Author: Caitlin H. Bell
Institution: Worcester Polytechnic Institute
Date: June 2005
Abstract
The ability of a bacterium to adhere to various surfaces is important in environmental and biomedical applications. While studying bacterial adhesion in the laboratory, unwanted artifacts can be caused by cell preparation and treatment protocols, which are used in virtually all experimental investigations. We investigated the effects of three cell separation methods (centrifugation, multiple rounds of centrifugation, and filtration) on the retention behavior of two Gram-negative bacteria: Pseudomonas putida KT2442 and Escherichia coli HB101. Their surfaces are different, since P. putida possesses extracellular polysaccharides (EPS), while the surface chemistry of E. coli HB101 is dominated by lipolysaccharides (LPS) and proteins. The retention of bacteria to glass was quantified in batch assays and described in terms of the collision efficiency (α), the ratio of attaching cells to the total number of cells that contact the substrate. Filtration as a separation technique can be considered a "control" treatment, and for both bacteria the α values were high for these experiments. For P. putida KT2442, filtration or gentle centrifugation results in similarly high α values. Centrifugation at a higher force decreased α, and centrifugation at this force multiple times further decreased α to zero, meaning that cells do not attach at all. For E. coli HB101, an initial decrease in α was seen for the centrifuged cells, but cells centrifuged one or multiple times had the same α values. In light of these findings, future researchers must be cautious to consider the adverse effects of centrifugation when used as part of bacterial treatment protocols.
Introduction
Bacterial adhesion is important in numerous biological applications, from oral biofilm formation leading to dental caries (Kohlenbrander 1997; Tang et al. 2004) to subsurface remediation of contaminants using bacteria capable of bioremediation (MacDonald et al. 1999). While studying these bacterial characteristics, cells are routinely subjected to a number of experimental protocols in laboratories. These protocols can affect the physiochemical and/or microbiological behavior of the bacteria, creating artifacts in the measurement of adhesion and other properties (Pembrey et al. 1999). Drying, freezing, exposure to ionic solutions, sonication, resuspension, and centrifugation are just some of the processes that need to be evaluated for their potential to cause artifacts. More specifically, the effects of centrifugation on a bacterium's ability to adhere to surfaces are of particular concern since multiple centrifugation steps are commonly found in cell preparation protocols. Centrifugation may alter cellular surface macromolecules, which are known to contribute to cellular adhesion (Sutherland 1983; Park et al. 2000; Tsuneda et al. 2003). These surface materials can be stripped off or compressed during centrifugation (Gilbert et al. 1991; Pembrey et al. 1999). Recent work (Makin and Beveridge 1996; Smets et al. 1999) suggests that the removal or damage to surface polysaccharides during centrifugation contribute to a decreased ability of the bacterium to adhere.
While some research has addressed the effects of centrifugation on cell properties, mixed results were obtained. Centrifugation can affect hydrophobicity and electrophoretic mobility (a measure of the surface potential or charge of the bacterium), as well as cause unpredictable adhesion behavior, as was seen for Escherichia coli ATCC 8739 (Pembrey et al. 1999). Such treatment also makes cells more susceptible to biocides, perhaps by destabilizing the cell envelope (Gilbert et al. 1991). In one study, centrifugation was employed to purposely reduce the adhesive ability of P. aeruginosa to buccal epithelial cells (Ferguson et al. 1992). Mechanical surface damage from extended centrifugation at 9,600 x g has been shown to reduce initial cell adhesion to silicone rubber, polymethylmethacrylate, and glass in P. aeruginosa strain #3 (Bruinsma et al. 2001). This damage also made cells less able to withstand detachment forces from the same surfaces after sonication (Bruinsma et al. 2001). Centrifugation of exponential phase cells at high force (10,000 x g) substantially reduced electrophoretic mobilities and subsequently the collision efficiencies (α; probability that collisions result in attachment) of Pseudomonas fluorescens in batch retention tests and column adhesion assays (Smets et al. 1999). Variability in past findings may be due to different centrifugation protocols and strain-to-strain differences in cell properties.
We investigated the role of centrifugation on the attachment and retention of bacteria with varying surface properties. Two types of Gram-negative bacteria were studied: Pseudomonas putida KT2442 and Escherichia coli HB101. KT2442 is a motile bacterium (possessing monotrichous flagella) known to have EPS on its surface that contribute to bacterial adhesion (Camesano and Abu-Lail 2002). HB101 is non-motile (has no flagella) and lacks the EPS layer on its surface, so LPS and surface proteins are more able to contact the interacting substrates. Previous work with P. putida KT2442 suggested that very low force centrifugation and separation from growth media by manual filtration were control cases, since neither affected bacterial retention, transport, or adhesion forces probed with an atomic force microscope (AFM) (Bell et al. 2005). Therefore, separation by filtration was treated as the control in the present study. We compared filtration with bacterial cells that were separated by single or multiple centrifugation steps before resuspension in ultrapure water. After treatment, the retention of bacteria in batch tests with glass was quantified. The results of this study will be useful to those designing bacterial adhesion tests in the laboratory.
Materials and Methods
Culture of Pseudomonas putida KT2442. P. putida KT2442, a Gram-negative, motile bacterial strain with EPS on its surface that contribute to bacterial adhesion (Camesano and Abu-Lail 2002), was chosen for experimentation. Culture of KT2442 bacteria to the late exponential phase was performed as described previously (Abu-Lail and Camesano 2002; Abu-Lail and Camesano 2003; Bell et al. 2005). Culture of Escherichia coli HB101. E. coli HB101 was obtained from the American Type Culture Collection (ATCC 33694). The bacterium is also Gram-negative, but it is non-motile and lacks an EPS layer. Cultures were grown in Luria Broth (10 g/l tryptone, 5 g/l yeast, 10 g/l NaCl) at 25°C and 50 rpm on a rotator for approximately 48 hours or until turbid. The pre-culture (1 ml) was transferred to 50 ml fresh Luria Broth at 37°C in a horizontal shaker bath until late exponential growth stage, which occurred after approximately 3 hours, corresponding to an optical density of 0.9-1.0 at 600 nm. Filtration. KT2442 and HB101 cells were each separated from their respective growth media by manual capturing onto a 0.2-_m syringe filter and back-washing into solution (Bell et al. 2005). This set of experiments was treated as the control case, for comparison with the centrifuged cells. Single-Step Centrifugation and Multiple-Step Centrifugation. To determine the effects of centrifugation on bacterial adhesion, cells were separated from growth media via a single centrifugation step. Cells were centrifuged for 10 minutes, at a relative force of 3,411 x g and resuspended in an equal amount of ultrapure water (milli-Q, Millipore Corp.). To determine the extended effect of centrifugation on bacterial adhesion, cells were separated from growth media via multiple centrifugation steps. Cells were centrifuged for 10 minutes at 3,411 x g and resuspended in an equal amount of ultrapure water. This centrifugation and resuspension procedure was repeated twice more so that the cells were subjected to a total of 30 minutes of centrifugation. Batch Retention Experiments. Filtered, single-centrifuged, and multiple-centrifuged P. putida and E. coli cells were allowed to adhere to glass slides to determine the number of cells retained on glass and to quantify the collision efficiency. If the surface coverage of bacteria to the slides increases linearly with t1/2, the dominant transport mechanism is molecular diffusion. This situation was verified previously for P. putida KT2442 by measuring the coverage at several time points (Bell et al. 2005). Subsequent to this verification, the time point of two hours was chosen for future batch reactors because it yielded a moderate number of attached cells to the glass, which could be easily and reproducibly counted under the microscope.
Glass microscope slides were cut, cleaned, and immersed in triplicate reaction beakers following a procedure we described previously (Bell et al. 2005). After the reaction time, the slides were rinsed, stained, and investigated under an epifluorescence microscope using an oil immersion objective (Bell et al. 2005). The average number of bacteria per microscopic field was translated to a retention count of cells per square centimeter by dividing the average number of bacteria per field by the size of the microscope field. The collision efficiency (α) was calculated using a published expression (Skoog et al. 1996). The radii of the bacteria were measured to be 0.4x10-6 m for P. putida and 0.75 x10-6 m for E. coli.
Results
Figure 1. Comparison of collision efficiencies (α for the filtered, single-step centrifuged, and multiple-step centrifuged conditions for Pseudomonas putida KT2442 and Escherichia coli HB101 in the batch retention tests. Mean collision efficiencies ± standard error for KT2442 are 1.20 ± 0.23 (n=40), 0.38 ± 0.010 (n=40), and essentially 0 (Not enough to accurately count the number retained, ie. _ = 0) for filtered, single-step centrifuged, and multiple-step centrifuged conditions respectively. For HB101, α values are 0.56 ± 0.06 (n=60), 0.29 ± 0.07 (n=60), and 0.26 ± 0.06 (n=60) respectively. All centrifugation steps were conducted for 10 minutes at 3,411 x g. Multi-step centrifugation refers to three rounds of centrifugation at these conditions, with washing in between with ultrapure water.
Pseudomonas putida KT2442. The collision efficiencies for P. putida KT2442 were 1.20 ± 0.23, 0.38 ± 0.01, and essentially 0, for filtered, single-step centrifuged and multiple-step centrifuged conditions, respectively, based on batch assays (Figure 1). In a previous study, centrifugation of this bacterium at a very low centrifugal force (190 x g) produced α values in the batch retention tests that were identical to the values obtained on filtered cells (1.19 ± 0.25) (Bell et al. 2005). Additionally, the effect of the enzymatic removal of the dominant surface polymer at the same centrifugal force (190 x g), cellulose, was investigated previously (Bell et al. 2005). It was determined that the removal of this compound had a detrimental effect on the ability of P. putida KT2442 by reducing the collision efficiency by 40% (α value of 0.69 ± 0.13). Escherichia coli HB101. For E. coli HB101 in batch retention assays, we also saw that filtered cells were more retained than the moderately centrifuged cells. The α values in the batch tests were 0.56 ± 0.06, 0.29 ± 0.07, and 0.26 ± 0.06 for the filtered, single-step centrifuged and multiple-step centrifuged conditions, respectively (Figure 1). In this case, single and multiple steps of centrifugation resulted in nearly equal α values for the batch tests.
Discussion and Conclusions
Behavior of Bacteria in Batch Retention Tests. As was seen in previous work (Bell et al. 2005), there is no significant difference between the ability of P. putida cells to retain to glass when separated from growth media via filtration and low-force centrifugation (190 x g). This means that in this and other future works, either low-force centrifugation or filtration can safely be used as "control" cell separation protocols.
A progressive decrease in α values was seen, however, when comparing cell retention for the filtered, single-step centrifuged and multiple-step centrifuged conditions across the two species (Figure 1). When subjected to 3,411 x g for 10 minutes, both species yielded similar collision efficiencies (Figure 1). These values were lower than the filtered α values by ~70% for the P. putida and ~50% for the E. coli. This reduction in adhesion of P. putida cells indicates that initial centrifugation may have affected the surface polymers, rendering the cell less able to attach. This can be surmised since a similar reduction in adhesion ability was seen after removal of surface cellulose polymers (Bell et al. 2005). Since there was also a reduction in E. coli's ability to adhere, initial centrifugation may affect more than just EPS, possibly causing cell envelope damage, as described by Gilbert et al. (1990).
After three washings at 3,411 x g for 10 minutes each, the E. coli cells showed very similar adhesion behavior to the cells from the single-step centrifuged run. This implies that there was no further damage done to the cells in this extended centrifugation time that would affect how they adhered, or that most of the damage done by centrifugation to E. coli cells is done in the first 10 minutes, as suggested by Gilbert et al. (1990). However, the P. putida cells showed such a remarkable reduction in adhesion ability that the collision efficiency was reduced to essentially zero. Even after repeated attempts at performing this experiment, no more than a few bacterial cells could be found on the entire glass slide. This shows that extended centrifugation indeed has a detrimental effect on the adhesion ability of P. putida KT2442. This may be due to the additional force exerted on the surface polymers. When surface polymers were removed or damaged, perhaps there were fewer contacts available between bacterial surface polymers and the glass substrate.
Species Comparison. The large difference in α values (for example, 53% for filtered cells) between P. putida and the E. coli can be attributed to the fact that E. coli cells do not have an EPS layer that aids in adhesion, while P. putida does have this layer (Goodacre and Berkeley 1991; Abu-Lail and Camesano 2002). Sometimes specific interactions between bacterial EPS and substrate surfaces facilitate bacterial adhesion (Simoni et al. 1998). If this polymeric layer is not present, these specific interactions cannot occur. This difference in retention could also be due to the motility of P. putida. In batch retention tests, the dominant transport mechanism the bacteria experience is diffusion, but bacterial swimming can compete with diffusion, leading P. putida cells to have a greater advantage in reaching and attaching to the glass surfaces (McLaine and Ford 2002). Conclusions. The adverse effects of centrifugation on a bacterium's ability to adhere were seen in batch retention tests for P. putida KT2442 and E. coli HB101. Single-step centrifugation reduced the adhesion of P. putida KT2442 cells to glass, presumably due to damage to the EPS layer. It may also have damaged the cell membrane or altered the LPS molecules embedded in this membrane, as was suggested by the E. coli data. Multiple centrifugation steps appeared to severely impact the EPS layer of P. putida, while having less of an effect on LPS, as the attachment of E. coli was less affected. In light of these findings, future researchers must be conscientious of the potentially adverse effects of centrifugation on cellular properties when used as part of treatment protocols.
Acknowledgements
This publication was made possible in part by the National Science Foundation (BES-0238627). We also acknowledge the donors of the Petroleum Research Fund of the American Chemical Society, for partial support of this work (grant PRF 38988-G2). CHB was partially funded by an Academic Excellence Award from the GE Fund at the Pennsylvania State University, through the Women in Engineering Program. We are grateful to Ms. Barbara Bogue for her assistance with this award. In addition, we would like to thank Dr. George Pins, Mr. Ray Emerson, Dr. Nehal Abu-Lail, and Mr. Brett Downing for their guidance and technical assistance during this study.
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