APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1990, p. 2600-2605
Vol. 56, No. 9
0099-2240/90/092600-06$02.00/0 Copyright C 1990, American Society for Microbiology
Hydrophobicity of Bacillus and Clostridium Sporest K. MARK WIENCEK,1 N. ARLENE KLAPES,2 AND P. M. FOEGEDINGl.2* Departments of Microbiology1 and Food Science,2 Box 7624, North Carolina State University, Raleigh, North Carolina 27695-7624 Received 30 January 1990/Accepted 10 June 1990
The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces. were
13), salt aggregation (36), and contact angle measurements (27). Because of the reported variability in the results obtained from these methods, several authors have suggested that more than one method should be used to study hydrophobicity (18, 29, 33). In this study we used two different methods, bacterial adherence to hexadecane (BATH) and HIC, to determine the relative hydrophobicities of spores of several Bacillus and Clostridium species and to evaluate the effects of heat and sporulation medium on spore hydrophobicity. (This work was presented in part at the 89th Annual Meeting of the American Society for Microbiology, New Orleans, La., 14 to 18 May 1989.)
The role of hydrophobic interactions in the adhesion of bacteria to the surfaces of inert materials has been addressed in recent studies (2, 22, 33). Both substratum hydrophobicity and bacterial cell surface hydrophobicity, as well as the related parameters surface tension and surface free energy, mediate a nonspecific, reversible interaction which can lead to permanent adhesion. Effective colonization by the bacteria can condition the surface, allowing the attachment of other organisms and the production of a complex biofilm (23, 25). Bacterial adhesion is a beneficial phenomenon in fixedfilm bioreactors, from which products are harvested without the removal of bacteria. Conversely, adhesion has been implicated as a possible virulence factor for several pathogenic microorganisms that are important in the medical, pharmaceutical, and food industries. Hydrophobic interactions have been associated with the adhesion of bacteria to surfaces in oral cavities (11, 34), contact lenses (26), surgical and dental biomaterials (12, 28), polymers targeted for food and pharmaceutical contact (39), and food (5). Although extensive data on vegetative cell hydrophobicity and adhesion exist (2, 33, 38), relatively few studies have thoroughly examined the surface hydrophobicity of bacterial spores or the adhesion of spores to inanimate substrata (4, 6, 9, 19, 31, 36). Bacillus and Clostridium spores are often implicated in food spoilage and food-borne illnesses. Because of their relative resistance to chemical and physical sterilization agents, spores of these bacteria are used as indicators of sterilization efficiency for treatments involving moist and dry heat, UV irradiation, and hydrogen peroxide (14). Understanding the surface properties of bacterial spores and their interactions with inanimate substrata is important for selection of packaging materials and for evaluation of the surface sterilization procedures used in the packaging of food, pharmaceutical agents, and medical supplies. The hydrophobicity of bacterial spores can be determined by applying methodologies used for measuring vegetative cell hydrophobicity. Established techniques for measuring surface hydrophobicity include adherence to hydrocarbons (1, 32), hydrophobic interaction chromatography (HIC) (6,
MATERIALS AND METHODS Bacterial strains and cultivation. The strains of Bacillus and Clostridium species used in this study are shown in Table 1, which also includes details concerning culture sources and sporulation conditions. Vegetative cell cultures of Bacillus spp. were grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.). Clostridium sporogenes ATCC 7955 and Clostridium putrefaciens ATCC 25786 were cultured in reinforced clostridial medium (Difco Laboratories, Detroit, Mich.). Clostridium botulinum 213B was grown in fluid thioglycolate medium (BBL). Overnight cultures of vegetative cells grown at the temperatures shown in Table 1 were used to inoculate sporulation media. When maximum sporulation had occurred (at the times indicated in Table 1), spores were harvested, washed four to six times by centrifugation (4,000 x g, 20 min, 4°C), and suspended in distilled water as described by Johnson et al. (15). Several of the spore preparations, including all Clostridium spp. preparations, required further cleaning until the concentration of intact vegetative cells was less than 5%. To remove vegetative cell debris, spore preparations in cold distilled water were sonicated with a probe sonicator (Branson S-110 Sonifier; Heat Systems-Ultrasonics, Inc., Plainview, N.Y.), centrifuged through 55% sucrose, and washed three times in cold distilled water. BATH assay. The relative hydrophobicities of bacterial spores and vegetative cells were measured by using the BATH assay of Rosenberg (32). This method involves partitioning a spore or vegetative cell suspension between an aqueous phase and the aqueous-hydrocarbon interface based on the degree of bacterial surface hydrophobicity. Spore
* Corresponding author. t Paper number 12497 of the Journal Series of North Carolina Agricultural Research Service, Raleigh, NC 27695-7643.
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TABLE 1. Sources of cultures and sporulation conditions Sporulation conditions Strain Strain
B. subtilis ATCC 6633 (crop I) B. subtilis ATCC 6633 (crop II)
B. B. B. B. B. B. B. B. B. B. B. C. C. C.
subtilis ATCC 6633 (crop III) subtilis ATCC 6633 (crop IV) subtilis ATCC 9372 (B. globigii)b subtilis ATCC 19221 subtilis A cereus T stearothermophilus ATCC 7953 coagulans ATCC 8038 coagulans FRR B666C megaterium ATCC 12872 megaterium ATCC 33729 botulinum 213Bcd sporogenes ATCC 7955 putrefaciens ATCC 25786
Medium
~~~~~~~~~~~~~~~~~~~~~~~~ sourcea
Temp
Time
Composition
Reference
(OC)
(days)
Fortified nutrient agar Fortified nutrient broth (fortified nutrient agar without agar) Glucose-salts Modified fortified nutrient agar Fortified nutrient agar Fortified nutrient agar Modified fortified nutrient agar Fortified nutrient agar Supplemented nutrient agar Brain heart infusion agar Soya peptone agar Tryptone yeast agar Tryptone yeast agar Trypticase peptone medium Cooked meat mediume Cooked meat medium
15
32 35
5 5
ATCC ATCC
10 7
35 35 35 32 30 30 55 43 50 32 32 30 37 25
3 4 4 2 3 2 5 3 3 3 3 6 3 7
ATCC ATCC AMSCO NCBS Lindsay NCSU NCSU ATCC Pflug ATCC ATCC Swift K-V ATCC
3 19 16 19 35
' ATCC, American Type Culture Collection, Rockville, Md.; AMSCO, American Sterilizer Co., Apex, N.C.; Lindsay, J. Lindsay, University of Florida, Gainesville; NCBS, North Carolina Biological Supply Co., Burlington, N.C.; NCSU, North Carolina State University, Raleigh; Pflug, I. J. Pflug, University of Minnesota, Minneapolis; Swift, Swift and Co. Research Center, Oakbrook, Ill.; K-V, KabiVitrum, Clayton, N.C. b This strain was identified as B. globigii by the American Sterilizer Co. Strain designation used by the culture source. d Spores of this strain were prepared by P.M.F. in 1981 by using previously described methods (35) and were stored for 8 years in distilled water at 4'C. e Obtained from BBL Microbiology Systems.
suspensions or cell suspensions (overnight cell cultures washed twice in 100 mM NaPO4 buffer, pH 6.8) at an A440 of 0.8 to 1.0 were incubated for 15 min in a 35°C water bath. Spores were suspended in distilled water, and vegetative cells were suspended in 100 mM NaPO4 buffer (pH 6.8). Then 0.1, 0.2, 0.6, or 1.0 ml of hexadecane (Fisher Scientific Co., Dallas, Tex.) was added to 3.0 ml of each spore or cell suspension. The mixture was vortexed (Vortex Genie 2; Fisher Scientific Co.; setting 5) for 1 min in round-bottom test tubes (15 by 100 mm), and the hexadecane and aqueous phases were allowed to partition for 15 min. The aqueous phase was carefully removed with a Pasteur pipette. The A440 of the aqueous suspension was measured with a model UV-260 spectrophotometer (Shimadzu Corp., Kyoto, Japan). The aqueous phase was monitored by phase-contrast microscopy for clumping or lysis of cells or spores that was due to hexadecane. The percent decrease in A440 for the aqueous suspension was calculated as follows: 100(AO Af)/AO, where Ao and Af were the initial absorbance and final absorbance, respectively. For each trial, the index of hydrophobicity was determined as the average percent decrease in absorbance for the four hexadecane volumes given above. The hydrophobicities for the spores and vegetative cells of each organism are reported below as the means + standard deviations for duplicate trials. Standard curves relating A440 and spore concentration (phase-contrast direct microscopic counts) were prepared for each strain of spores. These standard curves were used to convert absorbance values to spore concentrations to calculate the percentage of the spore population which was hydrophobic. HIC. Duplicate columns of Sepharose CL-4B (Sigma Chemical Co., St. Louis, Mo.) were prepared by using large-volume Pasteur pipettes (Fisher Scientific Co.) plugged with glass wool and packed to a height of 25 mm (1.7 ml) as described by Ismaeel et al. (13). Each column was washed several times with 4 M NaCl in 20 mM NaPO4 buffer (pH
6.85). The high ionic strength of the buffer was selected to mask the electrostatic repulsion between the spores and the charged groups of the Sepharose CL-4B, thus allowing hydrophobic interactions to occur. Spores were centrifuged twice and suspended in NaCl buffer at the desired concentration (A440, 0.3 to 0.6). Then 5 ml of the spore suspension, which was incubated at 35'C for 15 min, was passed through the column. The A440 of the eluent was measured. The percentage of adherence of spores to the hydrophobic gel was calculated as described above for the BATH assay by using averages from duplicate trials; these values are reported below as percent of hydrophobicity. The percent decreases in spore concentration were derived from standard curves of absorbance versus direct microscopic counts. Effects of heat on spore hydrophobicity. Concentrated spore suspensions (ca. 109 spores per ml) of Bacillus subtilis A, B. subtilis ATCC 9372 (Bacillus globigii), and Bacillus stearothermophilus ATCC 7953 in distilled water were heated in glass tubes at 75, 85, or 100°C for 10 min. After heating, the spores were rapidly cooled in an ice water bath and refrigerated overnight. The hydrophobicities of the heat-treated spores were measured by using the BATH assay described above and 0.1 ml of hexadecane; the values obtained were compared with the values obtained for the unheated controls. RESULTS Determination of spore and vegetative cell hydrophobicity. Figure 1 shows the percent hydrophobicity for spores and vegetative cells of each Bacillus and Clostridium strain at each hexadecane volume tested. The resulting curves show that spore hydrophobicity varied among species and strains. However, for each organism, the hydrophobicity of spores was greater than the vegetative cell hydrophobicity at each of the four hexadecane volumes. Most vegetative cell and spore populations exhibited constant hydrophobicity at each
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WIENCEK ET AL.
APPL. ENVIRON. MICROBIOL.
20
o
100
__
__
_
LL
-o 00
60-
4020 0 0.0
0.2
0.4
0.6
0.8
1.0 0.0
0.2
0.4
0.6
0.8
1.0
Hexadecane (ml) FIG. 1. Hydrophobicities of spores (closed symbols) and vegetative cells (open symbols) of Bacillus and Clostridium spp. as measured by the BATH assay. Error bars indicate the standard deviations from duplicate trials. The percent hydrophobicity was the percent decrease in the A440 of the aqueous phase that was due to partitioning with hexadecane. (A) Symbols: 1 and *, B. subtilis ATCC 6633 (crop I); A and A, B. megaterium ATCC 12872; 0 and 0, B. megaterium ATCC 33729. (B) Symbols: O and *, B. cereus T; A and A, B. stearothermophilus ATCC 7953; 0 and 0, B. subtilis A. (C) Symbols: 0 and *, B. subtilis ATCC 19221; A and A, B. coagulans ATCC 8038; 0 and 0, B. coagulans FRR B666; V and V, B. subtilis ATCC 9372 (B. globigii). (D) Symbols: 0 and *, C. botulinum 213B; A and A, C. sporogenes ATCC 7955; 0 and 0, C. putrefaciens ATCC 25786.
of the four hexadecane volumes. However, several vegetative cell and spore populations had increased hydrophobicities at the higher volumes of hexadecane. The spores of B. stearothermophilus ATCC 7953, Bacillus megaterium ATCC 33729, Bacillus coagulans ATCC 8038, B. coagulans FRR B666, and C. sporogenes ATCC 7955 increased at least 40% in hydrophobicity as the hexadecane volume was increased from 0.1 to 1.0 ml (Fig. 1). The mean hydrophobicity of each vegetative cell and spore population, as determined by the BATH method, is shown in Table 2. HIC with Sepharose CL-4B was also used to measure the hydrophobicities of bacterial spores. Although phenyl-Sepharose or octyl-Sepharose is commonly used as the gel matrix for HIC, the results of preliminary studies in which we used phenyl-Sepharose as the gel matrix did not allow us to differentiate among populations of spores with relatively high hydrophobicities (data not shown). The percent hydrophobicity as determined by HIC are shown in Table 2. In most cases, the spore hydrophobicity measured by HIC was somewhat lower than the hydrophobicity determined by adherence to hexadecane. However, the two methods generally produced similar results, especially with very hydrophobic spores. Effects of heat and sporulation conditions on bacterial spore hydrophobicity. The hydrophobicities of Bacillus spores were increased considerably by 10-min heat treatments in
distilled water (Fig. 2). Spores did not germinate during the heat treatment or the BATH assay as determined by phasecontrast microscopy. Each of the three strains tested showed increased hydrophobicities at higher temperatures. No apparent effect of sporulation medium composition or sporulation temperature on spore hydrophobicity was observed (Tables 1 and 2). For example, the relative hydrophobicities of four B. subtilis ATCC 6633 populations, each sporulated in different types of media at different temperatures and for different lengths of time (Table 1), differed by less than 2% when they were measured by the BATH method and by less than 10% when the HIC assay was used. DISCUSSION The hydrophobicities of spores of both Bacillus and Clostridium spp. were considerably greater than the hydrophobicities of the vegetative cells, as determined by the BATH assay. Several studies in which workers used adherence to hydrocarbons to examine bacterial spore hydrophobicity have produced similar observations. Koshikawa et al. (19) used the BATH method to measure the spore hydrophobicities of several Bacillus spp. and reported that the spores of B. megaterium QMB1551 were 80% more hydrophobic than the vegetative cells. The spores of Clostridium perfringens NCTC 8679 were found to be 82% hydrophobic, as measured
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TABLE 2. Hydrophobicities of spores and vegetative cells of Bacillus and Clostridium spp. Spores %
Hydrophobicity of
Strain BATH assay HIC Strain cells BAHasyHCvegetative (BATH assay)a % Hydro% Decrease % Hydro% Decrease
phobicitya B. B. B. B. B. B. B. B. B. B. B. B. B.
subtilis ATCC 6633 (crop I) subtilis ATCC 6633 (crop II) subtilis ATCC 6633 (cropII1) subtilis ATCC 6633 (crop IV) subtilis ATCC 19221 subtilis ATCC 9372 (B. globigii) subtilis A cereus T coagulans ATCC 8038 coagulans FRR B666 stearothermophilus ATCC 7953 megaterium ATCC 12872 megaterium ATCC 33729
C. botulinum 213B C. sporogenes ATCC 7955 C. putrefaciens ATCC 25786
in concnb
phobicityc
in concnb
(0.0) (0.0) (1.1) (0.2) (2.4) (3.2) (0.4) (1.1) (0.7) (2.5) (0.0) (1.8)
95 96 96 93 97 53 23 95 56 71 57 90 33
89 (1.5) 93 (0.0) 83 (5.1) 88 (0.8) 91(4.0) 20 (3.2) 23 (1.8) 98 (1.2) 29 (2.0) 50 (0.4) 41 (2.4) 25 (2.5) 41 (1.5)
90 94 84 89 92 23 27 98 36 57 46 26 45
7 (1.6) 8 (2.3) 5 (2.1) NDe 5 (4.6) 3 (1.4) 10 (5.2) 3 (1.3) 6 (5.1) 17 (3.5) 1 (0.3) 20 (0.7) 9 (9.9)
50 (0.2) 67 (1.1) 79 (2.6)
56 75 86
35 (1.0) 41(4.0) 52 (4.4)
41 50 60
11(3.4) 24 (1.0) 22 (4.6)
94 95 95 92 95 47 19 95 49 65 53 88 30
(0.3)d
Percent hydrophobicity is the average percent decrease in the A440 of the aqueous phases after partitioning with 0.1, 0.2, 0.6, and 1.0 ml of hexadecane. Average percent decrease in the spore concentration of the aqueous phase that was due to hexadecane partitioning (BATH assay) or retention in the Sepharose column (HIC). c Percent hydrophobicity is the average percent decrease in the A4,0 of the spore suspensions eluded from duplicate Sepharose columns. d The numbers in parentheses are standard deviations (n = 2). e ND, Not determined. a
b
by percentage of adherence to 0.1 ml of toluene (4). Foegeding and Fulp (8) reported a hydrophobic index of 67% for spores of Bacillus cereus T when the BATH assay was used. Doyle et al. (6) tested several Bacillus species by using the BATH method with 0.6 ml of hexadecane and found variable spore hydrophobicities, ranging from 13.2% for B. subtilis 168 to 63.8% for B. cereus T. All vegetative cells of the
m 0
80
-
60
-
40
-
A
0L
a00
I O'
I 0"_
S 20 -
U
UNHEATED CONTROL
80
90
1 00
TEMPERATURE (*C) FIG. 2. Effects of heat treatments on the hydrophobicities of Bacillus spores. Spore suspensions were heated for 10 min, cooled rapidly in an ice bath, and refrigerated overnight. Hydrophobicity was assayed with the BATH method by using 0.1 ml of hexadecane, as previously described. Error bars indicate the standard deviations from duplicate trials. Symbols: *, B. subtilis A; A, B. subtilis ATCC B. stearothermophilus ATCC 7953. The 9372 (B. globigii); percent hydrophobicity was the percent decrease in the A4,0 of the aqueous phase that was due to partitioning with hexadecane. 0,
Bacillus species tested were less than 6.0% adherent to hexadecane (6). Our observations indicate that the vegetative cells of Bacillus and Clostridium spp. are generally not hydrophobic when they are measured by the BATH assay. However, several Bacillus and Clostridium vegetative cell populations exhibited elevated hydrophobicities at the higher volumes of hexadecane (Fig. 1). HIC yielded results which indicated that there was increased spore hydrophobicity when spores were suspended in 4 M NaCl buffer. Without 4 M NaCl in the buffer, the spores were not retained in the Sepharose gel columns (data not shown), indicating that high ionic strength was necessary to overcome the electrostatic repulsion between the spores and Sepharose. In the only previous study in which HIC was used to measure bacterial spore hydrophobicity, Doyle et al. (6) measured the hydrophobicities of two Bacillus species by using octyl-Sepharose columns in 0.15 M NaCl and found that both species were more than 67% adherent to the columns. There was a good correlation between the BATH and HIC methods when they were used to measure bacterial spore hydrophobicities. While the absolute hydrophobicities for a specific test organism did not agree well between the two methods in several cases, the ranking of hydrophobic indices for species and strains of spores was consistent between methods. One notable exception was the spore population of B. megaterium ATCC 12872, which exhibited much greater affinity for the hexadecane in the BATH assays than for the Sepharose gel used in HIC. It has been suggested that the increased hydrophobicity of bacterial spores is due to the relative abundance of protein in the outer coats and exosporium compared with peptidoglycan on gram-positive vegetative cell surfaces (6, 24, 37). An association between spore hydrophobicity and the presence of an exosporium has been reported recently for several Bacillus species (18). Kutima and Foegeding (20) noted a
2604
WIENCEK ET AL.
decrease in the hydrophobicity of spores of B. cereus T when spore coats were removed by chemical treatments. Takubo et al. (36) and Koshikawa et al. (19) reported decreased hydrophobicities in outer-coat-negative mutants of B. megaterium QMB1551 (= ATCC 12872). The decreased adherence to hexadecane of an outer-coat-negative spore mutant, B. megaterium ATCC 33729, compared with wild-type strain B. megaterium ATCC 12872 is shown in Table 2, suggesting that the outer coats or the exosporium plays a role in spore hydrophobicity. We found evidence that heat applied to dormant spores can raise their surface hydrophobicities. Doyle et al. (6) reported considerable increases in spore hydrophobicity for two Bacillus species after 15-min treatments in water at 100°C, although the effects were not as pronounced as those in our study. Craven and Blankenship (4) noted an increase in the relative hydrophobicities of spores of C. perfringens NCTC 8679 after treatment at 75°C for 20 min, but showed that the effects could be negated by washing the spores after the heat treatment. Increases in the hydrophobicity of spores because of heat treatment may result from the disruption of outer coat or exosporium proteins (6). Li-Chan et al. (21) reported that increased temperatures can alter the structure of macromolecules, causing an unfolding of proteins and exposing internal hydrophobic moieties. Doyle et al. (6) have suggested that hydrophobic interactions are important in the attachment of spores to environmental proteins. Increased spore hydrophobicity because of heat activation might increase affinity of the spores for lipids or proteins, thus providing a nutritional source for outgrowth of the vegetative cells following germination of the spores. Therefore, the net effect of heat to increase spore hydrophobicity may represent a mechanism for increasing the chance for survival following germination of the spores. Several studies have shown that the hydrophobic property of vegetative cells is dependent upon conditions such as growth medium and culture age (1, 30) and that sporulation conditions can affect spore properties such as heat and chemical resistance, structure, and germination (8, 10, 17). However, Koshikawa et al. (19) have reported recently that sporulation medium does not affect Bacillus spore hydrophobicity. Data presented here also indicate that sporulation medium and sporulation temperature do not appear to affect Bacillus spore hydrophobicity. The physical methods (e.g., differential centrifugation, sonication) used in this study to produce spore crops which were free of vegetative cell material are recommended to minimize alteration of inherent spore hydrophobicity (data not shown). Thermal treatment is not recommended for the removal of vegetative cells from spore preparations which are to be used in hydrophobicity assays because of observed increases in spore hydrophobicity after sublethal heat treatment. In addition, the use of enzyme treatments, such as lysozyme or trypsin treatments, to clean spore preparations is not recommended because of reported effects on spore hydrophobicity (6). Thus, the spores of Bacillus and Clostridium spp. are relatively more hydrophobic than the vegetative cells. This suggests that hydrophobic interactions may play a role in the adhesion of spores to solid substrata. The increase in spore hydrophobicity because of exposure of spores to heat may play an important role in the increased adhesion of bacterial spores to materials following a sublethal thermal process. Further studies are needed to expand the base of knowledge in the area of bacterial spore hydrophobicity. Understanding the role of hydrophobic interactions between bacterial
APPL. ENVIRON. MICROBIOL.
spores and substrata in the adhesion of spores to surfaces is critical in the development of more efficient methods of surface sanitation or sterilization of equipment or packaging materials used in the medical, pharmaceutical, and food industries. ACKNOWLEDGMENTS This research was supported in part by the Center for Aseptic Processing and Packaging Studies and by North Carolina Agricultural Research Service project 2152. LITERATURE CITED 1. Beck, G., E. Puchelle, C. Plotkowski, and R. Peslin. 1988. Effect of growth on surface charge and hydrophobicity of Staphylococcus aureus. Ann. Inst. Pasteur/Microbiol. (Paris) 139:655664. 2. Busscher, H. J., and A. H. Weerkamp. 1987. Specific and non-specific interactions in bacterial adhesion to solid substrata. FEMS Microbiol. Rev. 46:165-173. 3. Cook, A. M., and M. R. W. Brown. 1964. The relation between heat activation and colony formation for the spores of Bacillus stearothermophilus. J. Pharm. Pharmacol. 16:725-732. 4. Craven, S. E., and L. C. Blankenship. 1987. Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat. Can. J. Microbiol. 33:773-776. 5. Dickson, J. S., and M. Koohmaraie. 1989. Cell surface charge characteristics and their relationship to bacterial attachment to meat surfaces. Appl. Environ. Microbiol. 55:832-836. 6. Doyle, R. J., F. Nedjat-Haiem, and J. S. Singh. 1984. Hydrophobic characteristics of Bacillus spores. Curr. Microbiol. 10: 329-332. 7. Edwards, J. L., Jr., F. F. Busta, and M. L. Speck. 1965. Thermal inactivation characteristics of Bacillus subtilis spores at ultrahigh temperatures. Appl. Microbiol. 13:851-857. 8. Foegeding, P. M., and M. L. Fulp. 1988. Comparison of coats and surface-dependent properties of Bacillus cereus T prepared in two sporulation environments. J. Appl. Bacteriol. 65:249259. 9. Ghione, M., D. Parrello, and C. Granucci. 1989. Adherence of bacterial spores to encrusted fabrics. J. Appl. Bacteriol. 67:371376. 10. Hodges, N. A., J. Melling, and S. J. Parker. 1980. A comparison of chemically defined and complex media for the production of Bacillus subtilis spores having reproducible resistance and germination characteristics. J. Pharm. Pharmacol. 32:126-130. 11. Hogg, S. D., and J. E. Manning. 1987. The hydrophobicity of "viridans" streptococci isolated from the human mouth. J. Appl. Bacteriol. 63:311-318. 12. Hogt, A. H., J. Dankert, J. A. De Vries, and J. Feien. 1983. Adhesion of coagulase-negative staphylococci to biomaterials. J. Gen. Microbiol. 129:2959-2968. 13. Ismaeel, N., J. R. Furr, W. J. Pugh, and A. D. Russell. 1987. Hydrophobic properties of Providencia stuartii and other Gramnegative bacteria measured by hydrophobic interaction chromatography. Lett. Appl. Microbiol. 5:91-95. 14. Ito, K. I., and K. E. Stevenson. 1984. Sterilization of packaging materials using aseptic systems. Food Technol. 38:60-62. 15. Johnson, K. M., C. L. Nelson, and F. F. Busta. 1982. Germination and heat resistance of Bacillus cereus spores from strains associated with diarrheal and emetic food-borne illnesses. J. Food Sci. 47:1268-1271. 16. Jones, A. T., and I. J. Pflug. 1981. Bacillus coagulans, FRR B666, as a potential biological indicator organism. J. Parenter. Sci. Technol. 35:82-87. 17. Khoury, P. H., S. J. Lombardi, and R. A. Slepecky. 1987. Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol. Curr. Microbiol. 15:15-19. 18. Kjelleberg, S. 1984. Adhesion to inanimate surfaces, p. 51-70. In K. C. Marshall (ed.), Microbial adhesion and aggregation. Springer-Verlag KG, Berlin. 19. Koshikawa, T., M. Yamazaki, M. Yoshimi, S. Ogawa, A. Yamada, K. Watabe, and M. Torii. 1989. Surface hydrophobicity
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39.
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growth rate on the adhesion of the oral bacteria Streptococcus mutans and Streptococcus milleri. Arch. Oral Biol. 29:147-150. Rosenberg, E., D. R. Brown, and A. L. Demain. 1985. The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores. Arch. Microbiol. 142:51-54. Rosenberg, M. 1984. Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. FEMS Microbiol. Lett. 22:289-295. Rosenberg, M., and S. Kjelleberg. 1986. Hydrophobic interactions: role in bacterial adhesion. Adv. Microbiol. Ecol. 9:353393. Rosenberg, M., E. Rosenberg, H. Judes, and E. Weiss. 1983. Bacterial adherence to hydrocarbons and to surfaces in the oral cavity. FEMS Microbiol. Lett. 20:1-5. Schmidt, C. F., and W. K. Nank. 1959. Radiation sterilization of food. I. Procedures for the evaluation of the radiation resistance of spores of Clostridium botulinum in food products. Food Res. 25:321-327. Takubo, Y., M. Atarashi, T. Nishihara, and M. Kondo. 1988. Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium. Microbiol. Immunol. 9:973979. Takumi, K., T. Kinouchi, and T. Kawata. 1979. Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A. Microbiol. Immunol. 28:443-454. Van Loosdrecht, M. C. M., J. Lyklema, W. Norde, G. Schraa, and A. J. B. Zehnder. 1987. The role of bacterial cell wall hydrophobicity in adhesion. Appl. Environ. Microbiol. 53:18931897. Van Pelt, A. W. J., A. H. Weerkamp, M. H. W. J. C. Uyen, H. J. Busscher, H. P. de Jong, and J. Arends. 1985. Adhesion of Streptococcus sanguis CH3 to polymers with different surface free energies. Appl. Environ. Microbiol. 49:1270-1275.
Vol. 56, No. 9
0099-2240/90/092600-06$02.00/0 Copyright C 1990, American Society for Microbiology
Hydrophobicity of Bacillus and Clostridium Sporest K. MARK WIENCEK,1 N. ARLENE KLAPES,2 AND P. M. FOEGEDINGl.2* Departments of Microbiology1 and Food Science,2 Box 7624, North Carolina State University, Raleigh, North Carolina 27695-7624 Received 30 January 1990/Accepted 10 June 1990
The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces. were
13), salt aggregation (36), and contact angle measurements (27). Because of the reported variability in the results obtained from these methods, several authors have suggested that more than one method should be used to study hydrophobicity (18, 29, 33). In this study we used two different methods, bacterial adherence to hexadecane (BATH) and HIC, to determine the relative hydrophobicities of spores of several Bacillus and Clostridium species and to evaluate the effects of heat and sporulation medium on spore hydrophobicity. (This work was presented in part at the 89th Annual Meeting of the American Society for Microbiology, New Orleans, La., 14 to 18 May 1989.)
The role of hydrophobic interactions in the adhesion of bacteria to the surfaces of inert materials has been addressed in recent studies (2, 22, 33). Both substratum hydrophobicity and bacterial cell surface hydrophobicity, as well as the related parameters surface tension and surface free energy, mediate a nonspecific, reversible interaction which can lead to permanent adhesion. Effective colonization by the bacteria can condition the surface, allowing the attachment of other organisms and the production of a complex biofilm (23, 25). Bacterial adhesion is a beneficial phenomenon in fixedfilm bioreactors, from which products are harvested without the removal of bacteria. Conversely, adhesion has been implicated as a possible virulence factor for several pathogenic microorganisms that are important in the medical, pharmaceutical, and food industries. Hydrophobic interactions have been associated with the adhesion of bacteria to surfaces in oral cavities (11, 34), contact lenses (26), surgical and dental biomaterials (12, 28), polymers targeted for food and pharmaceutical contact (39), and food (5). Although extensive data on vegetative cell hydrophobicity and adhesion exist (2, 33, 38), relatively few studies have thoroughly examined the surface hydrophobicity of bacterial spores or the adhesion of spores to inanimate substrata (4, 6, 9, 19, 31, 36). Bacillus and Clostridium spores are often implicated in food spoilage and food-borne illnesses. Because of their relative resistance to chemical and physical sterilization agents, spores of these bacteria are used as indicators of sterilization efficiency for treatments involving moist and dry heat, UV irradiation, and hydrogen peroxide (14). Understanding the surface properties of bacterial spores and their interactions with inanimate substrata is important for selection of packaging materials and for evaluation of the surface sterilization procedures used in the packaging of food, pharmaceutical agents, and medical supplies. The hydrophobicity of bacterial spores can be determined by applying methodologies used for measuring vegetative cell hydrophobicity. Established techniques for measuring surface hydrophobicity include adherence to hydrocarbons (1, 32), hydrophobic interaction chromatography (HIC) (6,
MATERIALS AND METHODS Bacterial strains and cultivation. The strains of Bacillus and Clostridium species used in this study are shown in Table 1, which also includes details concerning culture sources and sporulation conditions. Vegetative cell cultures of Bacillus spp. were grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.). Clostridium sporogenes ATCC 7955 and Clostridium putrefaciens ATCC 25786 were cultured in reinforced clostridial medium (Difco Laboratories, Detroit, Mich.). Clostridium botulinum 213B was grown in fluid thioglycolate medium (BBL). Overnight cultures of vegetative cells grown at the temperatures shown in Table 1 were used to inoculate sporulation media. When maximum sporulation had occurred (at the times indicated in Table 1), spores were harvested, washed four to six times by centrifugation (4,000 x g, 20 min, 4°C), and suspended in distilled water as described by Johnson et al. (15). Several of the spore preparations, including all Clostridium spp. preparations, required further cleaning until the concentration of intact vegetative cells was less than 5%. To remove vegetative cell debris, spore preparations in cold distilled water were sonicated with a probe sonicator (Branson S-110 Sonifier; Heat Systems-Ultrasonics, Inc., Plainview, N.Y.), centrifuged through 55% sucrose, and washed three times in cold distilled water. BATH assay. The relative hydrophobicities of bacterial spores and vegetative cells were measured by using the BATH assay of Rosenberg (32). This method involves partitioning a spore or vegetative cell suspension between an aqueous phase and the aqueous-hydrocarbon interface based on the degree of bacterial surface hydrophobicity. Spore
* Corresponding author. t Paper number 12497 of the Journal Series of North Carolina Agricultural Research Service, Raleigh, NC 27695-7643.
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TABLE 1. Sources of cultures and sporulation conditions Sporulation conditions Strain Strain
B. subtilis ATCC 6633 (crop I) B. subtilis ATCC 6633 (crop II)
B. B. B. B. B. B. B. B. B. B. B. C. C. C.
subtilis ATCC 6633 (crop III) subtilis ATCC 6633 (crop IV) subtilis ATCC 9372 (B. globigii)b subtilis ATCC 19221 subtilis A cereus T stearothermophilus ATCC 7953 coagulans ATCC 8038 coagulans FRR B666C megaterium ATCC 12872 megaterium ATCC 33729 botulinum 213Bcd sporogenes ATCC 7955 putrefaciens ATCC 25786
Medium
~~~~~~~~~~~~~~~~~~~~~~~~ sourcea
Temp
Time
Composition
Reference
(OC)
(days)
Fortified nutrient agar Fortified nutrient broth (fortified nutrient agar without agar) Glucose-salts Modified fortified nutrient agar Fortified nutrient agar Fortified nutrient agar Modified fortified nutrient agar Fortified nutrient agar Supplemented nutrient agar Brain heart infusion agar Soya peptone agar Tryptone yeast agar Tryptone yeast agar Trypticase peptone medium Cooked meat mediume Cooked meat medium
15
32 35
5 5
ATCC ATCC
10 7
35 35 35 32 30 30 55 43 50 32 32 30 37 25
3 4 4 2 3 2 5 3 3 3 3 6 3 7
ATCC ATCC AMSCO NCBS Lindsay NCSU NCSU ATCC Pflug ATCC ATCC Swift K-V ATCC
3 19 16 19 35
' ATCC, American Type Culture Collection, Rockville, Md.; AMSCO, American Sterilizer Co., Apex, N.C.; Lindsay, J. Lindsay, University of Florida, Gainesville; NCBS, North Carolina Biological Supply Co., Burlington, N.C.; NCSU, North Carolina State University, Raleigh; Pflug, I. J. Pflug, University of Minnesota, Minneapolis; Swift, Swift and Co. Research Center, Oakbrook, Ill.; K-V, KabiVitrum, Clayton, N.C. b This strain was identified as B. globigii by the American Sterilizer Co. Strain designation used by the culture source. d Spores of this strain were prepared by P.M.F. in 1981 by using previously described methods (35) and were stored for 8 years in distilled water at 4'C. e Obtained from BBL Microbiology Systems.
suspensions or cell suspensions (overnight cell cultures washed twice in 100 mM NaPO4 buffer, pH 6.8) at an A440 of 0.8 to 1.0 were incubated for 15 min in a 35°C water bath. Spores were suspended in distilled water, and vegetative cells were suspended in 100 mM NaPO4 buffer (pH 6.8). Then 0.1, 0.2, 0.6, or 1.0 ml of hexadecane (Fisher Scientific Co., Dallas, Tex.) was added to 3.0 ml of each spore or cell suspension. The mixture was vortexed (Vortex Genie 2; Fisher Scientific Co.; setting 5) for 1 min in round-bottom test tubes (15 by 100 mm), and the hexadecane and aqueous phases were allowed to partition for 15 min. The aqueous phase was carefully removed with a Pasteur pipette. The A440 of the aqueous suspension was measured with a model UV-260 spectrophotometer (Shimadzu Corp., Kyoto, Japan). The aqueous phase was monitored by phase-contrast microscopy for clumping or lysis of cells or spores that was due to hexadecane. The percent decrease in A440 for the aqueous suspension was calculated as follows: 100(AO Af)/AO, where Ao and Af were the initial absorbance and final absorbance, respectively. For each trial, the index of hydrophobicity was determined as the average percent decrease in absorbance for the four hexadecane volumes given above. The hydrophobicities for the spores and vegetative cells of each organism are reported below as the means + standard deviations for duplicate trials. Standard curves relating A440 and spore concentration (phase-contrast direct microscopic counts) were prepared for each strain of spores. These standard curves were used to convert absorbance values to spore concentrations to calculate the percentage of the spore population which was hydrophobic. HIC. Duplicate columns of Sepharose CL-4B (Sigma Chemical Co., St. Louis, Mo.) were prepared by using large-volume Pasteur pipettes (Fisher Scientific Co.) plugged with glass wool and packed to a height of 25 mm (1.7 ml) as described by Ismaeel et al. (13). Each column was washed several times with 4 M NaCl in 20 mM NaPO4 buffer (pH
6.85). The high ionic strength of the buffer was selected to mask the electrostatic repulsion between the spores and the charged groups of the Sepharose CL-4B, thus allowing hydrophobic interactions to occur. Spores were centrifuged twice and suspended in NaCl buffer at the desired concentration (A440, 0.3 to 0.6). Then 5 ml of the spore suspension, which was incubated at 35'C for 15 min, was passed through the column. The A440 of the eluent was measured. The percentage of adherence of spores to the hydrophobic gel was calculated as described above for the BATH assay by using averages from duplicate trials; these values are reported below as percent of hydrophobicity. The percent decreases in spore concentration were derived from standard curves of absorbance versus direct microscopic counts. Effects of heat on spore hydrophobicity. Concentrated spore suspensions (ca. 109 spores per ml) of Bacillus subtilis A, B. subtilis ATCC 9372 (Bacillus globigii), and Bacillus stearothermophilus ATCC 7953 in distilled water were heated in glass tubes at 75, 85, or 100°C for 10 min. After heating, the spores were rapidly cooled in an ice water bath and refrigerated overnight. The hydrophobicities of the heat-treated spores were measured by using the BATH assay described above and 0.1 ml of hexadecane; the values obtained were compared with the values obtained for the unheated controls. RESULTS Determination of spore and vegetative cell hydrophobicity. Figure 1 shows the percent hydrophobicity for spores and vegetative cells of each Bacillus and Clostridium strain at each hexadecane volume tested. The resulting curves show that spore hydrophobicity varied among species and strains. However, for each organism, the hydrophobicity of spores was greater than the vegetative cell hydrophobicity at each of the four hexadecane volumes. Most vegetative cell and spore populations exhibited constant hydrophobicity at each
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WIENCEK ET AL.
APPL. ENVIRON. MICROBIOL.
20
o
100
__
__
_
LL
-o 00
60-
4020 0 0.0
0.2
0.4
0.6
0.8
1.0 0.0
0.2
0.4
0.6
0.8
1.0
Hexadecane (ml) FIG. 1. Hydrophobicities of spores (closed symbols) and vegetative cells (open symbols) of Bacillus and Clostridium spp. as measured by the BATH assay. Error bars indicate the standard deviations from duplicate trials. The percent hydrophobicity was the percent decrease in the A440 of the aqueous phase that was due to partitioning with hexadecane. (A) Symbols: 1 and *, B. subtilis ATCC 6633 (crop I); A and A, B. megaterium ATCC 12872; 0 and 0, B. megaterium ATCC 33729. (B) Symbols: O and *, B. cereus T; A and A, B. stearothermophilus ATCC 7953; 0 and 0, B. subtilis A. (C) Symbols: 0 and *, B. subtilis ATCC 19221; A and A, B. coagulans ATCC 8038; 0 and 0, B. coagulans FRR B666; V and V, B. subtilis ATCC 9372 (B. globigii). (D) Symbols: 0 and *, C. botulinum 213B; A and A, C. sporogenes ATCC 7955; 0 and 0, C. putrefaciens ATCC 25786.
of the four hexadecane volumes. However, several vegetative cell and spore populations had increased hydrophobicities at the higher volumes of hexadecane. The spores of B. stearothermophilus ATCC 7953, Bacillus megaterium ATCC 33729, Bacillus coagulans ATCC 8038, B. coagulans FRR B666, and C. sporogenes ATCC 7955 increased at least 40% in hydrophobicity as the hexadecane volume was increased from 0.1 to 1.0 ml (Fig. 1). The mean hydrophobicity of each vegetative cell and spore population, as determined by the BATH method, is shown in Table 2. HIC with Sepharose CL-4B was also used to measure the hydrophobicities of bacterial spores. Although phenyl-Sepharose or octyl-Sepharose is commonly used as the gel matrix for HIC, the results of preliminary studies in which we used phenyl-Sepharose as the gel matrix did not allow us to differentiate among populations of spores with relatively high hydrophobicities (data not shown). The percent hydrophobicity as determined by HIC are shown in Table 2. In most cases, the spore hydrophobicity measured by HIC was somewhat lower than the hydrophobicity determined by adherence to hexadecane. However, the two methods generally produced similar results, especially with very hydrophobic spores. Effects of heat and sporulation conditions on bacterial spore hydrophobicity. The hydrophobicities of Bacillus spores were increased considerably by 10-min heat treatments in
distilled water (Fig. 2). Spores did not germinate during the heat treatment or the BATH assay as determined by phasecontrast microscopy. Each of the three strains tested showed increased hydrophobicities at higher temperatures. No apparent effect of sporulation medium composition or sporulation temperature on spore hydrophobicity was observed (Tables 1 and 2). For example, the relative hydrophobicities of four B. subtilis ATCC 6633 populations, each sporulated in different types of media at different temperatures and for different lengths of time (Table 1), differed by less than 2% when they were measured by the BATH method and by less than 10% when the HIC assay was used. DISCUSSION The hydrophobicities of spores of both Bacillus and Clostridium spp. were considerably greater than the hydrophobicities of the vegetative cells, as determined by the BATH assay. Several studies in which workers used adherence to hydrocarbons to examine bacterial spore hydrophobicity have produced similar observations. Koshikawa et al. (19) used the BATH method to measure the spore hydrophobicities of several Bacillus spp. and reported that the spores of B. megaterium QMB1551 were 80% more hydrophobic than the vegetative cells. The spores of Clostridium perfringens NCTC 8679 were found to be 82% hydrophobic, as measured
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TABLE 2. Hydrophobicities of spores and vegetative cells of Bacillus and Clostridium spp. Spores %
Hydrophobicity of
Strain BATH assay HIC Strain cells BAHasyHCvegetative (BATH assay)a % Hydro% Decrease % Hydro% Decrease
phobicitya B. B. B. B. B. B. B. B. B. B. B. B. B.
subtilis ATCC 6633 (crop I) subtilis ATCC 6633 (crop II) subtilis ATCC 6633 (cropII1) subtilis ATCC 6633 (crop IV) subtilis ATCC 19221 subtilis ATCC 9372 (B. globigii) subtilis A cereus T coagulans ATCC 8038 coagulans FRR B666 stearothermophilus ATCC 7953 megaterium ATCC 12872 megaterium ATCC 33729
C. botulinum 213B C. sporogenes ATCC 7955 C. putrefaciens ATCC 25786
in concnb
phobicityc
in concnb
(0.0) (0.0) (1.1) (0.2) (2.4) (3.2) (0.4) (1.1) (0.7) (2.5) (0.0) (1.8)
95 96 96 93 97 53 23 95 56 71 57 90 33
89 (1.5) 93 (0.0) 83 (5.1) 88 (0.8) 91(4.0) 20 (3.2) 23 (1.8) 98 (1.2) 29 (2.0) 50 (0.4) 41 (2.4) 25 (2.5) 41 (1.5)
90 94 84 89 92 23 27 98 36 57 46 26 45
7 (1.6) 8 (2.3) 5 (2.1) NDe 5 (4.6) 3 (1.4) 10 (5.2) 3 (1.3) 6 (5.1) 17 (3.5) 1 (0.3) 20 (0.7) 9 (9.9)
50 (0.2) 67 (1.1) 79 (2.6)
56 75 86
35 (1.0) 41(4.0) 52 (4.4)
41 50 60
11(3.4) 24 (1.0) 22 (4.6)
94 95 95 92 95 47 19 95 49 65 53 88 30
(0.3)d
Percent hydrophobicity is the average percent decrease in the A440 of the aqueous phases after partitioning with 0.1, 0.2, 0.6, and 1.0 ml of hexadecane. Average percent decrease in the spore concentration of the aqueous phase that was due to hexadecane partitioning (BATH assay) or retention in the Sepharose column (HIC). c Percent hydrophobicity is the average percent decrease in the A4,0 of the spore suspensions eluded from duplicate Sepharose columns. d The numbers in parentheses are standard deviations (n = 2). e ND, Not determined. a
b
by percentage of adherence to 0.1 ml of toluene (4). Foegeding and Fulp (8) reported a hydrophobic index of 67% for spores of Bacillus cereus T when the BATH assay was used. Doyle et al. (6) tested several Bacillus species by using the BATH method with 0.6 ml of hexadecane and found variable spore hydrophobicities, ranging from 13.2% for B. subtilis 168 to 63.8% for B. cereus T. All vegetative cells of the
m 0
80
-
60
-
40
-
A
0L
a00
I O'
I 0"_
S 20 -
U
UNHEATED CONTROL
80
90
1 00
TEMPERATURE (*C) FIG. 2. Effects of heat treatments on the hydrophobicities of Bacillus spores. Spore suspensions were heated for 10 min, cooled rapidly in an ice bath, and refrigerated overnight. Hydrophobicity was assayed with the BATH method by using 0.1 ml of hexadecane, as previously described. Error bars indicate the standard deviations from duplicate trials. Symbols: *, B. subtilis A; A, B. subtilis ATCC B. stearothermophilus ATCC 7953. The 9372 (B. globigii); percent hydrophobicity was the percent decrease in the A4,0 of the aqueous phase that was due to partitioning with hexadecane. 0,
Bacillus species tested were less than 6.0% adherent to hexadecane (6). Our observations indicate that the vegetative cells of Bacillus and Clostridium spp. are generally not hydrophobic when they are measured by the BATH assay. However, several Bacillus and Clostridium vegetative cell populations exhibited elevated hydrophobicities at the higher volumes of hexadecane (Fig. 1). HIC yielded results which indicated that there was increased spore hydrophobicity when spores were suspended in 4 M NaCl buffer. Without 4 M NaCl in the buffer, the spores were not retained in the Sepharose gel columns (data not shown), indicating that high ionic strength was necessary to overcome the electrostatic repulsion between the spores and Sepharose. In the only previous study in which HIC was used to measure bacterial spore hydrophobicity, Doyle et al. (6) measured the hydrophobicities of two Bacillus species by using octyl-Sepharose columns in 0.15 M NaCl and found that both species were more than 67% adherent to the columns. There was a good correlation between the BATH and HIC methods when they were used to measure bacterial spore hydrophobicities. While the absolute hydrophobicities for a specific test organism did not agree well between the two methods in several cases, the ranking of hydrophobic indices for species and strains of spores was consistent between methods. One notable exception was the spore population of B. megaterium ATCC 12872, which exhibited much greater affinity for the hexadecane in the BATH assays than for the Sepharose gel used in HIC. It has been suggested that the increased hydrophobicity of bacterial spores is due to the relative abundance of protein in the outer coats and exosporium compared with peptidoglycan on gram-positive vegetative cell surfaces (6, 24, 37). An association between spore hydrophobicity and the presence of an exosporium has been reported recently for several Bacillus species (18). Kutima and Foegeding (20) noted a
2604
WIENCEK ET AL.
decrease in the hydrophobicity of spores of B. cereus T when spore coats were removed by chemical treatments. Takubo et al. (36) and Koshikawa et al. (19) reported decreased hydrophobicities in outer-coat-negative mutants of B. megaterium QMB1551 (= ATCC 12872). The decreased adherence to hexadecane of an outer-coat-negative spore mutant, B. megaterium ATCC 33729, compared with wild-type strain B. megaterium ATCC 12872 is shown in Table 2, suggesting that the outer coats or the exosporium plays a role in spore hydrophobicity. We found evidence that heat applied to dormant spores can raise their surface hydrophobicities. Doyle et al. (6) reported considerable increases in spore hydrophobicity for two Bacillus species after 15-min treatments in water at 100°C, although the effects were not as pronounced as those in our study. Craven and Blankenship (4) noted an increase in the relative hydrophobicities of spores of C. perfringens NCTC 8679 after treatment at 75°C for 20 min, but showed that the effects could be negated by washing the spores after the heat treatment. Increases in the hydrophobicity of spores because of heat treatment may result from the disruption of outer coat or exosporium proteins (6). Li-Chan et al. (21) reported that increased temperatures can alter the structure of macromolecules, causing an unfolding of proteins and exposing internal hydrophobic moieties. Doyle et al. (6) have suggested that hydrophobic interactions are important in the attachment of spores to environmental proteins. Increased spore hydrophobicity because of heat activation might increase affinity of the spores for lipids or proteins, thus providing a nutritional source for outgrowth of the vegetative cells following germination of the spores. Therefore, the net effect of heat to increase spore hydrophobicity may represent a mechanism for increasing the chance for survival following germination of the spores. Several studies have shown that the hydrophobic property of vegetative cells is dependent upon conditions such as growth medium and culture age (1, 30) and that sporulation conditions can affect spore properties such as heat and chemical resistance, structure, and germination (8, 10, 17). However, Koshikawa et al. (19) have reported recently that sporulation medium does not affect Bacillus spore hydrophobicity. Data presented here also indicate that sporulation medium and sporulation temperature do not appear to affect Bacillus spore hydrophobicity. The physical methods (e.g., differential centrifugation, sonication) used in this study to produce spore crops which were free of vegetative cell material are recommended to minimize alteration of inherent spore hydrophobicity (data not shown). Thermal treatment is not recommended for the removal of vegetative cells from spore preparations which are to be used in hydrophobicity assays because of observed increases in spore hydrophobicity after sublethal heat treatment. In addition, the use of enzyme treatments, such as lysozyme or trypsin treatments, to clean spore preparations is not recommended because of reported effects on spore hydrophobicity (6). Thus, the spores of Bacillus and Clostridium spp. are relatively more hydrophobic than the vegetative cells. This suggests that hydrophobic interactions may play a role in the adhesion of spores to solid substrata. The increase in spore hydrophobicity because of exposure of spores to heat may play an important role in the increased adhesion of bacterial spores to materials following a sublethal thermal process. Further studies are needed to expand the base of knowledge in the area of bacterial spore hydrophobicity. Understanding the role of hydrophobic interactions between bacterial
APPL. ENVIRON. MICROBIOL.
spores and substrata in the adhesion of spores to surfaces is critical in the development of more efficient methods of surface sanitation or sterilization of equipment or packaging materials used in the medical, pharmaceutical, and food industries. ACKNOWLEDGMENTS This research was supported in part by the Center for Aseptic Processing and Packaging Studies and by North Carolina Agricultural Research Service project 2152. LITERATURE CITED 1. Beck, G., E. Puchelle, C. Plotkowski, and R. Peslin. 1988. Effect of growth on surface charge and hydrophobicity of Staphylococcus aureus. Ann. Inst. Pasteur/Microbiol. (Paris) 139:655664. 2. Busscher, H. J., and A. H. Weerkamp. 1987. Specific and non-specific interactions in bacterial adhesion to solid substrata. FEMS Microbiol. Rev. 46:165-173. 3. Cook, A. M., and M. R. W. Brown. 1964. The relation between heat activation and colony formation for the spores of Bacillus stearothermophilus. J. Pharm. Pharmacol. 16:725-732. 4. Craven, S. E., and L. C. Blankenship. 1987. Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat. Can. J. Microbiol. 33:773-776. 5. Dickson, J. S., and M. Koohmaraie. 1989. Cell surface charge characteristics and their relationship to bacterial attachment to meat surfaces. Appl. Environ. Microbiol. 55:832-836. 6. Doyle, R. J., F. Nedjat-Haiem, and J. S. Singh. 1984. Hydrophobic characteristics of Bacillus spores. Curr. Microbiol. 10: 329-332. 7. Edwards, J. L., Jr., F. F. Busta, and M. L. Speck. 1965. Thermal inactivation characteristics of Bacillus subtilis spores at ultrahigh temperatures. Appl. Microbiol. 13:851-857. 8. Foegeding, P. M., and M. L. Fulp. 1988. Comparison of coats and surface-dependent properties of Bacillus cereus T prepared in two sporulation environments. J. Appl. Bacteriol. 65:249259. 9. Ghione, M., D. Parrello, and C. Granucci. 1989. Adherence of bacterial spores to encrusted fabrics. J. Appl. Bacteriol. 67:371376. 10. Hodges, N. A., J. Melling, and S. J. Parker. 1980. A comparison of chemically defined and complex media for the production of Bacillus subtilis spores having reproducible resistance and germination characteristics. J. Pharm. Pharmacol. 32:126-130. 11. Hogg, S. D., and J. E. Manning. 1987. The hydrophobicity of "viridans" streptococci isolated from the human mouth. J. Appl. Bacteriol. 63:311-318. 12. Hogt, A. H., J. Dankert, J. A. De Vries, and J. Feien. 1983. Adhesion of coagulase-negative staphylococci to biomaterials. J. Gen. Microbiol. 129:2959-2968. 13. Ismaeel, N., J. R. Furr, W. J. Pugh, and A. D. Russell. 1987. Hydrophobic properties of Providencia stuartii and other Gramnegative bacteria measured by hydrophobic interaction chromatography. Lett. Appl. Microbiol. 5:91-95. 14. Ito, K. I., and K. E. Stevenson. 1984. Sterilization of packaging materials using aseptic systems. Food Technol. 38:60-62. 15. Johnson, K. M., C. L. Nelson, and F. F. Busta. 1982. Germination and heat resistance of Bacillus cereus spores from strains associated with diarrheal and emetic food-borne illnesses. J. Food Sci. 47:1268-1271. 16. Jones, A. T., and I. J. Pflug. 1981. Bacillus coagulans, FRR B666, as a potential biological indicator organism. J. Parenter. Sci. Technol. 35:82-87. 17. Khoury, P. H., S. J. Lombardi, and R. A. Slepecky. 1987. Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol. Curr. Microbiol. 15:15-19. 18. Kjelleberg, S. 1984. Adhesion to inanimate surfaces, p. 51-70. In K. C. Marshall (ed.), Microbial adhesion and aggregation. Springer-Verlag KG, Berlin. 19. Koshikawa, T., M. Yamazaki, M. Yoshimi, S. Ogawa, A. Yamada, K. Watabe, and M. Torii. 1989. Surface hydrophobicity
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