APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1991, P. 1969-1973 0099-2240/91/071969-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 57, No. 7
Characterization of Physicochemical Forces Involved in Adhesion of Listeria monocytogenes to Surfacest AKIER ASSANTA MAFU,1 DENIS ROY,2* JACQUES GOULET,1
AND
LUC SAVOIE2
Department of Food Science and Technology, Universite Laval, Ste-Foy, Quebec, Canada GJK 7P4,1 and Agriculture Canada Food Research and Development Centre, 3600, Casavant Boulevard West, St.-Hyacinthe, Quebec, Canada J2S 8E32 Received 23 January 1991/Accepted 7 May 1991
This study investigated the physicochemical forces involving the adhesion of Listeria monocytogenes to surfaces. A total of 22 strains of L. monocytogenes were compared for relative surface hydrophobicity with the salt aggregation test. Cell surface charges and hydrophobicity of L. monocytogenes Scott A were also determined by electrophoretic mobility, hydrophobic-interaction chromatography, and contact angle measurements. Electrokinetic measurements indicated that the strain Scott A has a negative electrophoretic mobility. Physicochemical characterization of L. monocytogenes by various methods indicates that this microorganism is hydrophilic. All L. monocytogenes strains tested with the salt aggregation test method aggregated at very high ammonium sulfate molarities. The hydrophobicity-interaction chromatography results show that L. monocytogenes Scott A cells do not adhere to octyl-Sepharose unless the pH is low. Results from contact angle measurements showed that the surface free energy of strain Scott A was 65.9 mJ m-2, classifying this microorganism as a hydrophilic bacterium. In addition, the interfacial free energy of adhesion of L. monocytogenes Scott A estimated for polypropylene and rubber was lower than that for glass and stainless steel. However, these theoretical implications could not be correlated with the attachment capabilities of L. monocytogenes. In recent years, Listeria monocytogenes has become a source of concern for the food industry because of outbreaks of food-borne illness associated with consumption of food products (8, 9). The psychotrophic nature of L. monocytogenes (7, 24) along with its ability to come into contact with inert surfaces and become attached to them makes its presence in food processing plants a major problem (10, 14,
of L. monocytogenes Scott A were also determined by electrophoretic mobility, hydrophobic interaction chromatography (HIC), and contact angle measurements. Calculations of the free energies of adhesion (derived from surface free energies from total, LW, and SR contributions) of L. monocytogenes cells onto stainless steel, glass, polypropylene, and rubber were made.
15). The capability of L. monocytogenes to adhere to surfaces represents a source of potential contamination for a wide variety of material coming in contact with solid surfaces (4). However, the mechanisms governing the adhesion of L. monocytogenes to inert surfaces are not understood and have not been defined. Several studies have shown that the adhesion of bacteria partly depends upon the nature of the inert surfaces, since bacteria become attached to different surfaces with various degrees of efficiency, and partly upon the bacterial surface properties (1, 11, 19). Several studies have described bacterial adhesion in terms of physicochemical parameters such as hydrophobicity (5, 17, 22), surface energy (1, 3), and electrostatic interactions of the cell particles with the supports (2, 21, 27). The major forces of attraction and repulsion between bacterial cells and solid substrata include long-range Lifshitz-van der Waals interactions (LW) and (SR) short-range interactions caused by hydrogen bonds (19, 28). The electrostatic repulsion can be compensated by the van der Waals attraction under some conditions, according to the Derjagouin-Landau-VerweyOverbeek theory (26). In this paper, the cell hydrophobicities of 22 isolates of L. monocytogenes were measured with the salt aggregation test (SAT). In addition, cell surface charges and hydrophobicity
MATERIALS AND METHODS
Microorganisms, media, and culture conditions. The bacterial strains used in this study are listed in Table 1. Working cultures of these microorganisms were maintained at 4°C on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) slants supplemented with 1% yeast extract (Difco Laboratories, Detroit, Mich.). Each stock culture was streaked on a Trypticase soy agar-yeast extract plate with 4% bovine blood and incubated for 24 h at 37°C. Colonies were picked and inoculated in a vial containing 5 ml of Trypticase soy broth (BBL) with 1% yeast extract (Difco) and incubated for 18 to 24 h at 37°C on a Lab-line environmental shaker (Lab-line, Melrose Park, Ill.) at 100 rpm. Cell suspensions were harvested by centrifugation (model DPR-6000; International Equipment Company, Division of Damon Corp., Needham Heights, Mass.) at 5,500 x g for 10 min, washed twice in salt-peptone buffer (0.85% NaCl, 0.05% Bacto-peptone [Difco]), and finally suspended in buffer according to the method tested. Viable counts were done with standard plate count agar (Difco). Serial dilutions were performed by adding 0.1 ml of sample to 9.9 ml of salt-peptone buffer. Five 0.1-ml samples of a given dilution were applied to the surfaces of dried agar plates. SAT. The SAT was performed essentially as described by Rozgonyi et al. (23). A 25-,ul sample of bacterial suspension in 0.02 M sodium phosphate buffer containing 0.85% NaCl (pH 6.8) was mixed with 40 RIu of 0.2, 1.0, 2.0, 3.0, or 4.0 M
Corresponding author. t Contribution no. 216 of the Food Research and Development *
Centre.
1969
1970
APPL. ENVIRON. MICROBIOL.
MAFU ET AL. TABLE 1. L. monocytogenes strains used in this study
Strain
Source
Clinical isolate Mexican cheese, California 88-53 Dairy plant, Halifax, Canada 88-56 Cheese, Germany 88-173 Dairy plant, Ontario, Canada CI-i Not determined 88-Lm-79 Sausage 88-Lm-84 Slaughterhouse 88-Lm-98 Environment 88-Lm-136 Smoked ham 88-PB-296 Smoked ham 88-PB-299 Smoked ham 88-PB-341 Chicken and veal loaf 88-PB-350 Cooked ham 88-PB-371 Smoked ham 88-PB-380 Pastrami 88-PB-483 Pastrami 88-PB-503 Frankfurter 88-PB-518 Garlic sausage 88-PB-520 Garlic sausage 88-PB-553 Smoked ham Scott A LA-5
Supplier
Health and Welfare Canada' Health and Welfare Canada Health and Welfare Canada
Health and Welfare Canada Health and Welfare Canada Health Laboratory Animalsb Animal Pathology Laboratoryc Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Animal Animal Animal Animal Animal Animal Animal
Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology
Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory
a Ottawa, Ontario, Canada. Guelph, Ontario, Canada.
b
c
Saint-Hyacinthe,
Quebec, Canada.
ammonium sulfate in 0.02 M sodium phosphate buffer (pH and placed on a glass depression slide. The bacteriumsalt solution mixture was gently rotated on a Titertek shaker (Flow Laboratories, Ontario, Canada) for 15 min at room temperature, and bacterial aggregation was observed with the naked eye under white-light illumination. The lowest concentration of ammonium sulfate giving visible aggregation was scored as the positive SAT hydrophobicity value (12). All strains of L. monocytogenes were tested four times. HIC. The HIC procedure was carried out as previously described by Dahlback et al. (5) and by Smyth et al. (25) with octyl-Sepharose or Sepharose CL-4B (Pharmacia, Uppsala, Sweden) gels. Two milliliters of gel was equilibrated overnight at 4°C in 3 ml of buffered sodium chloride solutions of 1.0, 2.0, 3.0, or 4.0 M. Solutions of sodium chloride were prepared by mixing an appropriate quantity of 0.1 M citric acid to 0.2 M Na2HPO4 to obtain the required pH value (3.5 or 7.0). Final pH values were adjusted with 1.0 M NaOH or 0.1 M citric acid. A 2.5-ml sample of equilibrated gel (octyl-Sepharose or Sepharose CL-4B) was added in a chromatographic column (Flex-Column, 40-mm length and 7-mm inside diameter; Mendel Scientific Co. Ltd, Guelph,
6.8)
Ontario, Canada) to obtain 0.8 ml of final gel bed volume. The gel bed was washed extensively with 10 bed volumes of sodium chloride solution to remove any trace of ethanol added as preservative. A sample of 0.1 ml of washed L. monocytogenes Scott A suspensions containing 2 x 1010 to 4 x 10'° CFU/ml, in appropriate buffer solution prepared as described above, was introduced on the gel bed; 4 ml of buffered sodium chloride solutions of different molarities (NaCl, 1.0 to 4.0 M [pH 3.5 and 7.0]) was allowed to pass through the gel (octyl-Sepharose or Sepharose CL-4B). The eluate (unadsorbed bacteria in 4 ml of salt solution) was collected and named e. The gel was also collected and named g. Viable counts of each fraction (eluate or gel) were
performed on standard plate count agar (Difco) as described above. The cell number of each fraction was estimated by the bioluminescence technique (16). The degree of hydrophobocity was expressed as the gle ratio; a log gle ratio of 1.5 mol/liter of ammonium sulfate) as defined by Ljungh et al. (13) and Mamo et al. (18). This test revealed that L. monocytogenes Scott A expressed low cell surface hydrophobicity. Mozes and Rouxhet (20) noted that the SAT lacks sensitivity and can be used satisfactory only for detecting very hydrophobic microorganisms. These authors reported that the HIC procedures performed with a pH close to the cell isoelectric point and a pH near neutrality allow the identification of hydrophilic microorganisms which are practically not retained by the gel. The HIC results indicate that the hydrophobicity of L. monocytogenes Scott A, as estimated by log gle, increases as the pH decreases, suggesting that the adsorption of strain Scott A to octyl-Sepharose may be due to modifications of surface potential. Mozes and Rouxhet (20) noted that the pH determines the sign and amplitude of the surface potential and indicated that the isoelectric point of microorganisms is in the pH range of 2 to 3.5. There exists on the cell surface charged groups such as capsules, lipopolysaccharides, and surface proteins with such pKa values that they become protonated when pH diminishes (2, 12). High ionic strengths also suppressed electrostatic interactions and favored hydrophobic interactions between L. monocytogenes cells and amphiphilic gels. The energy barrier was lowered by high ionic strengths and low pH (12, 20). SAT and HIC methods revealed the low hydrophobic nature of L. monocytogenes. The negative HIC values (log gle) obtained at pH 7.0 are in agreement with those of Dickson and Koohmaraie (6), who also studied the relative hydrophobicity of the strain Scott A. Our results also revealed that at neutral pH (7.3), L. monocytogenes Scott A exhibited a highly negative electrophoretic mobility value compared with those determined by Bayer and Sloyer (2) for gram-positive and gram-negative bacteria. The electrostatic charge of the cell surface is a net charge resulting from the combined charges of the molecules composing the cell surface and their counterions (2). At pH 3.5, the net charge of strain Scott A surface could approach zero, and this microorganism could be retained on the octyl-Sepharose gel (Table 1). Mozes and Rouxhet (20) noted that hydrophilic
cells do not adhere to amphiphilic gels unless the pH is very close to the isoelectric point. Adhesion of bacteria to surfaces can involve both specific interactions between complementary surface of microorganisms such as polymers or lectinlike interactions and physicochemical characteristics such as charge and surface free energies (3). In this study, contact angle measurements with water and ot-bromonaphthalene allow calculation of a surface free energy value of 65.9 + 0.7 mJ- m-2 for L. monocytogenes Scott A. Although Absolom et al. (1) used other methods to derive surface free energies, these authors calculated that surface free energy of L. monocytogenes was 66.3 + 0.6 mJ- m-2, as obtained from contact angle measurement with saline (26.1 ± 1.20) via the equation-of-state approach. Dickson and Koohmaraie (6) reported that the contact angle with phosphate buffer was 26.5° for L. monocytogenes Scott A. Our results are in agreement with these values of surface free energy or contact angle measurements, which is 26.30 with water. Finally, Absolom et al. (1) classified a microorganism as hydrophilic when the surface free energy was in the range of 65 to 69 mJ m2. The polar component (SR) of surface free energy (41.3 mJ m-2) of L. monocytogenes was lower than that of water (-ySR = 51.0 mJ. m-2). In the thermodynamic concept proposed by Absolom et al. (1) and Busscher et al. (3) for the approach toward surface free energies based on polar and dispersion components, adhesion of L. monocytogenes should be energetically more favorable as surface free energies of solid surfaces decrease. Polypropylene and rubber surfaces showed lower energy surfaces than glass and stainless steel (14). In addition, the interfacial free energy of adhesion of strain Scott A estimated for polypropylene and rubber was lower than that for glass and stainless steel. However, the interfacial free energy of octyl-Sepharose was quite similar to that of polymeric surfaces. The HIC results show that L. monocytogenes cells do not adhere to octylSepharose unless the pH is very close to the isoelectric
point.
Although theoretical premise predicts that polypropylene and rubber could allow better attachment than glass and stainless steel, Mafu et al. (14) did not find correlation between the low surface energy values of polypropylene and rubber and the ability of L. monocytogenes to attach to them. However, Mafu et al. (15) have found that L. monocytogenes Scott A was more resistant to sanitizing agents when attached to polypropylene and rubber surfaces than when attached to glass and stainless steel surfaces, although scanning electron microscopic examination revealed that L. monocytogenes could attach to low- and high-energy surfaces after short contact times (14). The physicochemical expression of hydrophobicity of L. monocytogenes Scott A is only apparent at low pH and at high ionic strengths. According to the attachment capabilities of L. monocytogenes reported earlier by Mafu et al. (14), factors other than cell surface hydrophobicity, such as surface charges and the presence of exopolymer, may be of importance in the adhesion process of this microorganism to stainless steel, glass, polypropylene, and rubber surfaces. ACKNOWLEDGMENTS The cooperation of Serge Messier and Andrd Gagnon, Animal Pathology Laboratory, St.-Hyacinthe, Quebec, Canada, is greatly appreciated. Thanks are also directed to Michel Britten for his scientific assistance. We also thank Lison Blanchette and MarieLine Desjardins for their technical assistance.
VOL. 57, 1991
PHYSICOCHEMICAL CHARACTERIZATION OF L. MONOCYTOGENES
REFERENCES 1. Absolom, D. R., F. V. Lamberti, Z. Policova, W. Zingg, C. J. van Oss, and A. W. Neumann. 1983. Surface thermodynamics of bacterial adhesion. Appl. Environ. Microbiol. 46:90-97. 2. Bayer, M. E., and J. L. Sloyer. 1990. The electrophoretic mobility of Gram-negative and Gram-positive bacteria: an electrokinetic analysis. J. Gen. Microbiol. 136:867-874. 3. Busscher, H. J., A. H. Weerkamp, H. C. van der Mei, A. W. J. van Pelt, H. P. de Jong, and J. Arends. 1984. Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion. Appl. Environ. Microbiol. 48:980-983. 4. Cox, L. J., T. Kleiss, J. L. Cordier, C. Crodelana, P. Konkel, C. Pedrazzini, R. Beuner, and A. Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6:49-61. 5. Dahlback, B., M. Hermansson, S. Kjellerberg, and B. Norkans. 1981. The hydrophobicity of bacteria-an important factor in their initial adhesion at the air-water interface. Arch. Microbiol. 128:267-270. 6. 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. 7. Donnelly, C. W., and E. H. Briggs. 1986. Psychrotrophic growth and thermal inactivation of Listeria monocytogenes as a function of milk composition. J. Food Prot. 49:994-998. 8. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407. 9. Griffiths, M. W. 1989. Listeria monocytogenes: its importance in the dairy industry. J. Sci. Food Agric. 47:133-158. 10. Herald, P. A., and E. A. Zottola. 1988. Attachment of Listeria monocytogenes to stainless steel surfaces at various temperatures and pH values. J. Food Sci. 53:1549-1562. 11. Kirtley, S. A., and J. McGuire. 1989. On differences in surface composition of dairy product contact materials. J. Dairy Sci. 72:1748-1753.
12. Lindahl, M., A. Faris, T. Wadstrom, and S. Hjerten. 1981. A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta 77: 471-476.
13. Ljungh, A., S. Hjertwn, and T. Wadstrom. 1985. High surface hydrophobicity of autoaggregating Staphylococcus aureus strains isolated from human infections studied with the salt aggregation test. Infect. Immun. 47:522-526. 14. Mafu, A. A., D. Roy, J. Goulet, and P. Magny. 1990. Attachment of Listeria monocytogenes to stainless steel, glass, polypropylene, and rubber surfaces after short contact times. J. Food Prot. 53:742-746.
15. Mafu, A. A., D. Roy, J. Goulet, L. Savoie, and R. Roy. 1990.
16.
17.
18.
19.
1973
Efficiency of sanitizing agents for destroying Listeria monocytogenes on contaminated surfaces. J. Dairy Sci. 73:3428-3432. Mafu, A. A., D. Roy, L. Savoie, and J. Goulet. 1991. Bioluminescence assay for estimating the hydrophobic properties of bacteria as revealed by hydrophobic interaction chromatography. Appl. Environ. Microbiol. 57:1640-1643. Magnusson, K. E. 1982. Hydrophobic interaction-a mechanism of bacterial binding. Scand. J. Infect. Dis. 33(Suppl.):32-36. Mamo, W., F. Rozgonyi, A. Brown, S. Hjerten, and T. Wadstrom. 1987. Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastisis. J. Appl. Bacteriol. 62:241-249. McEldowney, S., and M. Fletcher. 1986. Variability of -the influence of physicochemical factors affecting bacterial adhesion to polystyrene substrata. Appl. Environ. Microbiol. 52:
460-465. 20. Mozes, N., and P. G. Rouxhet. 1987. Methods for measuring hydrophobicity of microorganisms. J. Microbiol. Methods 6:99112. 21. Pedersen, K. 1980. Electrostatic interaction chromatography, a method for assaying the relative surface charges of bacteria. FEMS Microbiol. Lett. 12:365-367. 22. Rosenberg, M., and S. Kjellerberg. 1987. Hydrophobic interactions: Role in bacterial adhesion. p. 353-393. In K. C. Marshall (ed.), Advances in microbial ecology, vol. 9. Plenum Publishing Corp., New York. 23. Rozgonyi, F., K. R. Szitha, S. Hjerten, and T. Wadstrom. 1985. Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species. J. Appl. Bacteriol. 59:451-457. 24. Ryser, E. T., and E. H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. J. Food Prot. 51:615-621. 25. Smyth, C. J., P. Jonsson, E. Olsson, 0. Soderlind, J. Rosengren, S. Hjerten, and T. Wadstrom. 1978. Differences in hydrophobic surfaces characteristics of porcine enteropathogenic Escherichia coli with and without K88 antigen as revealed by hydrophobic interaction chromatography. Infect. Immun. 22:462-472. 26. Tadros, T. F. 1980. Particle-surface adhesion, p. 93-116. In R. C. W. Berkeley, J. M. Lynch, J. Melling, P. R. Rutter, and B. Vincent (ed.), Microbial adhesion to surfaces. Ellis Horwood, Ltd., Publisher, Chichester, United Kingdom. 27. van Loosdrecht, M. C. M., J. Lyklema, W. Norde, G. Schraa, and A. J. B. Zehnder. 1987. Electrophoretic mobility and hydrophobicity as a measure to predict the initial steps of bacterial adhesion. Appl. Environ. Microbiol. 53:1898-1901. 28. van Oss, C. J., R. J. Good, and M. K. Chaudhury. 1986. The role of van der Waals forces and hydrogen bonds in "hydrophobic interactions" between biopolymers and low energy surfaces. J. Colloid Interface Sci. 111:378-390.
Vol. 57, No. 7
Characterization of Physicochemical Forces Involved in Adhesion of Listeria monocytogenes to Surfacest AKIER ASSANTA MAFU,1 DENIS ROY,2* JACQUES GOULET,1
AND
LUC SAVOIE2
Department of Food Science and Technology, Universite Laval, Ste-Foy, Quebec, Canada GJK 7P4,1 and Agriculture Canada Food Research and Development Centre, 3600, Casavant Boulevard West, St.-Hyacinthe, Quebec, Canada J2S 8E32 Received 23 January 1991/Accepted 7 May 1991
This study investigated the physicochemical forces involving the adhesion of Listeria monocytogenes to surfaces. A total of 22 strains of L. monocytogenes were compared for relative surface hydrophobicity with the salt aggregation test. Cell surface charges and hydrophobicity of L. monocytogenes Scott A were also determined by electrophoretic mobility, hydrophobic-interaction chromatography, and contact angle measurements. Electrokinetic measurements indicated that the strain Scott A has a negative electrophoretic mobility. Physicochemical characterization of L. monocytogenes by various methods indicates that this microorganism is hydrophilic. All L. monocytogenes strains tested with the salt aggregation test method aggregated at very high ammonium sulfate molarities. The hydrophobicity-interaction chromatography results show that L. monocytogenes Scott A cells do not adhere to octyl-Sepharose unless the pH is low. Results from contact angle measurements showed that the surface free energy of strain Scott A was 65.9 mJ m-2, classifying this microorganism as a hydrophilic bacterium. In addition, the interfacial free energy of adhesion of L. monocytogenes Scott A estimated for polypropylene and rubber was lower than that for glass and stainless steel. However, these theoretical implications could not be correlated with the attachment capabilities of L. monocytogenes. In recent years, Listeria monocytogenes has become a source of concern for the food industry because of outbreaks of food-borne illness associated with consumption of food products (8, 9). The psychotrophic nature of L. monocytogenes (7, 24) along with its ability to come into contact with inert surfaces and become attached to them makes its presence in food processing plants a major problem (10, 14,
of L. monocytogenes Scott A were also determined by electrophoretic mobility, hydrophobic interaction chromatography (HIC), and contact angle measurements. Calculations of the free energies of adhesion (derived from surface free energies from total, LW, and SR contributions) of L. monocytogenes cells onto stainless steel, glass, polypropylene, and rubber were made.
15). The capability of L. monocytogenes to adhere to surfaces represents a source of potential contamination for a wide variety of material coming in contact with solid surfaces (4). However, the mechanisms governing the adhesion of L. monocytogenes to inert surfaces are not understood and have not been defined. Several studies have shown that the adhesion of bacteria partly depends upon the nature of the inert surfaces, since bacteria become attached to different surfaces with various degrees of efficiency, and partly upon the bacterial surface properties (1, 11, 19). Several studies have described bacterial adhesion in terms of physicochemical parameters such as hydrophobicity (5, 17, 22), surface energy (1, 3), and electrostatic interactions of the cell particles with the supports (2, 21, 27). The major forces of attraction and repulsion between bacterial cells and solid substrata include long-range Lifshitz-van der Waals interactions (LW) and (SR) short-range interactions caused by hydrogen bonds (19, 28). The electrostatic repulsion can be compensated by the van der Waals attraction under some conditions, according to the Derjagouin-Landau-VerweyOverbeek theory (26). In this paper, the cell hydrophobicities of 22 isolates of L. monocytogenes were measured with the salt aggregation test (SAT). In addition, cell surface charges and hydrophobicity
MATERIALS AND METHODS
Microorganisms, media, and culture conditions. The bacterial strains used in this study are listed in Table 1. Working cultures of these microorganisms were maintained at 4°C on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) slants supplemented with 1% yeast extract (Difco Laboratories, Detroit, Mich.). Each stock culture was streaked on a Trypticase soy agar-yeast extract plate with 4% bovine blood and incubated for 24 h at 37°C. Colonies were picked and inoculated in a vial containing 5 ml of Trypticase soy broth (BBL) with 1% yeast extract (Difco) and incubated for 18 to 24 h at 37°C on a Lab-line environmental shaker (Lab-line, Melrose Park, Ill.) at 100 rpm. Cell suspensions were harvested by centrifugation (model DPR-6000; International Equipment Company, Division of Damon Corp., Needham Heights, Mass.) at 5,500 x g for 10 min, washed twice in salt-peptone buffer (0.85% NaCl, 0.05% Bacto-peptone [Difco]), and finally suspended in buffer according to the method tested. Viable counts were done with standard plate count agar (Difco). Serial dilutions were performed by adding 0.1 ml of sample to 9.9 ml of salt-peptone buffer. Five 0.1-ml samples of a given dilution were applied to the surfaces of dried agar plates. SAT. The SAT was performed essentially as described by Rozgonyi et al. (23). A 25-,ul sample of bacterial suspension in 0.02 M sodium phosphate buffer containing 0.85% NaCl (pH 6.8) was mixed with 40 RIu of 0.2, 1.0, 2.0, 3.0, or 4.0 M
Corresponding author. t Contribution no. 216 of the Food Research and Development *
Centre.
1969
1970
APPL. ENVIRON. MICROBIOL.
MAFU ET AL. TABLE 1. L. monocytogenes strains used in this study
Strain
Source
Clinical isolate Mexican cheese, California 88-53 Dairy plant, Halifax, Canada 88-56 Cheese, Germany 88-173 Dairy plant, Ontario, Canada CI-i Not determined 88-Lm-79 Sausage 88-Lm-84 Slaughterhouse 88-Lm-98 Environment 88-Lm-136 Smoked ham 88-PB-296 Smoked ham 88-PB-299 Smoked ham 88-PB-341 Chicken and veal loaf 88-PB-350 Cooked ham 88-PB-371 Smoked ham 88-PB-380 Pastrami 88-PB-483 Pastrami 88-PB-503 Frankfurter 88-PB-518 Garlic sausage 88-PB-520 Garlic sausage 88-PB-553 Smoked ham Scott A LA-5
Supplier
Health and Welfare Canada' Health and Welfare Canada Health and Welfare Canada
Health and Welfare Canada Health and Welfare Canada Health Laboratory Animalsb Animal Pathology Laboratoryc Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Pathology Laboratory Animal Animal Animal Animal Animal Animal Animal Animal
Pathology Pathology Pathology Pathology Pathology Pathology Pathology Pathology
Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory
a Ottawa, Ontario, Canada. Guelph, Ontario, Canada.
b
c
Saint-Hyacinthe,
Quebec, Canada.
ammonium sulfate in 0.02 M sodium phosphate buffer (pH and placed on a glass depression slide. The bacteriumsalt solution mixture was gently rotated on a Titertek shaker (Flow Laboratories, Ontario, Canada) for 15 min at room temperature, and bacterial aggregation was observed with the naked eye under white-light illumination. The lowest concentration of ammonium sulfate giving visible aggregation was scored as the positive SAT hydrophobicity value (12). All strains of L. monocytogenes were tested four times. HIC. The HIC procedure was carried out as previously described by Dahlback et al. (5) and by Smyth et al. (25) with octyl-Sepharose or Sepharose CL-4B (Pharmacia, Uppsala, Sweden) gels. Two milliliters of gel was equilibrated overnight at 4°C in 3 ml of buffered sodium chloride solutions of 1.0, 2.0, 3.0, or 4.0 M. Solutions of sodium chloride were prepared by mixing an appropriate quantity of 0.1 M citric acid to 0.2 M Na2HPO4 to obtain the required pH value (3.5 or 7.0). Final pH values were adjusted with 1.0 M NaOH or 0.1 M citric acid. A 2.5-ml sample of equilibrated gel (octyl-Sepharose or Sepharose CL-4B) was added in a chromatographic column (Flex-Column, 40-mm length and 7-mm inside diameter; Mendel Scientific Co. Ltd, Guelph,
6.8)
Ontario, Canada) to obtain 0.8 ml of final gel bed volume. The gel bed was washed extensively with 10 bed volumes of sodium chloride solution to remove any trace of ethanol added as preservative. A sample of 0.1 ml of washed L. monocytogenes Scott A suspensions containing 2 x 1010 to 4 x 10'° CFU/ml, in appropriate buffer solution prepared as described above, was introduced on the gel bed; 4 ml of buffered sodium chloride solutions of different molarities (NaCl, 1.0 to 4.0 M [pH 3.5 and 7.0]) was allowed to pass through the gel (octyl-Sepharose or Sepharose CL-4B). The eluate (unadsorbed bacteria in 4 ml of salt solution) was collected and named e. The gel was also collected and named g. Viable counts of each fraction (eluate or gel) were
performed on standard plate count agar (Difco) as described above. The cell number of each fraction was estimated by the bioluminescence technique (16). The degree of hydrophobocity was expressed as the gle ratio; a log gle ratio of 1.5 mol/liter of ammonium sulfate) as defined by Ljungh et al. (13) and Mamo et al. (18). This test revealed that L. monocytogenes Scott A expressed low cell surface hydrophobicity. Mozes and Rouxhet (20) noted that the SAT lacks sensitivity and can be used satisfactory only for detecting very hydrophobic microorganisms. These authors reported that the HIC procedures performed with a pH close to the cell isoelectric point and a pH near neutrality allow the identification of hydrophilic microorganisms which are practically not retained by the gel. The HIC results indicate that the hydrophobicity of L. monocytogenes Scott A, as estimated by log gle, increases as the pH decreases, suggesting that the adsorption of strain Scott A to octyl-Sepharose may be due to modifications of surface potential. Mozes and Rouxhet (20) noted that the pH determines the sign and amplitude of the surface potential and indicated that the isoelectric point of microorganisms is in the pH range of 2 to 3.5. There exists on the cell surface charged groups such as capsules, lipopolysaccharides, and surface proteins with such pKa values that they become protonated when pH diminishes (2, 12). High ionic strengths also suppressed electrostatic interactions and favored hydrophobic interactions between L. monocytogenes cells and amphiphilic gels. The energy barrier was lowered by high ionic strengths and low pH (12, 20). SAT and HIC methods revealed the low hydrophobic nature of L. monocytogenes. The negative HIC values (log gle) obtained at pH 7.0 are in agreement with those of Dickson and Koohmaraie (6), who also studied the relative hydrophobicity of the strain Scott A. Our results also revealed that at neutral pH (7.3), L. monocytogenes Scott A exhibited a highly negative electrophoretic mobility value compared with those determined by Bayer and Sloyer (2) for gram-positive and gram-negative bacteria. The electrostatic charge of the cell surface is a net charge resulting from the combined charges of the molecules composing the cell surface and their counterions (2). At pH 3.5, the net charge of strain Scott A surface could approach zero, and this microorganism could be retained on the octyl-Sepharose gel (Table 1). Mozes and Rouxhet (20) noted that hydrophilic
cells do not adhere to amphiphilic gels unless the pH is very close to the isoelectric point. Adhesion of bacteria to surfaces can involve both specific interactions between complementary surface of microorganisms such as polymers or lectinlike interactions and physicochemical characteristics such as charge and surface free energies (3). In this study, contact angle measurements with water and ot-bromonaphthalene allow calculation of a surface free energy value of 65.9 + 0.7 mJ- m-2 for L. monocytogenes Scott A. Although Absolom et al. (1) used other methods to derive surface free energies, these authors calculated that surface free energy of L. monocytogenes was 66.3 + 0.6 mJ- m-2, as obtained from contact angle measurement with saline (26.1 ± 1.20) via the equation-of-state approach. Dickson and Koohmaraie (6) reported that the contact angle with phosphate buffer was 26.5° for L. monocytogenes Scott A. Our results are in agreement with these values of surface free energy or contact angle measurements, which is 26.30 with water. Finally, Absolom et al. (1) classified a microorganism as hydrophilic when the surface free energy was in the range of 65 to 69 mJ m2. The polar component (SR) of surface free energy (41.3 mJ m-2) of L. monocytogenes was lower than that of water (-ySR = 51.0 mJ. m-2). In the thermodynamic concept proposed by Absolom et al. (1) and Busscher et al. (3) for the approach toward surface free energies based on polar and dispersion components, adhesion of L. monocytogenes should be energetically more favorable as surface free energies of solid surfaces decrease. Polypropylene and rubber surfaces showed lower energy surfaces than glass and stainless steel (14). In addition, the interfacial free energy of adhesion of strain Scott A estimated for polypropylene and rubber was lower than that for glass and stainless steel. However, the interfacial free energy of octyl-Sepharose was quite similar to that of polymeric surfaces. The HIC results show that L. monocytogenes cells do not adhere to octylSepharose unless the pH is very close to the isoelectric
point.
Although theoretical premise predicts that polypropylene and rubber could allow better attachment than glass and stainless steel, Mafu et al. (14) did not find correlation between the low surface energy values of polypropylene and rubber and the ability of L. monocytogenes to attach to them. However, Mafu et al. (15) have found that L. monocytogenes Scott A was more resistant to sanitizing agents when attached to polypropylene and rubber surfaces than when attached to glass and stainless steel surfaces, although scanning electron microscopic examination revealed that L. monocytogenes could attach to low- and high-energy surfaces after short contact times (14). The physicochemical expression of hydrophobicity of L. monocytogenes Scott A is only apparent at low pH and at high ionic strengths. According to the attachment capabilities of L. monocytogenes reported earlier by Mafu et al. (14), factors other than cell surface hydrophobicity, such as surface charges and the presence of exopolymer, may be of importance in the adhesion process of this microorganism to stainless steel, glass, polypropylene, and rubber surfaces. ACKNOWLEDGMENTS The cooperation of Serge Messier and Andrd Gagnon, Animal Pathology Laboratory, St.-Hyacinthe, Quebec, Canada, is greatly appreciated. Thanks are also directed to Michel Britten for his scientific assistance. We also thank Lison Blanchette and MarieLine Desjardins for their technical assistance.
VOL. 57, 1991
PHYSICOCHEMICAL CHARACTERIZATION OF L. MONOCYTOGENES
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