4 °C, cellswere rinsed three times for 5 min in 0.1 M cacodylate buffer at pH 7and centrifuged at 1200 g. The pellet was suspended in 1 mL of thepost-fixative solution (2% osmium tetroxide, 0.1 M cacodylate bufferat pH 7, 500 ppm ruthenium red). After 15 min of incubation at 4 °C,the cells were rinsed three times for 5 min in water by centrifugationat 1200 g for 5 min. After dehydration in graded acetonitrile, thepellets were embedded in Epon 812 (Merck, Darmstadt, Germany).Thin sections were cut on a Reichert-Jung ultramicrotome andcollected on 100 or 150 mesh grids (H150-Cu, Euromedex, EMS,Hatfield, USA). After double staining with 2% uranyl acetate in 50%ethanol and lead citrate (Reynolds, 1963), the sections were observedon a Hitachi 600 electron microscope at 75 kV. The length of the hair-like nap surrounding the exosporiumwasmeasured on these sections.The measurements were done at three or four different points on aperfectly tangential section of a spore, on 10 spores per strain. Theprecision of the measure is about 5 nm due to the faintness of somebrushes.

2.3. Spore surface glycoproteins, composition in saccharidic residues

Before extraction, spores were washed twice in water. Spores(15 mL in PMSF (Sigma) 25 mM, around 109 spores/mL) were thensubjected to four successive passages through a French press at20,000 psi (SLM Instruments, Urbana, USA). Spores were eliminatedby centrifugation (3000 g, 30 min, 4 °C). The supernatant waslyophilized and boiled in a reducing buffer (125 mM Tris–HCl pH6.8, 4% w/v SDS, 10% v/v β-mercaptoethanol, 1 mM DTT, 0.05% w/vbromophenol blue, 10% v/v glycerol) for 8 min, as described bySteichen et al. (2003). Sampleswere centrifuged for 2 min at 8000 g toremove any insoluble materials. Glycoproteins were separated bySDS-PAGE (12% acrylamide) at 40 mA and patterns were observedafter staining gels with PAS (Sigma).

For the determination of monosaccharide composition of exosporia,spores were washed twice in water, suspended in water and subjectedto four successive passages through the French press at 20,000 psi.Spores were eliminated by centrifugation (3000 g, 30 min, 4 °C).Insolublematerials containing exosporia and solublematerials contain-ing material from the parasporal space were separated by ultracentri-fugation (120,000 g, 30 min, 4 °C). Monosaccharide composition ofinsoluble and soluble fractions of B. cereus ATCC 14579 was establishedby GC and GC/MS, both as heptafluoro-butyrated (Zanetta et al., 1999)and alditol acetate derivatives. Briefly, for generation of alditol acetatederivatives, samples were hydrolyzed in 4 M trifluoroacetic acid (TFA)

127C. Faille et al. / International Journal of Food Microbiology 143 (2010) 125–135

for 4 h at 100 °C and then reduced with sodium borohydride in 0.05 MNaOH for 4 h. Reduction was stopped by drop-wise addition of aceticacid until pH 6 was reached, and borate salts were co-distilled byrepetitive evaporation in dry methanol. Peracetylation was performedin acetic anhydride at 100 °C for 2 h. All monosaccharide derivativeswere identified according to their specific retention times and EI-MSfragmentation patterns.

2.4. Microbial physico-chemical properties

In order to characterize the spore hydrophobic property, apartitioning method was used, based on the affinity of spores to anapolar solvent, hexadecane (Sigma). Spore suspensions in saline wereadjusted to an absorbance of 0.5 to 0.6 at 600 nm (A0) in glass tubes(10 mm in diameter×75 mm). Three milliliter aliquots of the sporesuspension and 500 μL of hexadecanewere vortexed for times rangingfrom 5 s to 180 s and left to settle for 30 min to allow completeseparation of the two phases. The absorbance at 600 nm of theaqueous phasewasmeasured (At). [(At/A0)⁎100] was plotted againstthe vortexing time (s). The initial slope, giving the initial removal rate(R0) from the aqueous suspension, is related to the hydrophilic/hydrophobic spore character. A spore was considered to be hydro-phobic when −R0 fell between −4.0 and −6.0 and to be highlyhydrophobic for lower values.

The spore zeta potential was measured using a zetameterZetaCompact (CAD Instrumentation, France). This was determinedfrom the electrophoretic mobility using the Helmholtz–Smoluchowskiequation. For this purpose, spores were suspended in 1 mM KNO3 toobtain around 50 spores per analysis. pHwas adjusted to values rangingfrom 3 to 9, with HNO3 1 mM or KOH 1mM. Trials at pH 2.86 wereperformed directly in HNO3 1 mM. Each sample was analyzed induplicate.

2.5. Adhesion of spores to stainless steel coupons

Stainless steel (304 L, bright annealed) coupons (15 mm×45 mm)were fouled by vertical immersion for 4 h in an aqueous sporesuspension (105 spores/mL), and then quickly rinsed in sterile water.The fouled coupons were subjected to an ultrasonication step in10 mL Tween 80 2% v/v (5 min, Ultrasonic bath, Deltasonic, France).The detached spores following sonication were enumerated onnutrient agar (1.3% w/v nutrient broth (Biorad, France), 1.5% w/vbacteriological agar type E (Biokar Diagnostics, France)) after 48 h at30 °C.

2.6. Statistical analysis

Data were analyzed by general linear model procedures using SASV8.0 software (SAS Institute, Gary, NC, USA). Variance analysis wasfirst performed to determine the role of strain in the ability of sporesto adhere. This was followed by a multiple comparison procedureusing Tukey's test (Alpha level=0.05). Other sets of variance analyseswere performed to determine the influence i) of the exosporium onspore adhesion (B. cereus D17was not taken into account), ii) of sporeproperties (morphology vs physico-chemistry) on spore adhesion.

3. Results

3.1. Spore morphology

Spore morphology was first investigated by TEM on whole spores(between 30 and 65 spores) after negative staining. This methodallows the clear observation of an exosporium surrounding spores ofthe B. cereus group (Fig. 1). Out of the 12 strains from the B. cereusgroup, only one, B. cereus D17 (Fig. 1C) exhibited a high level of sporeswith an injured exosporium, that did not allow accurate measure-

ments. Exosporia of most of the other strains (8/10) were character-ized by an average length ranging from 2.0 μm to 3.0 μm (Table 1),except B. anthracis 9131 (Fig. 1D) and B. cereus 5832 (Fig. 1B),respectively surrounded by a small (1.51 μm) and a large (3.15 μm)exosporium. Furthermore, observations revealed a wide distributionin the size of the exosporia within spore batches: within a single sporebatch, the longest exosporium was sometimes twice as long as theshortest.

Appendages of about 10 nm in width were also found aroundexosporia of all the strains of the B. cereus group except B. anthracis9131 (Fig. 1D). Yet, their number varies among strains, between 2 andover 20 per spore (Table 1), but also within spore batches. Conversely,no appendage was observed on spores of distant species, except onfew spores of B. subtilis 7135 (Fig. 2E).

In order to better visualize the hair-like nap and any other superficialspore features, sections of spores previously stainedwith rutheniumred(specific for saccharidic residues) were observed. Irrespective of theB. cereus group strain, the exosporium was composed of a thin basallayer whichwas impervious to electrons (the paracrystalline layer) andan external hair-like nap (Fig. 2A), whose filament length ranged from25 nm to 40 nm (Table 1), except on B. anthracis spores (Fig. 2C) whosenap length reached60 nm.Anexternal layer not steadily adherent to theouter coat was sometimes observed around spores belonging to lessrelated species, as in the caseofB. pumilus (Fig. 2F) and to a lesser extent,that of B. sporothermodurans MB581 (Fig 2G) and B. subtilis 7135(Fig. 2I). Elsewhere, B. subtilis 98/7 spores were covered by a thick layerof tangled filaments strongly stained with ruthenium red (Fig. 2H),which is indicative of their saccharidic nature. This surroundingmaterialwas already observable by TEM after negative staining (Fig. 2F). Onsome spores, these filaments seemed to be longer at the spore tips.Lastly, spores of B. licheniformis (Fig. 2E) were devoid of exosporia,appendages, or any superficial features observed by TEM.

We then measured the length (L) and width (l) of the spores,without taking into account exosporia or polymers. The spore wasconsidered as an ellipsoid and its volume was estimated by thefollowing equation: 4/3⁎π⁎(L/2)⁎(l /2)2. Spores of the B. cereusgroup were voluminous (Table 1) compared to spores of the otherspecies. Their volumes were equal to or greater than 0.3 μm3, exceptfor B. cereus 14579. Spores of other species were mostly smaller, withvolumes equal to or lower than 0.3 μm3 (4 over 5 strains). It must alsobe noticed that B. cereus 98/4 spores were very large (0.56 μm3). Atthe other extreme, B. pumilus spores were very small (0.13 μm3), noteasily identifiable even by optical microscopy (data not shown).

3.2. Presence of superficial glycoproteins, composition in saccharidicresidues

PAS staining of SDS-PAGE gels revealed the presence of glycopro-tein complexes over 200 kDa in all strains belonging to the B. cereusgroup (Fig. 3). Glycoprotein bands exhibiting apparent molecularweight ranging from 60 to 85 kDa were also observed in every strainbut B. anthracis 9131. In strains belonging to other Bacillus species, thetop of the lane was faintly stained. Some areas of lower PM were alsostained, either as distinct bands (e.g. B. subtilis 7135) or as a smear(e.g. B. subtilis 98/7). One may conclude that every spore tested, withor without exosporium, in this study was surrounded by a wide arrayof glycoproteins or polysaccharides.

The monosaccharide composition of spore superficial fractions(soluble and insoluble), was determined by GC and GC/MS for allBacillus strains used in the present study. We first determined thesaccharidic profile of the insoluble fraction containing exosporia ofB. cereus ATCC 14579 that was used as standard strain. Based on bothretention times in comparison with authentic standards, andelectronic-impact mass spectrometry (EI/MS) fragmentation patternsof reduced per-acetylated monosaccharide derivatives, a wide rangeof major monosaccharides, including rhamnose (Rha) derivatives,

Fig. 1. Transmission electronmicroscopyonwhole sporesafternegative staining.A:B. cereus98/4,B:B. cereus5832, C:B. cereusD17,D:B. anthracis9131, E:B. subtilis7135, F:B. subtilis98/7.

128 C. Faille et al. / International Journal of Food Microbiology 143 (2010) 125–135

galactose (Gal), glucosamine, (GlcNH2) and galactosamine (GalNH2)was identified (Fig. 4D, Table 2). Considering the large batch to batchvariability of Gal quantities, this monosaccharide was, at least for alarge part, considered as a contamination from agarose (sporulationmedium), and was not included in further analysis. Along with thesemajor compounds, minor components that accounted for less than 5%of the total monosaccharides were also identified, including ribose,xylose and mannose. Of particular interest, three derivatives ofrhamnose residues were unambiguously assigned to Rha, 2-O-methyl-rhamnose (2-Me-Rha), and 3-O-methyl-rhamnose (3-Me-Rha) (Fig. 4A). A fourth prominent component was then identified asa 2,4-O-dimethyl-6-deoxyhexose, based on fragmentation pattern ofits associated GC signal (Fig. 4B). The lack of an authentic standardfor this rare component did not permit us to directly establish thering configuration of this component, but the presence of threeother derivatives of rhamnose, strongly suggest it is 2,4-O-dimethyl-rhamnose (2,4-diMe-Rha). This was confirmed by showing that itspermethylated derivative co-eluted with permethylated Rha deriva-tive (data not shown). On the other hand, composition analysis of thesoluble fraction, devoid of exosporium, did not show any significantquantity of rhamnose, but large quantities of glucosamine andmuramic acid (Mur) residues (data not shown). This result estab-lished that rhamnose derivatives were specifically associated to theexosporium, while Mur would be present in the parasporal space ofthe B. cereus 14579 strain.

The monosaccharide compositions of superficial fractions of allBacillus strains were then compared to those of B. cereus ATCC 14579.Among the B. cereus strains, three groups were identified based on theratios of rhamnose and hexosamine derivatives found in the insolublefraction (Table 2). Most of the strains tested exhibited monosaccha-ride compositions similar to B. cereus ATCC 14579 and were assignedto Group 1, characterized by the presence of all four rhamnosederivatives and GalNH2. Conversely, strains belonging to Group 2(B. cereusD6 and B. cereus 433) showed no Rha residues as well as lowamounts of or no GalNH2 residues. Lastly, B. cereus LM9 was devoid of2-Met-Rha and GalNH2 and was assigned to Group 3. It is noteworthythat B. thuringiensis strains belonged to Group 1, and B. anthracis wascharacterized by a specific profile with the absence of 2-Met-Rha and2,4-Me-Rha (Table 3). The latter strain was assigned to Group 4.

Among Bacillus species generally assumed to be devoid of anexosporium, B. licheniformis and B. sporothermodurans showed highmonosaccharides levels in the insoluble fraction with a differentcomposition than that of the exosporia of the B. cereus group(Table 3). B. licheniformis contained almost exclusively GlcNH2 andGalNH2, whereas B. sporothermodurans contained almost exclusivelyRha and GlcNH2. Conversely, only traces of monosaccharides wereobserved in the insoluble fraction of B. subtilis and B. pumilus spores(data not shown). In order to identify the nature of the polymericsubstance observed by TEM on B. subtilis 98/7 spores (Fig. 2H), we alsoanalyzed the saccharidic composition of the soluble fraction. The soluble

Table 1Surface properties of the Bacillus spores and adhesion to stainless steel coupons.

Exosporiumlength (μm)

Nap length(nm)

Spore volume(μm3)

Number ofappendages

Hydrophobocity(−R0)

Zetapotential

Adhesion

B. cereus 14579 2.01(0.48)⁎

25 0.25(0.04)

4.7(1.7)

4.8 −38.98 1.06E+04

B. cereus 98/4 2.59(0.43)

25 0.56(0.12)

18.3(11.2)

9 −26.33 2.03E+05

B. cereus 433 2.66(0.40)

30 0.45(0.07)

12.3(3.9)

3.6 −22.08 4.40E+04

B. cereus 5832 3.15(0.49)

35 0.41(0.09)

2.3(1.5)

3.1 −21.67 4.13E+03

B. cereus LM9 2.83(0.47)

40 0.40(0.09)

7.3(2.2)

5.5 −22.45 1.04E+04

B. cereus D1 2.66(0.34)

35 0.41(0.05)

9.9(6.9)

4.4 −29.75 1.75E+04

B. cereus D6 2.28(0.42)

40 0.31(0.06)

4.6(1.6)

5.4 −26.05 7.25E+04

B. cereus D17 nd nd 0.32(0.06)

nd 5.2 −34.66 1.06E+04

B. cereus D31 2.89(0.38)

35 0.45(0.10)

5.7(1.8)

4.2 −17.61 7.57E+04

B. anthracis 9131 1.54(0.17)

60 0.42(0.09)

0 5.1 −20.26 2.68E+04

B. thuringiensis 407 2.41(0.72)

30 0.31(0.07)

2.7(1.3)

6.4 −28.47 1.79E+04

B. thuringiensis 7138 2.13(0.53)

40 0.31(0.05)

1.6(0.9)

5.5 −26.00 1.78E+04

B. licheniformis 6933 − − 0.20(0.04)

0 5 −18.65 1.41E+04

B. pumilus 98/6 − − 0.13(0.02)

0 4.3 −44.80 1.88E+04

B. sporothermodurans MB581 − − 0.28(0.05)

0 1.1 −40.49 6.39E+03

B. subtilis 98/7 − − 0.33(0.08)

0 0.5 −24.26 1.56E+02

B. subtilis 7135 − − 0.23(0.04)

1 1 −46.81 2.67E+03

⁎ Numbers in brackets: standard deviations.

129C. Faille et al. / International Journal of Food Microbiology 143 (2010) 125–135

fraction contained large amounts ofmonosaccharides,mainly Rha and arelatively rare monosaccharide, the 6-deoxy-glucose or quinovose(Qui).

3.3. Spore physico-chemical properties

The spore hydrophobic/hydrophilic character was estimated byMATH assay. In the B. cereus group, the spore surfacewas hydrophobicto highly hydrophobic, as indicated by the initial removal rate rangingfrom −3.1 s−1 (B. cereus 5832) to −6.4 s−1 (B. thuringiensis 407),and B. cereus 98/4 spores were distinguishable by a very high affinityto hexadecane (−9.0 s−1), indicating an even more hydrophobiccharacter. More contrasting results were obtained with spores of theother species. B. licheniformis 6933 and B. pumilus 98/6 exhibited R0close to those obtained with the B. cereus group. B. subtilis 98/7 washighly hydrophilic with an initial removal rate of −0.5 s−1, followedby B. subtilis LMG 7235 and B. sporothermodurans MB581 with initialremoval rates around −1.0 s−1.

The spore electric charge was characterized by the zeta potential.The spore electrophoretic motilities indicate a clear electronegativecharacter of all strains at pH 7.0, even if wide variations were observedbetween strains (zeta potential ranging from−17.61 to−46.81 mV).The lowest values were attained by strains of species distant to theB. cereus group.

3.4. Spore adhesion to stainless steel coupons, role of exosporia and sporeproperties

For adhesion experiments, stainless steel coupons were verticallyimmersed for 4 h in a spore suspension and the number of adherent

spores was determined. Adhesion varied greatly among strains(Table 1), with a number of adherent spores per coupon rangingfrom 1.5·102 to 2.0·105, but only from 4.1·103 to 2.0·105 betweenspores of the B. cereus group. The variance analysis showed that thestrain accounted for 86% of the variability in the spore adhesion.According to Tukey's grouping, the 17 strains tested were groupedinto ten classes (Table 4). Shortly, most of the highly adherent sporesbelonged to the B. cereus group, except B. pumilus 98/6, and most ofthe poorly adherent spores belonged to other Bacillus species, exceptB. cereus 5832 which is characterized by a large exosporium. Howeverthe presence/absence of a wholly-structured exosporium (character-istic of the B. cereus group) only accounted for 24% of the differencesobserved in the number of adherent spores (p-value of 0.0013),indicating that the exosporium is not the main criteria for explainingadhesion variability.

We then investigated the role of morphological (spore volume,number of appendages) and physico-chemical (hydrophobic charac-ter, zeta potential at pH 7.0) properties, on the ability of spores toadhere to stainless steel coupons. The variance analysis of the wholepanel of spores indicated that only 51% of the variability in thenumber of adherent spores was explained by the four parameterstested, but the model was accepted (pb0.0001). Further analyses ofvariance were performed in order to determine which of theseparameters really accounted for the differences in the number ofadherent spores: each parameter was withdrawn sequentially fromthe variance analyses. Two parameters, hydrophobicity and thenumber of appendages, still accounted for 50% of the variability: thenumber of adherent spores was mainly affected by hydrophobicity(pb0.0001) and to a lesser extent by the number of appendages(p=0.0450).

Fig. 2. Transmission electron microscopy on spore sections after ruthenium staining. A: B. cereus 98/4 (bl: basal layer, nap: hair-like nap); B: B. cereus 433; C: B. anthracis 9131;D: B. thuringiensis 407; E: B. licheniformis 6933; F: B. pumilus 98/6; G: B. sporothermodurans MB581; H: B. subtilis 98/7; I: B. subtilis 7135.

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We then investigated if spores surrounded by a wholly-structuredexosporium could also be discriminated on the basis on their ability toadhere and which were the predominant factors affecting the numberof adherent spores. According to Tukey's grouping, strains of theB. cereus group were grouped into seven groups depending on theirability to adhere (Table 5). To explain the differences observed, threenew parameters were analyzed, along with the four previousparameters: exosporium length, nap length, and superficialsaccharidic composition. The variance analysis indicated that theseven parameters account for 73% of the variability in spore adhesion(p-valueb0.0001). The same approach as previously mentioned wasused to determine the most relevant parameters. Three of the sevenfactors still account for 54% of the variability: the number ofappendages (pb0.0001), exosporium length (p=0.0004) and to alesser extent, zeta potential (p = 0.0087).

4. Discussion

B. cereus spores present a remarkable ability to adhere firmly tovarious inert materials (Faille et al., 2002; Seale et al., 2008) and thisproperty is suspected of being related to the presence of an externalloose-fitting outermost membrane called exosporium, common to allmembers of the B. cereus group (Henriques and Moran, 2007). Inorder to characterize the potential role of the exosporium on spore

adhesion, we analyzed surface properties and the ability to adhere ofspores of the B. cereus group, as well as of spores of other Bacillusspecies such as B. subtilis or B. licheniformis, reported to be devoid of anexosporium (DesRosier and Lara, 1981; Hachisuka et al., 1984; Kimet al., 2009).

We first confirmed the presence of an exosporium around sporesof every strain of the B. cereus group. Surprisingly, one spore (of B.anthracis 9131) was found to be surrounded by two exosporia(Fig. 2C), both exhibiting a clear hair-like nap, but the internal one didnot wholly surround the spore. As far as we know, only one formerstudy reported a similar observation on one B. megaterium spore(Beaman et al., 1972), but no clear hair-like nap was observed on theinner exosporium. One can hypothesize that the exosporiumassembly control may be defective in this particular spore. Inaccordance with previous reported works (Hachisuka et al., 1984),no exosporium could be observed on B. licheniformis spores, whichwere surrounded by a lamellar inner coat and a thick, dense outer coat(Kim et al., 2009). On some spores of other Bacillus species reported tobe devoid of the classical exosporium found in the B. cereus group, anexternal layer, more or less tightly bound to the underlying coat, wasobserved, such as in B. pumilus and to a lesser extent B. sporothermo-durans MB581 and B. subtilis 7135. This thin outermost layer isreminiscent of the layer sometimes observed on B. megaterium spores(Beaman et al., 1972). The latter authors suggested this layer was an

Fig. 3. Electrophoretic profiles of Bacillus extracts (separation on a 12% acrylamide gel, PAS staining).

131C. Faille et al. / International Journal of Food Microbiology 143 (2010) 125–135

exosporium, but conflicting results were also reported (Hachisukaet al., 1984; Wang et al., 2007). In this study, we observed that thisexternal layer was made of more or less impervious layers, whoselamellae structure suggested it was composed of coat layers. However,other data would suggest the presence of an exosporium in B. pumilusand B. licheniformis. Indeed, several sequences of putative glycopro-teins with a structure characteristic of the BclA-like proteins werefound in the genomes of B. pumilus (BPUM 0706, BPUM 1662, BPUM1804) and B. licheniformis (BL03072, BL03073) strains, but not ofB. subtilis, which spores are supposed to be devoid of exosporium.Moreover, the BclA-like proteins identified in the B. cereus groupwereonly found in exosporia (Henriques and Moran, 2007). However,these glycoproteins only shared between 14.4 and 54.9% similitudewith at least one of the glycoproteins Bcla, ExsH and BclB, alreadyidentified in the exosporium in B. cereus or B. anthracis (programneedle open gap penalty 1, gap extension penalty 0.5, matrix blosum62), that would suggest different locations and/or functions. There-fore, the question whether the outermost spore layer observed indifferent Bacillus species is an exosporium or not is still open. Lastly, B.subtilis 98/7 spores were surrounded by a thick layer with projectionswhich were strongly stained with ruthenium red, suggesting it waslargely composed of saccharidic fractions. This layer was sometimesmore easily observed at the spore tips, as previously reported (Carrollet al., 2008). The observation of a saccharidic layer has been reportedin the literature (Waller et al., 2004), but its structure was moreregular without protrusions, suggesting a different composition. Thesame authors hypothesized that the glycosylated surface layer mightbe a rudimentary exosporium, while others supposed it was thestructural and functional equivalent of the exosporium (Henriquesand Moran, 2007), though no BclA-like glycoprotein genes could beidentified in B. subtilis.

We then investigated the morphological properties of sporesbelonging to the B. cereus group and to less related species. First,strains belonging to the B. cereus group could be distinguished onthe basis of the exosporium size. For example, B. anthracis 9131 andB. cereus D6 spores were characterized by a very small exosporiumand B. cereus 5832 spores by a large exosporium. According to previousreported works, such diversity was also found within the B. anthracisspecies (Hachisuka and Kozuka, 1981). Furthermore, a wide distribu-tion in the size of the exosporium within spore batches was alsorevealed whatever the strain in use: the longest exosporium was oftentwice as long as the shortest (Plomp et al., 2005b). Elsewhere,numerous parasporal inclusions were observed in some species. Thelarge planar inclusions, mainly observed in most of the B. cereus 433spores, resulted in exosporium deformities. This phenomenon has beenpreviously described in B. megaterium spores (Beaman et al., 1972). Wealso considered the properties of the hair-like nap, surrounding theexosporium crystalline basal layer, whose filament length varies moreamong B. anthracis strains (Sylvestre et al., 2003), than among B. cereusisolates (Tauveron et al., 2006). In accordance with these results, wefound similar filament lengths in the panel of spores tested in thisstudy, except for B. anthracis whose nap was longer.

The size of the inner spore inside the exosporium would also be ofconcern in B. cereus spore adhesion, as is the case for spores notsurrounded by an exosporium. Several works mentioned sporelengths of around 1.40–1.50 μm for B. thuringiensis (Westphal et al.,2003), B. anthracis and B. cereus (Wang et al., 2007), and B. subtilis(Leuschner and Lillford, 2001). Dimensions of the same order havebeen obtained in this study, but spores of the B. cereus group weremostly larger than those belonging to other species in concordancewith previous works (Carrera et al., 2007). We also observed cleardifferences between strains of a given species as shown by these

Fig. 4. Composition analysis of exosporium from B. cereus ATCC 14579. A: detail of the deoxysugar region of the GC-FID-chromatogram from alditol acetate derivative analysis; B: GC-EI-MS and C: fragmentation spectrum and analysis of major fragments of 2,4-O-dimethyl-rhamnose alditol acetate derivative; D: monosaccharide composition analysis.

132 C. Faille et al. / International Journal of Food Microbiology 143 (2010) 125–135

authors on B. anthracis spores. Furthermore, it is noteworthy that theB. pumilus strain used throughout this study produced very smallspores, contrary to what has been previously reported (Wang et al.,2007).

The spores belonging to the B. cereus group often has moreappendages, with a spiral structure (Faille et al., 2010) and a diameterof around 10 nm (Plomp et al., 2005a). As previously shown (Stalheimand Granum, 2001; Tauveron et al., 2006), their length and numbergreatly vary within strains, as well as their number per spore. Weobserved such appendages on the surface of spores of every strain ofthe B. cereus group, except that of B. anthracis spores, as previouslyshown on many B. anthracis strains (Hachisuka and Kozuka, 1981,

Table 2Exosporial saccharidic composition of spores of the B. cereus group.

% Bc 145791 Bc 98/41 Bc 4332 Bc

2,4-Me-Rha 15.3 25.1 19.5 252-Me-Rha 11.7 5.4 6.8 53-Me-Rha 9.4 14.5 13.3 14Rha 32.8 25.5 Nd 25GlcNH2 9.4 11.6 51.3 11GalNH2 21.4 18.0 9.1 18

Superscripted numbers: saccharidic groups.

1984). Moreover, as expected from previous works (DesRosier andLara, 1981), no appendage could be observed around spores of B.sporothermodurans, B. licheniformis, and B. pumilus even if somecontradictory results have been also reported on the latter species(Hachisuka et al., 1984). Lastly, as far as we know, it is the firstobservation reported on the presence of appendages on spores of a B.subtilis strain (B. subtilis 7135), in contradiction to what is generallyadmitted (DesRosier and Lara, 1981; Hachisuka et al., 1984).

We also identified differences in the physico-chemical properties ofspores. Spores of the B. cereus group were moderately to highlyhydrophobic, in accordance with previous results (Andersson andRönner, 1998; Simmonds et al., 2003; Tauveron et al., 2006). This

58321 Bc LM93 Bc D11 Bc D62 Bc D311

.1 2.9 5.7 11.4 14.7

.4 nd 5.9 1.9 7.9

.5 23.5 11.5 7.2 17.0

.5 33.1 23.7 nd 32.9

.6 40.6 6.4 79.6 7.1

.0 nd 46.8 nd 20.5

Table 5Number of adherent spores of the B. cereus group for three trials, expressed in logcfu/coupon, and class levels for strains studied (p-value of 0.05).

Tukey grouping⁎ Mean Strain

A 5.1967 B. cereus 98/4A B 4.9100 B. cereus D31A B C 4.7667 B. cereus D6A B C 4.6367 B. cereus 433A B C 4.3700 B. anthracis 9131

B C 4.2500 B. thuringiensis 7138B C 4.1767 B. thuringiensis 407B C 4.1500 B. cereus D1

C 3.9600 B. cereus LM9C D 3.9167 B. cereus ATCC 14579

D 3.0100 B. cereus CIP 5832

⁎ Groups with common letters are not significantly different; group A is the mostadherent whereas group D is the less adherent.

Table 3Exosporial saccharidic composition of spores of Bacillus species distant from theB. cereus group.

% Bc 14579 Ba 91314 Bt 4071 Bt 71381 B sporo MB581 Bl 6933

2,4-Me-Rha 15.3 nd 11.4 4.2 nd 0.32-Me-Rha 11.7 Nd 13.4 5.2 nd nd3-Me-Rha 9.4 13.6 12.5 6.7 1.4 ndRha 32.8 39.1 23.5 11.8 38.8 0.7GlcNH2 9.4 15.8 7.0 50.1 59.8 46.0GalNH2 21.4 28.7 32.1 22.0 nd 52.9

Superscripted numbers: saccharidic groups.

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hydrophobic character was supposed to be linked to the presence of anexosporium (Koshikawa et al., 1989), but our results indicated thatB. licheniformis and B. pumilus spores, though devoid of a wholly-structured exosporium,were alsomoderately hydrophobic. Concerningthe spore electric charge, no clear correlationwas observed between theelectronegativity and the Bacillus species, except that the lowest valueswere observed on spores of strains distant from the B. cereus group:B. pumilus, B. sporothermodurans, and B. subtilis. Data obtained in thisstudy are in the range (Ahimou et al., 2001), or lower (Ankolekar andLabbe, 2010) than results previously reported.

At a molecular level, BclA proteins are highly conserved (from 70.1%to 99.3%) for 17 sequenced genomes of B. cereus, B. weihenstephanensis,B. cytotoxis, B. anthracis, and B. thuringiensis (Lequette, personalcommunication). Conversely, the saccharidic composition of BclAdiffered greatly among Bacillus species (Fox et al., 2003). In agreementwith previous works (Fox et al., 2003; Garcia-Patrone and Tandecarz,1995), carbohydrate analysis of Bacillus exosporium, essentially BclA,confirmed the presence of 3-Me-Rha on every strain of B. anthracis, B.cereus, and B. thuringiensis. GalNH2, Rha, 2-Me-Rha and a newlyidentified rhamnose derivative, the 2,4-Me-Rha were also identified inmost of the strains belonging to theB. cereusgroup. Only B. anthracis andthree B. cereus strains (D6, LM9, and 433) exhibited different saccharidicprofiles, with the absence of at least one of the three rhamnosederivatives, leading to the identification of four groups based on theirsaccharidic composition. It must also be kept in mind that themethodology used in this study did not permit the detection ofanthrose, the sugar specific to B. anthracis. Therefore, it is notpossible to draw any conclusion from our results as to the presence orabsence of this N-acylated-dideoxy-glucose derivative in B. cereus andB. thuringiensis spores. Nevertheless, the differences noted in this studysuggest the presence of different glycosylation patterns within B. cereus

Table 4Number of adherent spores (the 17 strains were taken into account) for three trials,expressed in log cfu/coupon, and class levels for strains studied (p-value of 0.05).

Tukey grouping⁎ Mean Strain

A 5.1967 B. cereus 98/4A B 4.9100 B. cereus D31A B C 4.7667 B. cereus D6A B C 4.6367 B. cereus 433A B C 4.3700 B. anthracis 9131A B C D 4.2500 B. thuringiensis 7138A B C D 4.2433 B. pumilus 98/6A B C D 4.1767 B. thuringiensis 407

B C D 4.1500 B. cereus D1B C D E 4.0233 B. cereus D17B C D E 3.9600 B. cereus LM9B C D E 3.9167 B. cereus ATCC 14579

C D E 3.8567 B. licheniformis 6933C D E 3.7933 B. sporothermodurans MB581

D E 3.3300 B. subtilis 7135E F 3.0100 B. cereus CIP 5832

F 2.2333 B. subtilis 98/7

⁎ Groups with common letters are not significantly different; group A is the mostadherent whereas group F is the less adherent.

strains. We also checked that these monosaccharidic residues werespecifically associated to exosporia. Conversely, muramic acid residues,not found in exosporium, were identified in the soluble fractionobtained after passage of spores through a French press. Althoughpreviously identified in whole spores (Wunschel et al., 1994), the exactorigin of these monosaccharides, traditionally associated with cortexpeptidoglycan, is still unknown. Our results strongly suggest that thisresidue was located in the parasporal space. Large amounts ofsaccharidic residues were also detected in the insoluble fractions ofspores of B. licheniformis and B. sporothermodurans spores, suggestingthe presence of an outermost layer, more or less observable by MET butquite different from the classical exosporium of the B. cereus group(absence of an hair-like nap, specific saccharidic profiles). In contrast tothese fractions, the thick layer observed on B. subtilis 98/7 spores washighly soluble, loosely attached to their surfaces and rich in rhamnoseand quinovose. The differential analysis of insoluble and solublefractions of material associated with the exosporium, allowed discrim-ination between the Rha containing exosporium glycoproteins inB. cereus group and the Rha/Qui containing soluble polysaccharide inB. subtilis. The presence of quinovose in B. subtilis spores has alreadybeen documented (Wunschel et al., 1994), but its origin was not clearlyidentified by the authors.

In order to determine the relationship between the presence of anexosporium and adhesion properties, spores were analyzed for theirability to adhere to stainless steel coupons in static conditions.Surprisingly, the presence of a wholly-structured exosporium, typicalof the B. cereus group, only weakly accounted for the spore's ability toadhere onto stainless steel coupons. The wide diversity of the sporesurface properties among B. cereus strains and/or of the similaritiesobserved between some spore properties of B. cereus and otherspecies might explain the slight involvement of exosporium. We theninvestigated if spore adhesion could be related to any other sporeproperties. Two sets of statistical analyses were performed, takinginto account: i) all the Bacillus strains tested, regardless of the species,ii) only strains with a wholly-structured, non-injured exosporium(stains of the B. cereus group, except B. cereus D17).

First of all, hydrophobicity was shown to play a major role in sporeadhesion, but only when spores of all species (hydrophilic tohydrophobic)were taken into account. Conversely, differences betweenthehydrophobic spores of theB. cereus groupare not substantial enoughto explain differences in the adhesion process. These results confirmprevious ones: if the main role of hydrophobicity in spore adhesion isnowadays generally admitted (Faille et al., 2002; Rönner et al., 1990;Wiencek et al., 1991), the few results restricted to B. cereus strains havebeen less than (Hüsmark and Rönner, 1992) or even not conclusive(Tauveron et al., 2006). Surface electrical charge, however, was slightlyrelated to spore adhesion, as previously shown (Flint et al., 2000;Hüsmark and Rönner, 1990), but only when the strains of the B. cereusgroup were taken into account.

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The ability of spores to adherewas also significantly affected by thepresence of appendages for spores of the B. cereus group, but only to aslight extent when all species are considered. The presence ofappendages would promote spore adhesion by facilitating sporepenetration of the potential barrier, as hypothesized by the DLVOtheory (van Loosdrecht et al., 1989). However, at this time, apart fromprevious results from our laboratory (Tauveron et al., 2006), their rolein adhesion has not been clearly ascertained (Klavenes et al., 2002;Stalheim and Granum, 2001), evenwithin the B. cereus group. The sizeof exosporium was also determinant in the adhesion process, inagreement with previous results on adhesion and resistance tocleaning of B. cereus spores (Tauveron et al., 2006). The lack ofrelevance of the spore volume could be the result of the dispersion inspore size within batches, or of this parameter playing a moredominant role in dynamic conditions (Sharma et al., 1992). Lastly,although logically suspected of being involved in the adhesionphenomenon (Brahmbhatt et al., 2007), we failed to demonstrateany involvement of the hair-like nap in spore adhesion, in theexperimental conditions used throughout this study. For example,despite great similarities in the BclA of B. cereus 14579 and 98/4,which share 94.3% of their properties and have identical length andsaccharidic composition, spores of B. cereus 98/4 are much moreadherent than those of B. cereus 14579.

In conclusion, we showed that B. cereus group is less homogeneousthan expected, in terms of exosporium properties such as exosporiumsize, hair-like nap length and saccharidic composition (e.g. inthe presence of a newly identified rhamnose derivative: the 2,4-diMe-Rha). At the same time, spores of some more distant speciesshare with B. cereus some of the properties supposed to affectspore adhesion, such as appendages, hydrophobicity or zetapotential. A thin external layer was also sometimes observed (e.g.on B. sporothermodurans and B. pumilus spores) whose nature(exosporium?) is still to be determined, while a thick saccharidic-type layer (mainly composed of rhamnose and quinovose) was clearlyobserved around B. subtilis 98/7 spores. The ability of spores to adhereto stainless steel was not clearly affected by the presence of anexosporium per se, but rather by the spore's hydrophobicity andnumber of appendages. Within the B. cereus group, whose spores aresurrounded by a wholly-structured exosporium, the differences in thespore's ability to adhere were essentially related to the differences inthe number of appendages, the exosporium length and to a lesserextent, the zeta potential.

Acknowledgements

This work has been financed by the «Agence Nationale de laRecherche» under the «Programme National de Recherche enAlimentation et Nutrition Humaine» project «ANR-07-PNRA-009,InterSpore». We are grateful to Etienne Dewailly, from INSERM U800, and Gilles Ronse, from INRA UR638 for technical assistance. Wealso thank ARCELOR-MITTAL (Isbergues, France) for providingstainless steel coupons.

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