<I>Bacillus Cereus</I>

2346

Journal of Food Protection, Vol. 70, No. 10, 2007, Pages 2346–2353 Copyright ᮊ, International Association for Food Protection

Occurrence of Bacillus cereus Spores with a Damaged Exosporium: Consequences on the Spore Adhesion on Surfaces of Food Processing Lines

C. FAILLE,1* G. TAUVERON,1 C. LE GENTIL-LELIE` VRE,1 AND C. SLOMIANNY2,3

1INRA-UR638, 369 rue Jules Guesde, BP 20039, F-59651 Villeneuve d’Ascq Cedex, France; 2INSERM-LPC, U-800, F-59655 Villeneuve d’Ascq Cedex, France; and 3Laboratoire de Physiologie Cellulaire, Universite´ des Sciences et Technologies de Lille 1,

F-59655 Villeneuve d’Ascq Cedex, France Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021

MS 07-195: Received 10 April 2004/Accepted 14 May 2007

ABSTRACT

This study was designed to evaluate the occurrence of Bacillus cereus spores with a damaged exosporium and the consequences of such damages on spore adhesion. The analysis of nine strains sporulated under optimal conditions (Spo8- agar, 30ЊC) revealed that damaged exosporia were systematically found in one strain (B. cereus D17) and occasionally in two others (B. cereus ATCC 14579T and B. cereus D6). The prevalence of spores with damaged exosporia increased when sporulation occurred under less favorable conditions (Spo8-broth or high temperature); for example, more than 50% of the B. cereus ATCC 14579T spores were damaged when sporulation occurred at 40ЊC on Spo8-agar or at 30ЊC in Spo8-broth. Furthermore, when subjected to shear stresses by circulation of spore suspensions through a peristaltic pump, the exosporium of a signiﬁcant amount of spores became partially or totally shorn off (for example, 40% of the B. cereus ATCC 14579T spores). The ability of damaged spores to adhere to inert surfaces and to resist cleaning under shear stress was signiﬁcantly affected when compared with intact spores, resulting in a decreased number of adhering spores (P Յ 0.004) and enhanced resistance to cleaning (P Յ 0.008). This study provides evidence that, under various conditions, the exosporium of B. cereus spores can be partly or wholly damaged, thereby affecting the ability of spores to contaminate the surfaces of food processing lines. The presence of spores devoid of exosporium will be of importance in determining the risk associated with B. cereus spores adherent to food processing line surfaces.

Bacillus cereus spores are widespread in environments ed results remain controversial, and for the time being, it such as soil, air, and work surfaces or in food and are would seem difficult to conclude that appendages would known to be responsible for foodborne illness. These spores either inhibit or promote adhesion (16, 26). Conversely, the are highly resistant to most of the treatments devised by exosporium’s length has recently been shown to affect both food producers to limit bacterial survival and growth during the resistance of adherent spores to a cleaning procedure food processing and storage: spores can survive pasteuri- and, to a lesser extent, the number of adherent spores (29). zation or disinfection procedures (6, 14, 21) and adhere Most of these previous studies have been carried out firmly to inert surfaces (12), and some psychrotrophic on spores produced under favorable conditions, far from strains can even germinate and grow under refrigerated con- the conditions probably encountered by bacteria in the real ditions. environment, such as high or low temperatures and desic- Spores from B. cereus strains are enclosed in a loose- cation. However, many studies have dealt with the influence fitting outermost integument, called the exosporium, and of growth conditions on their surface properties and adhe- surrounded by appendages. The remarkable ability of B. sion of various microorganisms (4, 10, 22) or have reported cereus spores to adhere to inert surfaces and to resist clean- on the influence of sporulation conditions on other spore ing in place (CIP) procedures may arise from the specific properties, such as thermal resistance (18, 20), density (17), surface properties of exosporia and the presence of ap- or coat and cortex composition (19). Conversely, no find- pendages. Many studies have reported that some spore sur- ings have as yet been reported on the effect of sporulation face properties have an influence on the adhesion ability of conditions on spore surface and adhesion. Moreover, it has spores. The hydrophobic character of spores from strains belonging to the B. cereus species has often been reported not been demonstrated that the exosporium is strong to play an important part in their ability to firmly adhere to enough to resist those shear or friction stresses encountered stainless steel and other inert surfaces (8, 12). The presence by some mature spores in food processing lines. Indeed, of appendages has also been suggested to have an important few studies have been carried out on the influence of shear role in spore adhesion (1, 29). However, previously report- on cells; these studies have mainly dealt with biotechnology or medicine, and no data have been reported on bacterial * Author for correspondence. Tel: ϩ33 (0)3-20-43-54-04; Fax: ϩ33 (0)3- cells. For example, the damage to microalgal cells (13) or 20-43-54-26; E-mail: [email protected]. to plant cells (15) due to shear inside a pump was found J. Food Prot., Vol. 70, No. 10 DAMAGE TO B. CEREUS EXOSPORIUM 2347 to be dependent on the pump technology and on the time ultramicrotome (Applied Biosystems, Foster City, Calif.) and col- of exposure. lected on 100- or 150-mesh grids. After double-staining with 2% The aim of the present study was to demonstrate the uranyl acetate in 50% ethanol and lead citrate (23), the sections occurrence of B. cereus spores with a damaged exosporium were observed under a Hitachi H600 electron microscope at 75 when spores were produced under various sporulation con- kV. ditions (e.g., temperature sporulation, liquid-agar medium) Mechanical damage to spore exosporia through a test rig. or subjected to shear stresses such as those encountered by To evaluate the consequence of shear stress on the integrity of the mature spores during food processing. We then inves- exosporia, aqueous suspensions of 106 spores per ml (B. cereus tigated the incidence of the lack of exosporium on the abil- CUETM 98/4, 5832, and ATCC 14579T) were circulated through ity of spores to adhere to stainless steel surfaces and to a peristaltic pump (704S, Watson-Marlowe Limited, Falmouth, Њ Ϫ1 resist a cleaning procedure. Cornwall, UK) at 20 C for 4 h at a flow rate of 300 liters h . The test rig consisted of a vessel containing the spore suspension, the pump, a surge tank to limit pulse flow rate variations, and MATERIALS AND METHODS Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021 pipes made of Marpren (Watson-Marlowe) and silicon. For trans- Bacillus strains and spore production. Nine strains be- mission electron microscopy observations and determination of longing to the B. cereus species were analyzed in this study: the the percentage of spores with lost or injured exosporium, samples type strain (ATCC 14579T), three strains isolated from the envi- were taken after 4 h of circulation, and spores were pelleted by ronment (748, 5832, and LM9), one strain isolated from a dairy centrifugation at 3,500 ϫ g for 30 min at 4ЊC. processing line (CUETM 98/4), and four strains from patients with diarrheic symptoms (D1, D6, D17, and D23). Spores from Production of batches of spores devoid of exosporium. the nine strains were produced at 30ЊC on Spo8-agar consisting Spores from B. cereus CUETM 98/4 and 5832 (15 ml of an aque- 9 of 8 g literϪ1 nutrient broth (Biokar Diagnostics, Beauvais, ous spore suspension of 10 spores per ml) were subjected to three Ϫ1 Ϫ1 successive passages through a French press at 20,000 psi (SLM France), 0.51 g liter MgSO4·7H2O, 0.97 g liter KCl, 0.2 g literϪ1 CaCl ·2H O, 3 ϫ 10Ϫ3 g literϪ1 MnCl ·4H O, 0.55 ϫ 10Ϫ3 Instruments, Urbana, Ill.). The detached exosporia and appendages 2 2 2 2 ϫ g literϪ1 FeSO ·7H O, and 1.5% agar. When over 95% of spores were separated from spores by two centrifugations (3,000 g, 4 2 Њ were obtained, they were harvested by scraping the surface, 30 min, 4 C). The pellet, containing 100% spores devoid of intact Њ washed five times in chilled water (by centrifugation at 1,200 ϫ exosporium, was resuspended in chilled water and stored at 4 C g for 15 min), and stored at 4ЊC until use. until use. Within spore batches passed through the French press, Spores from B. cereus CUETM 98/4, 5832, and ATCC many spores were still surrounded with fragments of exosporium, 14579T were also produced under other conditions. Sporulation suggesting that the underlying coat remained intact. was performed on the Spo8-agar at the following temperatures: Microbial affinity to hexadecane. A partitioning method 15, 20, 40, and 45ЊC, in the way described above. Spores from was used, based on the affinity of spores to an apolar solvent, the three strains were also produced in Spo8-broth (same com- hexadecane (Sigma, St. Louis, Mo.) (3), and slightly modified in position as Spo8-agar, except that no agar was added) at 30ЊC. our laboratory. Briefly, a saline spore suspension was adjusted to When sporulation occurred in liquid medium, cells were activated an absorbance of 0.5 to 0.6 at 600 nm (A0) in glass tubes (10 mm by two successive transfers to nutrient agar slants and incubated in diameter by 75 mm). Three-milliliter aliquots of the spore sus- for24hat30ЊC. A final transfer was carried out in 100 ml of pension and 500 ␮l of hexadecane were vortexed for times rang- Spo8-broth, and cells were incubated at 30ЊC under shaking (200 ing from 5 to 180 s and left to settle for 30 min to allow a com- rpm). When over 95% of spores were obtained, they were har- plete separation of the two phases. The absorbance of the aqueous vested by centrifugation at 1,200 ϫ g. Before each experiment, ϫ phase was measured (At). The log[(At/A0) 100] was plotted two further washes in water were carried out. All experiments against the vortexing time (in seconds), and the initial slope was were done on two independent spore batches. calculated. This measured the initial removal rate (R0) of spores from the aqueous suspension, which is related to the hydrophilic- Transmission electron microscopy. Whole spores were ad- hydrophobic spore character. A spore was considered hydrophobic sorbed on Formvar-coated grids and examined after negative when ϪR fell between 4.0 and 6.0 and was considered highly staining with 1% uranyl acetate on a Hitachi H600 electron mi- 0 hydrophobic for higher values. croscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV. The prevalence of damaged spores was estimated on at least Adhesion and resistance to a CIP procedure. Stainless 50 spores from at least two different batches, and several pictures steel (304L, bright annealed) coupons (15 by 45 mm) were soiled were taken. by vertical immersion for 4 h in an aqueous spore suspension (105 To observe the external nap composed of glycoproteins (27), spores per ml) and then rinsed in sterile water. To determine the a ruthenium red staining procedure previously described (25), and number of adhering spores, half of the fouled coupons were sub- slightly modified in our laboratory, was performed. Briefly, cells jected to an ultrasonication step in 10 ml of Tween 80, 2% (vol/ were fixed in a solution of 2.5% glutaraldehyde, 0.1 mol literϪ1 vol) (5 min; Ultrasonic bath, Deltasonic, Meaux, France). The cacodylate buffer, pH 7.0, and 500 ppm of ruthenium red. After detached spores following sonication were enumerated on nutrient a 15-min incubation at 4ЊC, spores were rinsed three times for 5 agar (1.3% [wt/vol] nutrient broth [Bio-Rad, France], 1.5% [wt/ min in 0.1 mol literϪ1 cacodylate buffer, pH 7.0, and centrifuged vol] bacteriological agar type E [Biokar]) after 48 h at 30ЊC. The at 1,200 ϫ g. The pellet was again suspended in 1 ml of the other soiled coupons were submitted to a CIP procedure, i.e., they postfixative solution (2% osmium tetroxide, 0.1 mol lϪ1 cacodyl- were placed in test pipes, which were inserted into a previously ate buffer, pH 7.0, and 500 ppm of ruthenium red). After a 15- described CIP rig (9), and the following mild CIP procedure was min incubation at 4ЊC, the cells were rinsed three times for 5 min carried out: (i) a 5-min rinse with softened water at a mean ve- in water and centrifuged at 1,200 ϫ g. After dehydration in graded locity of 0.5 m sϪ1; (ii) a 10-min cleaning with a Galor 7/32 acetonitrile, the pellets were embedded in Epon 812 (Merck, alkaline detergent (Penngar, France), 1% (vol/vol) at 50ЊCata Darmstadt, Germany). Thin sections were cut on a Reichert-Jung mean velocity of 1.0 m sϪ1; and (iii) a 5-min rinse in softened 2348 FAILLE ET AL. J. Food Prot., Vol. 70, No. 10 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021

FIGURE 1. Transmission electron microscopy of negatively stained B. cereus spores produced under optimal conditions (Spo8-agar at 30ЊC): B. cereus ATCC 14579T (a), B. cereus 5832 (b), B. cereus CUETM 98/4 (c), B. cereus 748 (d), B. cereus D6 (e), B. cereus D17 (f). Bars ϭ 1 ␮m. water at a mean velocity of 0.5 m sϪ1. The rig was dismantled, ditions known to promote a high rate of sporulation (Spo8- and the number of residual adhering spores was estimated as de- agar, 30ЊC), most of the spores from the nine strains tested scribed above. The results from two independent experiments exhibited intact exosporium (Fig. 1). B. cereus D17 was the (batches of spores) were analyzed. To evaluate the resistance of only strain to be systematically damaged, with around 80% spores to a CIP procedure, the percentage of residual spores after of its spores having either a malformed exosporium or none cleaning was then calculated. at all (see Figs. 1 through 4, particularly Figs. 1f and 4b). Statistical analysis. Data were analyzed by general linear Two more strains (B. cereus D6 and B. cereus ATCC model procedures by means of SAS V8.0 software (SAS Institute, 14579T) presented a low percentage of spores with a dam- Cary, N.C.). Variance analyses were performed to determine the aged exosporium (Ͻ20%) in some batches. inﬂuence of the removal of exosporium (intact versus damaged B. cereus 98/4 spores; intact versus damaged B. cereus 5832 spores) Role of sporulation conditions on the occurrence of on adhesion and resistance to CIP, taking into account the vari- damaged spores. Experiments were performed on the B. ability between replicates. This was followed by a multiple com- T parison procedure by Tukey’s test (alpha level ϭ 0.05). cereus ATCC 14579 (small exosporium and approximate- ly ﬁve appendages) and on two strains previously shown to RESULTS exhibit an intact exosporium when sporulated under optimal Occurrence of injured spores produced under op- conditions (Spo8-agar, 30ЊC): B. cereus 98/4, characterized timal conditions. When spores were produced under con- by a small exosporium with up to 30 appendages, and B. J. Food Prot., Vol. 70, No. 10 DAMAGE TO B. CEREUS EXOSPORIUM 2349 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021

FIGURE 2. Transmission electron microscopy of negatively stained B. cereus spores produced under various conditions: on Spo8-agar at 40ЊC (a, c, e) or in Spo8-broth at 30ЊC (b, d, f). B. cereus ATCC 14579T (a, b), B. cereus 5832 (c, d), B. cereus CUETM 98/4 (e, f). Bars ϭ 1 ␮m. cereus 5832, characterized by a large exosporium and very versely, only a few damages were observed on B. cereus few short appendages. The effect of sporulation temperature 98/4 spore exosporia when sporulation occurred at 40 or (ranging from 15 to 45ЊC) was first investigated. Sporula- 45ЊC. A concomitant decrease in the exosporium size was tion of B. cereus 98/4 was less sensitive to temperature than also clearly observed. For example, the length of the B. B. cereus 5832 and ATCC 14579T. Indeed, sporulation of cereus 5832 spores decreased from 3.5 ␮m(Ϯ0.5) at 30ЊC B. cereus 98/4 still occurred at a high rate at 15 and 45ЊC, (Fig. 1b) to 1.9 ␮m(Ϯ0.3) at 40ЊC (Fig. 2c). with around 80 and 90% mature spores, respectively. With The exosporium integrity of spores produced in broth B. cereus 5832, only 50% mature spores were obtained was then investigated. When suspended in Spo8-broth, the when sporulation occurred at 15ЊC, and no spores were ob- time for complete sporulation significantly increased (up to served at 45ЊC. Lastly, with B. cereus ATCC 14579T, spor- 9 days) when compared with sporulation on agar, and the ulation did not occur at these temperatures. Observation of sporulation yield of the three strains decreased from 95% spores by transmission electron microscopy revealed major to less than 90%. Sporulation in Spo8-broth led to the in- changes in the exosporium integrity when sporulation oc- jury of 53% of the B. cereus ATCC 14579T spores, 45% curred at high temperature (Ͼ30ЊC). At 40ЊC, for example of the B. cereus 98/4 spores, and only 26% of the B. cereus (Fig. 2), 19 and 54% of the B. cereus 5832 and ATCC 5832 spores (Fig. 2). Figure 4c shows that the damaged 14579T spores, respectively, were either surrounded with exosporia of B. cereus 98/4 produced in Spo8-broth did not fragments of exosporium or devoid of exosporium. Con- exhibit any specific morphological features, such as a thin- 2350 FAILLE ET AL. J. Food Prot., Vol. 70, No. 10

Consequence of the exosporium removal on the ability of spores to adhere. Spores produced on agar at 30ЊC were passed through a French press and subjected to an adhesion step and a CIP procedure. As shown on Table 1, despite great differences in the number of adherent spores between spore batches, the removal of exosporium induced for both strains a decrease in the number of spores adhering to stainless steel, and the decrease was more marked on the B. cereus 98/4 spores. Variance analysis indicated that 87% of the variability in the number of adherent B. cereus 98/4 spores was explained by the trial and the intact or damaged state of the exosporium (P values of 0.0348 and 0.004, respectively). A similar value (81%) was Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021 obtained for B. cereus 5832 spores (P values of 0.4731 and 0.0019, respectively). Differences were also observed in the percentage of residual spores after cleaning, with damaged spores from both strains being more resistant than intact spores. Variance analysis indicated that 80 and 94% of the variability in the percentage of residual spores after cleaning was explained by the trial and the intact or damaged state of the exosporium of B. cereus 98/4 and B. cereus 5832 spores, respectively. Contrary to what was observed on the number of adherent spores, the variability between trials was negligible (P Ͼ 0.05) when compared with the variability between intact and damaged spores (P ϭ 0.0081 and Ͻ0.0001 on B. cereus 98/4 and B. cereus 5832, respectively). The consequences of the exosporium removal on the spore surface were investigated. The release of exosporium induced a clear increase in spore hydrophobicity: the treated spores were strongly hydrophobic, with an initial removal rate of Ϫ11.6 and Ϫ9.5 sϪ1, for B. cereus 98/4 and B. cereus 5832, respectively, when compared with initial rates of around Ϫ7.0 and 6.0 sϪ1 for intact spores from the same strains. Where the morphology of the treated spores is con- cerned, the removal of exosporium resulted in a lack of appendages and an average length of 1.70 and 1.42 ␮m for FIGURE 3. Transmission electron microscopy of negatively B. cereus 98/4 and B. cereus 5832 spores, respectively. stained B. cereus spores produced under optimal conditions (Spo8-agar at 30ЊC) and subjected to shear stress: B. cereus DISCUSSION ATCC 14579T (a), B. cereus 5832 (b), B. cereus CUETM 98/4 The investigation of spore damage was ﬁrst performed (c). Bars ϭ 1 ␮m. on spores produced under favorable conditions known to promote a high sporulation rate (Spo8-agar at 30ЊC). Under these conditions, only a few strains exhibited some spores ner or an irregular crystalline layer, when compared with with a damaged exosporium. Such an injury could be the the exosporia of spores produced on agar at 30ЊC (Fig. 4a). result of exosporium fragility or of incomplete exosporium Sporulation in Spo8-broth also resulted in a marked de- formation. However, on thin sections of the B. cereus D17 crease in the exosporium length. B. cereus 5832 spore damaged spores observed by transmission electron micros- length, for example, was 2.2 Ϯ 0.7 ␮m when sporulated in copy, we did not observe any feature that could be related broth (Fig. 2d) and 3.5 Ϯ 0.5 ␮m when sporulated on agar to a possible fragility of the exosporium, such as a thinner (Fig. 1b). or an irregular crystalline layer (electron-dense layer at the Role of shear stresses on the integrity of exosporia. inner part of the exosporium). It could then be hypothesized The effect of shear stresses on the exosporium integrity of that such damage would be the result of a difference in the mature spores produced on Spo8-agar was studied. The exosporium’s chemical composition—for example, in the shear stress induced by a 4-h passage through a rig at 300 ExsFA and ExsFB proteins, which are essential for the sta- liters hϪ1 gave rise to only slight injury of B. cereus 98/4 bility of the basal crystalline layer (28), or in the ExsA (22% damaged spores), whereas batches of B. cereus ATCC protein, essential for the assembly of the exosporium of B. 14579T and 5832 spores were greatly affected with 40 and cereus spores (2). 51% of damaged spores, respectively (Fig. 3). It is important to note that in natural environments, J. Food Prot., Vol. 70, No. 10 DAMAGE TO B. CEREUS EXOSPORIUM 2351 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021

FIGURE 4. Transmission electron microscopy of thin sections of spores produced under various conditions: B. cereus CUETM 98/4 (a) and B. cereus D17 (b) produced on Spo8-agar at 30ЊC, B. cereus CUETM 98/4 produced in broth at 30ЊC (c) and B. cereus CUETM 98/4 produced on agar at 30ЊC and subjected to shear stress (d). Bars ϭ 1 ␮m. conditions encountered by bacteria during growth and spor- cereus sporulation on tryptic soy agar and in tryptic soy ulation could be far from our experimental conditions. As broth. Because sporulation has been reported to be triggered a consequence, the proportion of damaged spores might be by starvation (5), these results suggest that different factors, higher. such as the availability of oxygen or nutrients, are to the To test this hypothesis, the effect of changes in spor- advantage of sporulation on the surface of solid media. ulation conditions on the proportion of damaged spores was Moreover, sporulation in liquid medium was stressful for investigated. First, as previously noted by Ryu et al. (24), the exosporium: together with a decrease in their size, some the temperature range required for sporulation is narrower exosporium damages were observed on the three strains than for growth. When sporulation occurred at a tempera- tested. ture higher than the optimal sporulation temperature, the In natural environments, some conditions encountered exosporium was much smaller and sometimes damaged. by bacteria in spore form may also be responsible for ex- Such modifications in the exosporium size and integrity, osporium damage. No findings have so far been reported following changes in the sporulation temperature, have not in the literature as to the shear effect on the integrity of been previously shown in the literature. However, Melly et bacterial cells. Only Horikawa et al. (11) have reported that al. (19) have reported that an increase in sporulation tem- conditions of growth influence the sensitivity of osteoblas- perature induces various changes in the cortex composition tic cells to shear. In this study, we demonstrated a strain- (percentage of muramic acid with a cross-link) and mainly dependent effect of shear stresses, close to those encoun- in the coat composition (reduced intensity of some Cot pro- tered during food processing, on the exosporium integrity teins). It seems likely that similar modifications could occur of mature spores. These great differences observed in the in the exosporium composition, either as a consequence of exosporium susceptibility to shear stresses among strains differences in gene expression or in protein stability and suggest differences in the exosporium structure or chemical assembly. The sporulation in Spo8-broth was also less ef- composition. ficient than on Spo8-agar. Analogous observations have re- To summarize, many environmental conditions en- cently been reported by Ryu et al. (24), who compared B. countered by B. cereus during growth or sporulation or in 2352 FAILLE ET AL. J. Food Prot., Vol. 70, No. 10

TABLE 1. Number of adherent spores per coupon after the soil- exosporium would account for a lower resistance of spores ing step (NADH) and percentage of residual spores after CIP of to cleaning under dynamic conditions. two batches of spores, intact or passed through the French press In conclusion, for the first time, it clearly appears that ϭ a (damaged), and class levels for strains studied (P 0.05) under various conditions (e.g., sporulation conditions, shear B. cereus CUETM 98/4 B. cereus 5832 stresses), the exosporium of B. cereus spores can be partly or wholly damaged. Therefore, it can be assumed that dam- % residual % residual age to or loss of exosporium is a common feature in natural log(NADH) spores log(NADH) spores environments. Such damage will induce significant modi- Dam- Dam- Dam- Dam- fications in the adherent behavior of B. cereus spores (ad- Intact aged Intact aged Intact aged Intact aged hesion and resistance to cleaning). As a consequence, the presence of spores devoid of exosporium must be taken into Tukey’s grouping A B B A A B B A account when assessing the efficiency of cleaning procedures. Moreover, because of the prevalence of damaged Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2346/1677253/0362-028x-70_10_2346.pdf by guest on 24 September 2021 Batch 1 spores, the risk associated with B. cereus spores adherent Replicate 1 6.34 5.50 3.47 1.12 5.00 4.28 0.167 8.8 to surfaces of food processing lines might be greatly un- Replicate 2 6.31 5.77 1.76 8.73 5.17 4.75 0.181 7.1 derestimated. Further studies will be required to determine Replicate 3 6.84 5.48 0.79 5.25 5.14 4.81 0.387 5.6 the properties of spores devoid of exosporium and to in- Replicate 4 7.55 5.73 1.44 3.31 5.12 4.51 0.253 3.3 vestigate the structure and protein composition of ‘‘fragile’’ Batch 2 exosporia, e.g., after sporulation in broth. Replicate 5 5.83 5.78 2.42 10.49 4.93 4.82 6.05 10.42 Replicate 6 5.16 3.96 1.96 10.00 4.94 4.67 0.76 7.50 ACKNOWLEDGMENTS Replicate 7 5.71 5.48 1.82 6.72 4.93 4.53 0.86 7.33 This study has been partially financed by the ‘‘Agence Nationale de Replicate 8 5.78 4.91 7.55 11.74 4.87 4.76 1.09 10.00 la Recherche’’ under the ‘‘Programme National de Recherche en Alimen- tation et Nutrition Humaine,’’ project ‘‘ANR-05-PNRA-013, B. cereus,’’ a Following Tukey’s grouping, groups with common letters are not by a grant from the Re´gion Nord-Pas de Calais (France) and by FEDER significantly different; group A is the most adherent and most funds. S. Parent, A. Ronse, J. F. Migdal, J. Jacquemont, and A. Khiati are resistant to CIP, whereas group B is the least adherent and least thanked for their valuable assistance in the laboratory. We are also grateful resistant to CIP. to E. 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