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Direct Infrared Spectroscopic Evidence of pH- and Ionic Strength-Induced Changes in Distance of Attached Pseudomonas aeruginosa from ZnSe Surfaces Michael J. McWhirter,† Philip J. Bremer,‡ and A. James McQuillan*,† Departments of Chemistry and Food Science, University of Otago, and New Zealand Institute for Crop & Food Research, P.O. Box 56, Dunedin, New Zealand Received June 19, 2001. In Final Form: September 25, 2001 Control of bacterial adhesion is important in many industrial and medical applications. Ionic strength and pH are known to affect the process of attachment; however their influence on bacteria/surface distances has been inferred but not quantitatively established. The distances between attached bacteria and surfaces have only recently been measured. In this paper ATR-IR spectroscopy has been used to study the influence of pH and ionic strength on the distance of freshly attached Pseudomonas aeruginosa from ZnSe surfaces. Reversible changes in the absorbance of P. aeruginosa were observed for changes of pH between 4 and 10 at a constant ionic strength of 0.003 mol L-1 and for changes of ionic strength between 0 and 0.15 mol L-1 at a constant pH of 6.3. An increase in ionic strength from 0 to 0.003 mol L-1 led to an increase in amide II absorbance of 30%, which corresponds to an average movement of 120 nm toward the surface. A movement of this magnitude is likely to be due to changes in the length of bacterial surface polymers. The influence on bacterial surface polymer lengths of ionic strengths in this range has not been widely considered in models of bacterial attachment.
Introduction While it is well established that bacteria will readily attach to a wide range of abiotic surfaces, the details of the attachment process remain unclear.1,2 It has been hypothesized that cell attachment takes place over various stages: a reversible adsorption step governed by longrange dispersion and electrostatic forces acting on individual bacteria, followed by irreversible adhesion of bacteria to surfaces, and in some cases communication and cooperation between adsorbed bacteria to form a biofilm.3,4 The behavior of the bacteria once attached is a significant, but often unconsidered, aspect of the attachment phenomenon.5 The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, originally used to account for the stability of colloidal dispersions, is the principal model used for bacterial attachment. This theory is based on a combination of van der Waals attractive forces and electrical double layer repulsive interactions. For bacterial interaction with a surface the DLVO theory,6 and its extension to include hydrophobic interactions,7 describe a secondary energy minimum on the order of 10 nm from the surface, where bacteria are postulated to accumulate. On this basis the behavior of attached bacteria is expected to respond to changes in ionic strength and charge as these variables impact on the position of the secondary energy minimum. * To whom correspondence may be addressed. E-mail:
[email protected]. Phone: +64 3 479 7928. Fax: +64 3 479 7906. † Department of Chemistry, University of Otago. ‡ New Zealand Institute for Crop & Food Research and Department of Food Science, University of Otago. (1) Mittleman, M. W. J. Dairy Sci. 1998, 81, 2760. (2) Ong, Y.; Razatos A.; Georgiou G.; Sharma M. M. Langmuir 1999, 15, 2719. (3) Marshall, K. C.; Stout, R.; Mitchell, R. J. Gen. Microbiol. 1971, 68, 337. (4) Watnick, P.; Kolter, R. Mol. Microbiol. 1999, 34, 586. (5) Otto, K.; Norbeck, J.; Larson, T.; Karlson, K.-A.; Hermansson, M. J. Bacteriol. 2001, 183, 2445. (6) Hermansson, M. Colloids Surf., B 1999, 14, 105. (7) van Oss, C. J. Cell Biophys. 1989, 14, 1.
Total internal reflection aqueous fluorescence microscopy, where fluorescein-dextran is present with the bacteria, has been used to measure the position of attached bacteria relative to surfaces. Distances of 54-103 nm between attached Escherichia coli and quartz surfaces have been reported.8 Evidence for a distance of attachment larger than 10 nm has also been provided by atomic force microscopy. Using this technique repulsion between bacteria covalently fixed to a surface and approaching silicon nitride tips has been measured over distances of hundreds of nanometers.9 The repulsion was attributed to electrosteric interactions due to bacterial surface polymers, whose length changed as pH varied.9 Cell surface molecules are thought to be important in attachment,10 and other work has indicated that ionic strength can affect the length of bacterial polymers. For example, extracellular polysaccharides have configurations that are influenced by ionic strength.11 Further, ionic strength has been shown by interference reflectance microscopy to influence the position of cells relative to surfaces due to condensation of bacterial surface polymers.12 To elucidate the impact of pH and ionic strength on the position of attached cells a technique that can study attached bacteria in-situ and without the presence of added complex chemicals or covalent bonding is desirable. Attenuated total reflection infrared (ATR-IR) spectroscopy is one such technique. ATR-IR spectroscopy has previously been used to study the rates of cell attachment and growth on surfaces.13-15 (8) Vigeant, M. A., Wagner, M., Tamm, L. K., and Ford, R. M. Langmuir 2001, 17, 2235. (9) Camesano, T. A.; Logan B. E. Environ. Sci. Technol. 2000, 34, 3354. (10) Rijnaarts, H. H. M.; Norde, W.; Lyklema, J.; Zehnder, A. J. B. Colloids Surf., B 1999, 14, 179. (11) Frank, B. P.; Belfort, G. Langmuir 1997, 13, 6234. (12) Marshall, P. A.; Loeb, G. I.; Cowan, M. M.; Fletcher, M. Appl. Environ. Microbiol. 1989, 55, 2827. (13) Bremer, P. J.; Geesey, G. G. Biofouling 1991, 3, 89. (14) Schmitt, J.; Nivens, D.; White, D. C.; Flemming, H. Wat. Sci. Technol. 1995, 32, 149.
10.1021/la010928k CCC: $22.00 © 2002 American Chemical Society Published on Web 01/29/2002
Distance of Attached Pseudomonas aeruginosa
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In this paper the influence of changes in the pH and ionic strength of the bulk aqueous phase on the behavior of Pseudomonas aeruginosa attached to ZnSe was studied following a 1 h period of attachment in the presence of 0.003 M KCl. Resulting ATR-IR spectroscopic evidence for the influence of pH and ionic strength on the distance of attached P. aeruginosa from the ZnSe surface is presented and discussed. Methods Preparation of IRE. Before each experiment, the ZnSe internal reflection element (IRE) was polished for 2 min on a Buehler polishing microcloth with an aqueous 0.015 µm Al2O3 slurry (BDH). The IRE was sonicated in ethanol for 5 min and then in water (MilliQ) for a further 5 min. Preparation of Bacterial Inoculum. The P. aeruginosa strain used was obtained from the New Zealand Culture Collection (strain NZRM918). The isolate was cultured and maintained on Tryptic Soy Agar or Tryptic Soy Broth (Difco, Fort Richard, New Zealand) supplemented with 0.6% yeast extract (Difco) (TSAYE or TSBYE, respectively). Stock cultures were maintained with the Microbank cryovial system (Pro-lab Diagnostics, Richmond Hill, Ontario, Canada) at -80 °C. To prepare a culture to attach to the IRE, a single colony from a streak plate was inoculated into 10 mL of a defined medium (Vogel’s)16 and incubated in a 500 mL conical flask on a rotary shaker (170 rpm) at 25 °C for 24 h. Fresh Vogel’s media (190 mL) was added, and the culture was incubated for a further 24 h. The suspension was centrifuged (10 min, 7520 g, 4 °C), and the pellet was resuspended in water and washed a further 2 times before being resuspended in an aqueous solution of 0.003 mol L-1 KCl to obtain a concentration of bacteria of ∼1010 cfu mL-1. Bacterial numbers in the inoculating suspension were confirmed by diluting (peptone, 0.1%, Oxoid) and plating onto TSAYE using the drop plate technique. Flow Procedures. A flow cell with an internal volume of 0.22 mL and a surface contact area of 2.2 cm2 was clamped onto the surface of the IRE and sealed via a silicon rubber gasket. The flow cell assembly was placed in the sample chamber of the infrared spectrometer (BioRad, FTS 60) and the chamber purged with dry air for 120 min. During this time the ZnSe surface was washed for 60 min with 10-2 mol L-1 NaOH and for 60 min with water to provide a background for the subsequent bacterial spectra. All solutions were flowed across the surface of the IRE at a flow rate of 2 mL min-1, which gave a residence time within the flow cell of 6.6 s. Spectra Acquisition. All spectra were constructed from 64 scans taken at a resolution of 4 cm-1 using Win-IR software (BioRad, Cambridge, MA). The baseline was corrected by setting the absorbances at 1800 and 900 cm-1 to zero and assuming a straight line between these points. Attachment of P. aeruginosa to the IRE. The suspension containing bacterial cells was passed over the surface of the IRE for 60 min in a 3 × 10-3 mol L-1 KCl solution. This resulted in the development of an attached layer of bacteria. After this 60 min period the flow of bacteria was stopped and all subsequent flowed solutions were bacteria-free. Impact of pH on Absorbance of Attached Bacteria. To study the influence of the pH of the surrounding aqueous solution on the absorbance of the attached bacteria, the attached layer was washed sequentially with cell-free solutions of different pH. Aqueous solutions of pH ) 4, 6.3, and 10 all with an ionic strength of 0.003 mol L-1 were used. All solutions were washed over the attached cells for 10 min. Readings were taken throughout this period with steady absorbances obtained within 1 min. The pH of solutions was adjusted to either pH ) 4 or 10 by the addition of 10-2 mol L-1 NaOH or HCl, and the ionic strength was made up to 0.003 mol L-1 by the addition of KCl. The effluent was checked to confirm that the number of bacteria in the bulk medium was too low to affect the measured absorbance. (15) Nivens, D. E.; Ohman, D. E.; Williams, J.; Franklin, M. J. J. Bacteriol. 2001, 183, 1047. (16) Vogels, H.; Bonner, D. J. Biological Chem. 1956, 218, 97.
Figure 1. Dependence on solution pH, at ionic strength ) 0.003 mol L-1, of the ATR-IR spectra of P. aeruginosa attached to ZnSe: (a) spectra from pH ) 4, 6.3, and 10 solutions; (b) amide II absorbances from duplicate experiments after 10 min at solution pH ) 4, 6.3, 10, 4, 10, and 6.3 recorded in sequence. Note that the time scale indicates the sequence of the experiment and steady absorbances were obtained within 1 min of each solution change. Impact of Ionic Strength on Absorbance of Attached Bacteria. An experiment similar to that outlined above, but with constant pH (6.3) and varying ionic strength, was conducted to determine the influence of surrounding solution ionic strength on the absorbance of the attached bacteria. The ionic strengths of the solutions were adjusted with KCl to 0, 0.003, 0.030, or 0.150 mol L-1. As before, the number of bacteria in the effluent was checked. Impact of Ionic Strength and pH on Absorbance of Adsorbed BSA Protein. The influence of pH and of ionic strength of the surrounding aqueous solution on the absorbance of attached pure protein was also investigated. BSA protein was adsorbed by washing the ZnSe surface with 10-5 mol L-1 BSA, and experiments identical to those with attached P. aeruginosa were carried out. Spectral Features. Spectral features of bacteria have been assigned.17 The infrared spectra of bacteria are dominated by the amide I (1650 cm-1) and amide II (1550 cm-1) absorptions of protein and a composite band about 1080 cm-1 due mainly to polysaccharide. The absorbance of the amide II band has been previously used as a measure of the number of bacteria on a surface, as the absorption of water can affect amide I absorbances.13-15
Results and Discussion The absorbance of P. aeruginosa cells attached to ZnSe changed as the pH of the surrounding bulk aqueous phase was varied at constant ionic strength (0.003 mol L-1) in a flow system (Figure 1). Figure 1a shows that the spectral profile of attached bacteria is unchanged as absorbance increases. Thus, the variation in the absorbance of the amide II band can be used to represent that of the whole spectrum. The absorbance ratio of protein to polysaccha(17) Le Gal, J.; Manfait, M.; Theophanides, T. J. Mol. Struct. 1991, 242, 397.
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ride bands remains constant (data not shown), indicating that the contribution from any protein conditioning film is insignificant. Figure 1b shows the changes of amide II absorbance with pH in the sequence that they were recorded during two typical experiments, illustrating the reversibilty of the absorbance changes. Solutions of different ionic strength but constant pH (6.3) also affected the absorbances of the attached P. aeruginosa cells (Figure 2). In the two sets of data shown the amide II absorbance increases by 30% and by 64% with an increase in ionic strength from 0 to 0.003 mol L-1 (measurements at 10 and 20 min). There was a further absorbance increase as the ionic strength changed to 0.030 mol L-1, but with a change in ionic strength from 0.030 to 0.15 mol L-1 the absorbance decreased. It is important to note that the solutions of different pH or ionic strength were free of bacteria. This shows that the reversible changes in absorbance could not have been due to cells detaching from, or more cells attaching to, the surface. The rapid absorbance changes (less than 1 min) after each solution change precludes cell replication and osmotic factors as causes. Also when BSA, rather than P. aeruginosa, was the adsorbed species, there was no change in absorbance with pH or ionic strength changes in the surrounding solution (data not shown). This indicates that attached P. aeruginosa behave differently compared to adsorbed protein in response to changes in surrounding solution composition. With ATR-IR spectroscopy, infrared light undergoes total internal reflection at the interface of the IRE with the aqueous phase. The internal reflection has an associated evanescent wave that is propagated in the less dense medium where bacteria absorb the radiation. The electric field amplitude of this wave (E) decays exponentially with distance (z) from the surface of the IRE18 as described by
E ) E0e-z/dp
(1)
E0 is the field amplitude at the surface of the IRE, and the penetration depth (dp) is the distance required for the electric field amplitude to decay to E0/e. The penetration depth18 is given by
dp )
(
λ
( ))
n2 2πn1 sin θ n1 2
2 1/2
(2)
In eq 2, λ is the wavelength of the incident radiation, n1 is the refractive index of the denser medium, θ is the incident angle, and n2 is the refractive index of the less dense medium. In our experiment θ ) 45°, n1 ) 2.42 (for ZnSe), and λ is equal to 6.45 µm (at 1550 cm-1). The refractive index of bacterial components is slightly higher than that of water.19 We have used n2 ) 1.33 (for water) as this difference is not significant in the calculations. Thus, eq 2 gives dp ) 0.93 µm. For an increase in amide II absorbance there must be an increase in the amount of protein within the evanescent wave or the protein that is already present must move closer to the surface. In the latter case it would interact with the evanescent wave where its electric field amplitude is greater. An increase in cell density (cells per unit volume) is effectively a movement of material toward the surface. Figures 1 and 2 show that even when there were no bacteria flowing through the flow cell, cyclical changes in (18) Mirabella, F. J. Appl. Spectrosc. Rev. 1985, 21, 45. (19) Heavens, O. S. J. Cell Sci. 1990, 95, 175.
Figure 2. Dependence on solution ionic strength, at pH ) 6.3, of the ATR-IR spectra of P. aeruginosa attached to ZnSe. Amide II absorbances from duplicate experiments after 10 min at solution ionic strengths of 0.003, 0, 0.003, 0.03, 0.15, 0.03, and 0 mol L-1 were recorded in sequence. Note that the time scale indicates the sequence of the experiment and steady absorbances were obtained within 1 min of each solution change.
the amide II absorbance were observed. The return of the absorbance to its original value when the original conditions were restored indicated that there was no significant change in the number of bacteria on the surface. Therefore, the absorbance changes indicate that there must have been movement of the bacteria in a direction normal to the surface. This movement can be estimated from the change in absorbance (A), where A is proportional to E2.18 Assuming the size, composition, and orientation of the bacteria are constant, a proportional relationship between the distance of the bacteria from the surface (z) and A can be obtained:
A ∝ e-2z/dp
(3)
Using this relationship it becomes apparent that the 30% increase in absorbance measured when the ionic strength increases from 0 to 0.003 mol L-1 corresponds to an average bacterial movement of 120 nm in a direction toward the substratum/liquid interface. To account for the variation with pH of the spectral data the pH dependence of the charge of both the surface and of the bacteria need to be considered. The isoelectric point of ZnSe occurs at about pH ) 4,20 and it is expected to have increasing negative charge with pH increase above the isoelectric point. The P. aeruginosa isoelectric point has been measured at 2.7, and electrophoretic mobility measurements show that the negative charge increases significantly as pH increases.21 Thus, the observed increase in absorbance with pH decrease from 10 to 4 (at constant ionic strength) may be due to the decrease in electrostatic repulsion between the solid surface and the bacteria. This is described by the DLVO theory of bacterial adhesion. In the theory ionic strength also plays a part, as the presence of ions in solution shields the opposing charges from one another. An increase in ionic strength would lead to a smaller distance between the bacteria and the surface, which could explain the larger absorbance signal with increased ionic strength. This shows that qualitatively the DLVO theory provides an explanation for the observed results. However, quantitatively the DLVO theory only predicts movements of tens of nanometers.6 It has been reported that increases in pH can increase bacterial surface polymer length by hundreds of (20) Tickanen, L.; Tejedor-Tejedor, M.; Anderson, M. Langmuir 1991, 7, 451. (21) Collins, Y. E.; Stotzky, G. Appl. Environ. Microbiol. 1992, 58, 1592.
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nanometers due to increased repulsion between polymer units.9 In that study changes in polymer length with a change in ionic strength were not observed and the authors suggested the use of organic, rather than inorganic, buffers as a reason for this. However, conformational changes in bacterial extracellular polymers11 and surface polymers12 with ionic strength have been reported. If surface polymers on the bacteria changed length, this would alter the density of bacteria near the surface. So the changes in absorbance with change in environmental pH and ionic strength reported here could be due to changes in length of bacterial surface polymers. Figure 2 shows that when the ionic strength was increased from 0.03 to 0.15 mol L-1 the absorbance decreased, while for the increases at lower ionic strength the absorbance increased. There is a change in the trend of P. aeruginosa attachment rates at an ionic strength of 0.1 mol L-1.22,23 This result and similar ones for other bacteria are considered to be evidence of a transition from a situation described by DLVO theory to one where salt concentration affects bacterial surface molecules.6,10 The changes in absorbance reported here show that bacterial (22) Stanley, P. Can. J. Microbiol. 1983, 29, 1493. (23) McWhirter, M. J.; McQuillan, A. J.; Bremer P. J. Colloid Surf., B, in press.
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surface molecules are also influenced at lower ionic strengths, and this confirms and extends previous work of others.12 In conclusion, changes in amide II absorbance are seen when attached P. aeruginosa are subjected to independent changes in solution pH and ionic strength. The reversible nature of the absorbances changes indicates that the causes are movements on the order of 100 nm in a direction normal to the surface. The mechanism causing this movement may involve changes in surface polymer lengths related to charge affects qualitatively described by the DLVO theory. If such charge effects apply to the distance between parts of the surface polymers, this may account for the DLVO theory qualitatively describing the movement observed. Acknowledgment. This work was supported by the New Zealand New Economy Research Fund Grant No. CO8X9903: Functional Interfaces and Materials. We acknowledge fruitful discussions with other groups funded by this grant and with Prof. Gill Geesey, Center for Biofilm Engineering/Microbiology Department, Montana State University, Bozeman, MT. LA010928K
