- Email: [email protected]
A kinetic approach to bacterial adherence to hydrocarbon
Journal of Microbiological Methods 4 (1985) 141- 146
i 41
Elsevier JMM 00125
A kinetic approach to bacterial adherence to hydrocarbon Dov Lichtenberg a, Mel Rosenberg h,c,*, Nellie Sharfman b and Itzhak
Ofek b
aDepartment o f Physiology an d Pharmacology, bDepartment o f Human Microbiology. and CSchool of Dental Medicine. Suckler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978 (Israel)
(Received 19 March 1985)(Revised versionreceived 17 June 1985)(Accepted 19 June 1985)
Summary Although bacterialadherence to hydrocarbonshas been widelyemployed to study bacterial hydrophobicity, quantitative criteria for the assay are not well established. In the present report, adherence of Streptococcus p.vogenes to hexadecane was studied as a function of time, and water: hexadecane ratio. Adherenceof cells as a function of mixingtime was exponential,yieldinga slope for each hexadecane volumeemployed.The values for slopes of the curves obtained increasedlinearlywith increasinghydrocarbon:water ratios. The slope of the latter curve, referred to as the 'removal coefficient'was 376 rain-I for S. pyogenes. The data suggest that the proposed kineticapproach may providea quantitativemeasurementof bacterialadherence to hydrocarbons.
Key words: Adherence - Cell surface - Hexadecane - Hydrocarbons - Hvdrophobicity - Streptococcus pyogenes
Introduction The ability of certain bacteria to partition at the oil: water interface has been k n o w n for over 60 years [1]. Several years ago bacterial adherence to hydrocarbons was proposed as a convenient technique for studying hydrophobic surface properties of microbial cells [2]. In the assay, washed bacterial suspensions are vortexed in the presence of test hydrocarbons (e.g., hexadecane, octane, xylene). Following separation of the phases, which enables cell-coated oil droplets to rise, the extent of adherence is determined from the decrease in cell density in the lower aqueous phase, observed through turbidimetric measurements. During the past few years, this method has been applied to the investigations of bacterial hydrophobicity in a wide range of species, and as a function of various bacterial growth conditions and treatments [3, 4]. Moreover, this assay * To whom correspondenceshould be addressed. 0167-7012/85/$03.30 © 1985 ElsevierSciencePublishersB.V. (BiomedicalDivision)
142 has helped elucidate the possible role of hydrophobic interactions in mediating bacterial adherence to surfaces of ecological and medical interest [3] and for isolation of mutants deficient in cell surface hydrophobicity [~-6]. In some cases, cell surface components which help mediate adherence to hydrocarbons have been identified [4, 6-8]. One of the major limitations of this method is that quantitative criteria for adherence to hydrocarbons have not been well established: rather, semi-quantitative results, obtained by altering the oil: water ratio [2], or duration of the mixing procedure [3, 9], have usually been reported. In some cases, clear differences exist between bacteria which adhere in high proportions to hydrocarbons, and those which are nonadherent [2, 3]. However, among the many bacterial strains which do adhere, at least to some extent, comparison of their relative affinities is difficult. Thus, vague terms, such as 'pronounced', 'intermediate' or 'slight', have commonly been used to describe adherence to hydrocarbons. In order to use this method to compare the cell-surface hydrophobicity of various microbial cells in a more exact fashion, a quantitative criterion must be established to better describe their affinity for the oil: water interface. In the present communication, we offer such a criterion, based on the kinetics of adherence to hydrocarbon, as a function of hydrocarbon-to-water ratio. Method Provided that a steady state distribution of bacteria between the bulk aqueous phase and the oil :water interface can be established, the oil: water steady state distribution can serve as a criterion for bacterial hydrophobicity. Such a distribution would depend linearly on the oil: water volume ratio, the slope of such a dependence constituting the oil:water distribution coefficient of the bacterial cells. However, preliminary data indicated that this experimental approach would be problematic; mainly it is difficult to determine whether the extent of adherence, as a function of duration of mixing, reaches a plateau which represents a true steady state distribution. Moreover, for a steady state distribution to occur, binding of bacteria at the interface during the mixing ought to be reversible. Thus, increasing the water: oil ratio following the mixing procedure should result in release of cells into the aqueous phase. As we were unable to demonstrate such a phenomenon, an alternative quantitative approach, based on the rate, rather than extent, of adherence, was adopted. Prior to testing the kinetics of adherence by turbidity measurements, two problems had to be addressed. First, when low hexadecane volumes are employed, it is conceivable that the droplets created by the mixing procedure possess a high specific gravity due to the adherence of large numbers of bacteria. This might prevent them from floating following mixing. Adherence experiments employing l09 bacteria (Streptococcus pyogenes M-5), 10 #1 hexadecane and D20, rather than H20, revealed that the extent of decrease in turbidity following mixing was not affected by the increased specific gravity of the aqueous phase (not shown). This suggested that the flotation of oil droplets following mixing is not hampered by the adherence of bacteria, even at high bacteria: hydrocarbon ratios. Microscopic observation of the upper phase following the mixing demonstrated that the diameter of the cell-coated droplets was about the same (ca. 50 ~m in diameter), irrespective of the hexadecane volume employed in our tests.
143 This observation helps explain the previous conclusion that all the hydrocarbon-associated bacteria float following the mixing procedure. It also assists in tackling a second potential problem, i.e., that the extremely low hexadecane volumes required might not provide sufficient surface area for adherence. The surface area of 1 #1 (10 9/.tm 3) hexadecane droplets of a radius of 25 #m is about 1.2 × 108 #m 2. Thus, the lowest hexadecane volume employed (10 ~tl) suffices to bind 1.2 × 109 bacteria of a diameter of 1 #m, if each cell occupies 1 #m 2 on the droplet surface. Therefore, all the experiments were carried out with less than 109 bacteria present in the assay. The assay itself involves mixing of the bacterial suspension to be tested with a given volume of hydrocarbon for fixed, consecutive time periods. Following each agitation, the phases are allowed to separate, and the turbidity of the lower aqueous phase measured. We employed test tubes which can be read directly in the spectrophotometer without removal and transfer of the lower aqueous phase. Following measurement, the same mixture is then subjected to subsequent agitations. Results and Discussion
The results of a typical experiment, using Streptococcus pyogenes M-5, previously reported to adhere to hexadecane [4, 11], are presented in Fig. 1. We observed that the decrease in turbidity as a function of time was exponential. Thus, linear results were obtained by plotting the logarithm of the percentage of cells remaining in the aqueous phase as a function of time. The slopes of the curves obtained (k) were observed to
.J" iQt 1~0~
~
tog[~, o115.
111
0
-%
~o .,~
s
~ _
0.5
10
(V,//Vw).IO.~
1.0 time(rain)
Fig. 1. The dependence of bacterial removal on the time of agitation. To 3 ml of bacterial suspension of an optical density (a0) in the range of 0.35~).40 as measured in a Junior Coleman, 10, 15, 20 or 25 #1 of hexadecane were added. After each agitation period at a multivortex (SMI) setting of 2, the tubes were allowed to stand for t5 min for phase separation, and optical density of the aqueous phase in the same tube was then directly measured (at). Results obtained for 10 p.I (e) and 20 p.l (',) are shown in the figure, in terms of the agitation time dependency of log (at/a0) • 100 (results obtained for other volumes are excluded for sake of clarity). The insert describes the rate of removal (slopes of the curves obtained with different hexadecane volumes) as a function of the hexadecane: water ratio. Each determination represents the average of triplicate tubes. All curves showed linear regression with correlation coefficients in excessof 0.95. Bacteria were grown, washed and suspended in phosphate-buffered saline as previously reported [4].
144
Fig. 2.
Adhesion to hexadecane. Adhesion of S. pyogenes to large hexadecane droplet.
145 increase linearly with increasing hexadecane volume employed in the test. A similar kinetic approach has been employed by Kawabata et al. [11] in their studies on removal of bacteria from aqueous media by insoluble polymers. These authors studied the dependency of the rate constants of removal on the ratio of weight of polymer to the water volume and coined the term 'removal coefficient' for the slope of such dependencies. Thus, in our case, 'removal coefficient' refers to the change in the rate constant of removal of bacteria from the bulk aqueous phase to the hydrocarbon:water interface, as a function of the ratio of hydrocarbon to water in the assay. The insert in Fig. 1 presents the slopes obtained as a function of hydrocarbon:water ratio. We submit that the slope of the curve is a quantitative expression of the affinity of the ceils tested for the water: oil interface, and denote it as the removal coefficient (K) of the cells from the bulk aqueous phase by the hydrocarlaon. For S. pyogenes, K was 376 min -1 . Adherence of the cells to hexadecane droplets is shown in Fig. 2. As Gibbons and Etherden pointed out [10], bacterial adherence to hydrocarbons yields reproducible results only when the conditions employed are carefully controlled (i.e., test tube dimensions). The kinetic approach suggested here should enable investigators to compare results obtained in their own laboratory, e.g., adherence of various strains, effects of growth conditions and treatments, in a quantitative fashion. Moreover, relative removal coefficients should be independent of experimental conditions as long as the identical conditions are employed to derive the removal coefficient. Furthermore, in preliminary studies, we repeated the experiment shown in Fig. 1 by employing faster agitation while maintaining all other parameters identical. Although the rate constants were much higher than those described in Fig. 1, a similar removal coefficient (414 min -j) was obtained. It is thus possible that the removal coefficient is independent of agitation conditions. Preliminary experiments in our laboratories suggest the general applicability of the quantitative approach proposed in the present manuscript: (i) S. pyogenes cells, harvested from logarithmic growth phase, yielded results similar to those presented here for stationary phase cells, but with coefficients several orders of magnitude lower; (ii) other bacterial species (Acinetobacter calcoaceticus, Serratia rnarcescens) exhibit linear dependencies of the removal rate constants on the hexadecane: water ratio, enabling derivation of their removal coefficients, in the manner described here.
Acknowledgement We thank Yardena Mazor for excellent technical assistance.
References 1 2 3 4
Mudd, S. and Mudd, E.B.H. (1924) The penetration of bacteria through capillary spaces. IV. A kinetic mechanism in interfaces. J. Exp. Med. 40, 633-645. Rosenbcrg, M., Gutnick, D. and Roscnbcrg, E. (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol. Lett. 9, 29-33. Rosenbcrg, M. (1984) Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. FEMS Microbiol. Lett. 22, 289-295. Ofek, I., Whimack, E. and Bcachey, E. H. (1983) Hydrophobic interactions of group A streptococci with hexadccane droplets. J. Bacteriol. 154, 139-145.
146 5 6 7 8 9 10 I1
Rosenberg, M. and Rosenberg, E. (1981) Role of adherence in growth of Acinetobacter calcoaceticus RAG-I on hexadecane. J. Bacteriol. 148, 51-57. Gibbons, R.J., Etherden, I. and Skobe, J. (1983) Association of fimbriae ~vith the hydropbobicity of Streptococcus sanguis FC-I and adherence to salivary pellicles. Infect. lmmun. 41,414-417. Rosenberg. M. (1984) Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity. J. Bacteriol, 160, 480-482. Rosenberg, M., Bayer. E.A., Delarea, J. and Rosenberg, E. (1982) Role of thin fimbriae in adherence and growth of Acinetobacter calcoaceticus on hexadecane. Appl. Environ. Microbiol. 44, 929-937. Olsson, J. and Westergren, G. (1982) Hydrophobic surface properties of oral streptococci. FEMS Microbiol. Lett. 15, 319-323. Gibbons, R.J. and Etherden, I. (1983) Comparative hydrophobicities of oral bacteria and their adherence to salivary pellicles. Infect. Immun. 41, 1190-1196. Kawabata, N., Hayashi, T. and Matsumoto, T. (1983) Removal of bacteria from water by adhesion to cross-linked poly (vinylpyridinium halide). Appl. Environ. Microbiol. 46, 203-210.
i 41
Elsevier JMM 00125
A kinetic approach to bacterial adherence to hydrocarbon Dov Lichtenberg a, Mel Rosenberg h,c,*, Nellie Sharfman b and Itzhak
Ofek b
aDepartment o f Physiology an d Pharmacology, bDepartment o f Human Microbiology. and CSchool of Dental Medicine. Suckler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978 (Israel)
(Received 19 March 1985)(Revised versionreceived 17 June 1985)(Accepted 19 June 1985)
Summary Although bacterialadherence to hydrocarbonshas been widelyemployed to study bacterial hydrophobicity, quantitative criteria for the assay are not well established. In the present report, adherence of Streptococcus p.vogenes to hexadecane was studied as a function of time, and water: hexadecane ratio. Adherenceof cells as a function of mixingtime was exponential,yieldinga slope for each hexadecane volumeemployed.The values for slopes of the curves obtained increasedlinearlywith increasinghydrocarbon:water ratios. The slope of the latter curve, referred to as the 'removal coefficient'was 376 rain-I for S. pyogenes. The data suggest that the proposed kineticapproach may providea quantitativemeasurementof bacterialadherence to hydrocarbons.
Key words: Adherence - Cell surface - Hexadecane - Hydrocarbons - Hvdrophobicity - Streptococcus pyogenes
Introduction The ability of certain bacteria to partition at the oil: water interface has been k n o w n for over 60 years [1]. Several years ago bacterial adherence to hydrocarbons was proposed as a convenient technique for studying hydrophobic surface properties of microbial cells [2]. In the assay, washed bacterial suspensions are vortexed in the presence of test hydrocarbons (e.g., hexadecane, octane, xylene). Following separation of the phases, which enables cell-coated oil droplets to rise, the extent of adherence is determined from the decrease in cell density in the lower aqueous phase, observed through turbidimetric measurements. During the past few years, this method has been applied to the investigations of bacterial hydrophobicity in a wide range of species, and as a function of various bacterial growth conditions and treatments [3, 4]. Moreover, this assay * To whom correspondenceshould be addressed. 0167-7012/85/$03.30 © 1985 ElsevierSciencePublishersB.V. (BiomedicalDivision)
142 has helped elucidate the possible role of hydrophobic interactions in mediating bacterial adherence to surfaces of ecological and medical interest [3] and for isolation of mutants deficient in cell surface hydrophobicity [~-6]. In some cases, cell surface components which help mediate adherence to hydrocarbons have been identified [4, 6-8]. One of the major limitations of this method is that quantitative criteria for adherence to hydrocarbons have not been well established: rather, semi-quantitative results, obtained by altering the oil: water ratio [2], or duration of the mixing procedure [3, 9], have usually been reported. In some cases, clear differences exist between bacteria which adhere in high proportions to hydrocarbons, and those which are nonadherent [2, 3]. However, among the many bacterial strains which do adhere, at least to some extent, comparison of their relative affinities is difficult. Thus, vague terms, such as 'pronounced', 'intermediate' or 'slight', have commonly been used to describe adherence to hydrocarbons. In order to use this method to compare the cell-surface hydrophobicity of various microbial cells in a more exact fashion, a quantitative criterion must be established to better describe their affinity for the oil: water interface. In the present communication, we offer such a criterion, based on the kinetics of adherence to hydrocarbon, as a function of hydrocarbon-to-water ratio. Method Provided that a steady state distribution of bacteria between the bulk aqueous phase and the oil :water interface can be established, the oil: water steady state distribution can serve as a criterion for bacterial hydrophobicity. Such a distribution would depend linearly on the oil: water volume ratio, the slope of such a dependence constituting the oil:water distribution coefficient of the bacterial cells. However, preliminary data indicated that this experimental approach would be problematic; mainly it is difficult to determine whether the extent of adherence, as a function of duration of mixing, reaches a plateau which represents a true steady state distribution. Moreover, for a steady state distribution to occur, binding of bacteria at the interface during the mixing ought to be reversible. Thus, increasing the water: oil ratio following the mixing procedure should result in release of cells into the aqueous phase. As we were unable to demonstrate such a phenomenon, an alternative quantitative approach, based on the rate, rather than extent, of adherence, was adopted. Prior to testing the kinetics of adherence by turbidity measurements, two problems had to be addressed. First, when low hexadecane volumes are employed, it is conceivable that the droplets created by the mixing procedure possess a high specific gravity due to the adherence of large numbers of bacteria. This might prevent them from floating following mixing. Adherence experiments employing l09 bacteria (Streptococcus pyogenes M-5), 10 #1 hexadecane and D20, rather than H20, revealed that the extent of decrease in turbidity following mixing was not affected by the increased specific gravity of the aqueous phase (not shown). This suggested that the flotation of oil droplets following mixing is not hampered by the adherence of bacteria, even at high bacteria: hydrocarbon ratios. Microscopic observation of the upper phase following the mixing demonstrated that the diameter of the cell-coated droplets was about the same (ca. 50 ~m in diameter), irrespective of the hexadecane volume employed in our tests.
143 This observation helps explain the previous conclusion that all the hydrocarbon-associated bacteria float following the mixing procedure. It also assists in tackling a second potential problem, i.e., that the extremely low hexadecane volumes required might not provide sufficient surface area for adherence. The surface area of 1 #1 (10 9/.tm 3) hexadecane droplets of a radius of 25 #m is about 1.2 × 108 #m 2. Thus, the lowest hexadecane volume employed (10 ~tl) suffices to bind 1.2 × 109 bacteria of a diameter of 1 #m, if each cell occupies 1 #m 2 on the droplet surface. Therefore, all the experiments were carried out with less than 109 bacteria present in the assay. The assay itself involves mixing of the bacterial suspension to be tested with a given volume of hydrocarbon for fixed, consecutive time periods. Following each agitation, the phases are allowed to separate, and the turbidity of the lower aqueous phase measured. We employed test tubes which can be read directly in the spectrophotometer without removal and transfer of the lower aqueous phase. Following measurement, the same mixture is then subjected to subsequent agitations. Results and Discussion
The results of a typical experiment, using Streptococcus pyogenes M-5, previously reported to adhere to hexadecane [4, 11], are presented in Fig. 1. We observed that the decrease in turbidity as a function of time was exponential. Thus, linear results were obtained by plotting the logarithm of the percentage of cells remaining in the aqueous phase as a function of time. The slopes of the curves obtained (k) were observed to
.J" iQt 1~0~
~
tog[~, o115.
111
0
-%
~o .,~
s
~ _
0.5
10
(V,//Vw).IO.~
1.0 time(rain)
Fig. 1. The dependence of bacterial removal on the time of agitation. To 3 ml of bacterial suspension of an optical density (a0) in the range of 0.35~).40 as measured in a Junior Coleman, 10, 15, 20 or 25 #1 of hexadecane were added. After each agitation period at a multivortex (SMI) setting of 2, the tubes were allowed to stand for t5 min for phase separation, and optical density of the aqueous phase in the same tube was then directly measured (at). Results obtained for 10 p.I (e) and 20 p.l (',) are shown in the figure, in terms of the agitation time dependency of log (at/a0) • 100 (results obtained for other volumes are excluded for sake of clarity). The insert describes the rate of removal (slopes of the curves obtained with different hexadecane volumes) as a function of the hexadecane: water ratio. Each determination represents the average of triplicate tubes. All curves showed linear regression with correlation coefficients in excessof 0.95. Bacteria were grown, washed and suspended in phosphate-buffered saline as previously reported [4].
144
Fig. 2.
Adhesion to hexadecane. Adhesion of S. pyogenes to large hexadecane droplet.
145 increase linearly with increasing hexadecane volume employed in the test. A similar kinetic approach has been employed by Kawabata et al. [11] in their studies on removal of bacteria from aqueous media by insoluble polymers. These authors studied the dependency of the rate constants of removal on the ratio of weight of polymer to the water volume and coined the term 'removal coefficient' for the slope of such dependencies. Thus, in our case, 'removal coefficient' refers to the change in the rate constant of removal of bacteria from the bulk aqueous phase to the hydrocarbon:water interface, as a function of the ratio of hydrocarbon to water in the assay. The insert in Fig. 1 presents the slopes obtained as a function of hydrocarbon:water ratio. We submit that the slope of the curve is a quantitative expression of the affinity of the ceils tested for the water: oil interface, and denote it as the removal coefficient (K) of the cells from the bulk aqueous phase by the hydrocarlaon. For S. pyogenes, K was 376 min -1 . Adherence of the cells to hexadecane droplets is shown in Fig. 2. As Gibbons and Etherden pointed out [10], bacterial adherence to hydrocarbons yields reproducible results only when the conditions employed are carefully controlled (i.e., test tube dimensions). The kinetic approach suggested here should enable investigators to compare results obtained in their own laboratory, e.g., adherence of various strains, effects of growth conditions and treatments, in a quantitative fashion. Moreover, relative removal coefficients should be independent of experimental conditions as long as the identical conditions are employed to derive the removal coefficient. Furthermore, in preliminary studies, we repeated the experiment shown in Fig. 1 by employing faster agitation while maintaining all other parameters identical. Although the rate constants were much higher than those described in Fig. 1, a similar removal coefficient (414 min -j) was obtained. It is thus possible that the removal coefficient is independent of agitation conditions. Preliminary experiments in our laboratories suggest the general applicability of the quantitative approach proposed in the present manuscript: (i) S. pyogenes cells, harvested from logarithmic growth phase, yielded results similar to those presented here for stationary phase cells, but with coefficients several orders of magnitude lower; (ii) other bacterial species (Acinetobacter calcoaceticus, Serratia rnarcescens) exhibit linear dependencies of the removal rate constants on the hexadecane: water ratio, enabling derivation of their removal coefficients, in the manner described here.
Acknowledgement We thank Yardena Mazor for excellent technical assistance.
References 1 2 3 4
Mudd, S. and Mudd, E.B.H. (1924) The penetration of bacteria through capillary spaces. IV. A kinetic mechanism in interfaces. J. Exp. Med. 40, 633-645. Rosenbcrg, M., Gutnick, D. and Roscnbcrg, E. (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol. Lett. 9, 29-33. Rosenbcrg, M. (1984) Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. FEMS Microbiol. Lett. 22, 289-295. Ofek, I., Whimack, E. and Bcachey, E. H. (1983) Hydrophobic interactions of group A streptococci with hexadccane droplets. J. Bacteriol. 154, 139-145.
146 5 6 7 8 9 10 I1
Rosenberg, M. and Rosenberg, E. (1981) Role of adherence in growth of Acinetobacter calcoaceticus RAG-I on hexadecane. J. Bacteriol. 148, 51-57. Gibbons, R.J., Etherden, I. and Skobe, J. (1983) Association of fimbriae ~vith the hydropbobicity of Streptococcus sanguis FC-I and adherence to salivary pellicles. Infect. lmmun. 41,414-417. Rosenberg. M. (1984) Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity. J. Bacteriol, 160, 480-482. Rosenberg, M., Bayer. E.A., Delarea, J. and Rosenberg, E. (1982) Role of thin fimbriae in adherence and growth of Acinetobacter calcoaceticus on hexadecane. Appl. Environ. Microbiol. 44, 929-937. Olsson, J. and Westergren, G. (1982) Hydrophobic surface properties of oral streptococci. FEMS Microbiol. Lett. 15, 319-323. Gibbons, R.J. and Etherden, I. (1983) Comparative hydrophobicities of oral bacteria and their adherence to salivary pellicles. Infect. Immun. 41, 1190-1196. Kawabata, N., Hayashi, T. and Matsumoto, T. (1983) Removal of bacteria from water by adhesion to cross-linked poly (vinylpyridinium halide). Appl. Environ. Microbiol. 46, 203-210.