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J Dent Res 85(3):231-234, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Interactive Forces between Co-aggregating and Non-co-aggregating Oral Bacterial Pairs

F. Postollec1, W. Norde1,2, J. de Vries1, H.J. Busscher1, and H.C. van der Mei1,*

1 Department of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; and
2 Laboratory of Physical Chemistry and Colloid Science, Wageningen University, PO Box 8038, 6700 EK Wageningen, The Netherlands

* corresponding author, h.c.van.der.mei{at}med.umcg.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The temporo-spatial development of plaque is governed by adhesive interactions between different co-aggregating bacterial strains and species. Physico-chemically, these interactions are due to attractive Lifshitz-Van der Waals and acid-base forces, and occur despite electrostatic repulsion and with a critical influence of temperature. The forces between co-aggregating and non-co-aggregating pairs have never been measured, however. The aim here, thus, is to investigate, by atomic force microscopy, whether there is a difference in interactive forces between co-aggregating and non-co-aggregating bacterial pairs at 10°C, 22°C, and 40°C. Actinomyces naeslundii 147 was immobilized on poly-L-lysine-coated tipless AFM cantilevers, while streptococci were immobilized on poly-L-lysine-coated glass surfaces. Upon approach, a repulsive force was measured, regardless of whether a co-aggregating or non-co-aggregating pair was involved. However, upon retraction, the co-aggregating pair exhibited larger adhesive forces and energies than did the non-co-aggregating pair. Adhesive interactions between the co-aggregating pair were smallest at 40°C.

KEY WORDS: co-aggregation • streptococci • actinomyces • atomic force microscopy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recognition between a localized receptor and ligand plays a central role in microbial aggregation and adhesion. Specific recognition and interactions between bacteria from different species in suspension are defined as co-aggregation. Co-aggregation between different bacterial strains and species has been described for aquatic biofilms (Rickard et al., 2003), and for human biofilms in the urogenital tract (Reid et al., 1988), and has been reviewed by Kolenbrander (1989) for the oral cavity. Recently, co-aggregation between actinomyces and streptococci was shown to be directly involved in the temporo-spatial development of dental plaque in vivo (Palmer et al., 2003).

Macroscopically, co-aggregation is evaluated qualitatively by the mixing of 2 bacterial suspensions. In the case of a co-aggregating pair, large visible co-aggregates are immediately formed, leaving a clear supernatant, referred to as the ’maximal score of four’. For a non-co-aggregating pair, the suspension remains evenly turbid, with no visible binding of cells, and a score of zero is given (Kolenbrander, 1989). Semi-quantitative measures of oral bacterial co-aggregation have been obtained from turbidimetric measurements (Handley et al., 1987). From a physico-chemical point of view, co-aggregation has been demonstrated to be due to attractive Lifshitz-Van der Waals and acid-base forces, while occurring despite electrostatic repulsion and with a critical influence of temperature on this balance of forces (Bos et al., 1996a,b). These forces are always operative between 2 cell surfaces and act pair-wise among all atoms involved in the entire cell body, in which case they are referred to as ‘non-specific forces’. Alternatively, forces may act between highly localized and stereo-chemically complementary cell-surface groups when present, in which case they are usually referred to as ’specific forces’ (Busscher et al., 1992). The magnitude of the interactive forces between co-aggregating and non-co-aggregating oral bacterial pairs, including both specific and non-specific contributions, has never been directly measured, however.

Atomic Force Microscopy (AFM) has emerged as a powerful tool for the measurement of interactive forces associated with biological systems in an aqueous environment. After proper immobilization of a bacterium, either to the tip of the AFM, or to an appropriately prepared substratum, or both, two interacting surfaces can be brought together. The force-distance curves measured upon approach usually show a repulsive barrier that has to be overcome prior to adhesion, while, upon retraction, adhesive forces are revealed (Razatos et al., 1998; Van der Mei et al., 2000; Lower et al., 2001; Tang et al., 2004). Functionalized probes have been used for the sensing of specific forces on living microbial cells (Bustanji et al., 2003) and the mapping of the cell surface for specific receptors (Gad et al., 1997; Grandbois et al., 2000). Adhesive forces between eukaryotic cells have been measured by AFM (Benoit et al., 2000), but, to our knowledge, the interactive forces between co-aggregating and non-co-aggregating oral bacterial pairs have never been measured directly, because their smaller size poses experimental problems and fewer options for, e.g., force-volume measurements to improve statistical analysis. Statistically, the application of AFM to microorganisms is difficult in any case, since the number of organisms that can realistically be studied in an experiment is always low compared with the number of organisms involved in the formation of a biofilm, like dental plaque.

The aim of this study was to compare the interactive forces between a co-aggregating (Actinomyces naeslundii 147 with Streptococcus oralis J22) and non-co-aggregating (A. naeslundii 147 with Streptococcus sanguis PK1889) oral bacterial pair, as measured by AFM at different temperatures (10°C, 22°C, and 40°C).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Culture Conditions, and Harvesting
S. oralis J22 and S. sanguis PK1889 were cultured in Todd-Hewitt broth (OXOID, Basingstoke, UK) at 37°C under aerobic conditions. A. naeslundii 147 was cultured in Schaedler’s broth at 37°C in an anaerobic atmosphere of 80% N2, 10% CO2, and 10% H2 (Concept 400, Ruskinn Technology Limited, West Yorkshire, UK). For each experiment, the strains were inoculated from blood agar in a batch culture for 24 hrs. This pre-culture was used to inoculate a main culture which was allowed to grow for 16 hrs. Bacteria were harvested by centrifugation (5 min at 5000 x g), washed twice, and re-suspended in demineralized water.

A. naeslundii 147 and S. oralis J22 constitute a co-aggregating pair (score four), while A. naeslundii 147 and S. sanguis PK1889 were used as a non-co-aggregating pair (score zero). A. naeslundii 147 is a completely bald strain that loses its ability to co-aggregate with S. oralis J22 when streptococci are pre-heated (80°C, 30 min), but not when the actinomyces are preheated. This suggests that co-aggregation between A. naeslundii 147 and S. oralis J22 is mediated by a heat-sensitive adhesin on the streptococcal cell surface.

Atomic Force Microscopy
V-shaped silicon nitride tipless cantilevers (Veeco Instruments Inc., Woodbury, NY, USA) were coated with A. naeslundii 147 after being coated with 0.01% poly-L-lysine (Vadillo-Rodriguez et al., 2004). Spring constants were experimentally determined by measurement of the resonance frequency of each cantilever used, from which the spring constant could be calculated according to


Formula 1(1)

where k is the spring constant, f is the resonance frequency, and a is a proportionality constant provided by Veeco (see APPENDIX 1 for resonance frequency determination). Repeated measurements of the spring constant of the same cantilever were highly reproducible (SD less than 0.3%). Since the spring constant denotes the proportionality constant in Hooke’s law between deflection and applied force, the spring constant is not affected by the weight of the bacterial coating, which merely yields an additional deflection accounted for during AFM measurements as an offset, with no influence on the forces measured. Streptococci were immobilized on poly-L-lysine-coated (Vadillo-Rodriguez et al., 2004) glass surfaces (Fig. 1Go).


Figure 1
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  		Figure 1. Micrographs of bacteria immobilized on a tipless cantilever and a poly-L-lysine-coated glass surface, together with a schematic presentation of the force-distance curves recorded by AFM. (a) Scanning electron micrograph of the tipless cantilever with immobilized actinomyces (upper panel) and light micrograph of a glass surface with an immobilized streptococcal partner (lower panel). The bars used are 5 µm. (b)finally broken and interaction is no longer detected.

 
We used a nanoscope III AFM (Digital Instruments, Santa Barbara, CA, USA), operating in contact mode, to measure interactive forces between the actinomyces and streptococcal surfaces. Measurements were taken in buffer (2 mM potassium phosphate, 50 mM potassium chloride, 1 mM calcium chloride, pH 6.8) at three different temperatures (10°C, 22°C, and 40°C). Force-distance curves were recorded after the cantilever was positioned over an individual streptococcus (Fig. 1Go; note the presence of single cells in the electron micrograph). Localization of an individual streptococcus was achieved with the actinomyces-coated cantilever used as a tip for imaging with the AFM (see APPENDIX 2 for an example of such an image). Forty-five force-distance curves were recorded for each pair, involving, on average, 3 cantilevers with actinomyces from different cultures and 15 different streptococci, also obtained from 3 different cultures. Due to the short range of the interactive forces, we believe that the force-distance curves measured represent the contact between 1 streptococcus and 1 actinomyces (see APPENDIX 2).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A repulsive force, sometimes extending up to 150 nm, was observed upon approach between the actinomyces and streptococcal surfaces, regardless of temperature or the pair involved (Fig. 2Go). This force must be overcome for co-aggregation to occur, but no qualitative differences in approach curve characteristics were observed for co-aggregating and non-co-aggregating pairs. Upon retraction, multiple adhesive interactions appeared until detachment of the interacting cell surfaces. Qualitatively, retracting force-distance curves invariably showed a higher adhesion force extending over a longer distance for the co-aggregating than for the non-co-aggregating pair. However, a large variation existed in numerical features among the different curves recorded for each pair. For statistical analyses, the spread of the data in the different features has been considered to be normal (see APPENDIX 3 for examples of the distribution of the repulsive force at zero separation distance F0), justifying the use of a one-sided Student t test.


Figure 2
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  		Figure 2. Examples of force-distance curves at 10°C, 22°C, and 40°C between A. naeslundii 147 and streptococci, including approach (upper line) and retracting (lower line) curves. (left panel) Co-aggregating pair, involving S. oralis J22. (right panel) Non-co-aggregating pair, involving S. sanguis PK1889.

 
The approach curve was fitted to a negative exponential to obtain the repulsive force at zero separation distance, F0, and its decay length ({Lambda}) (TableGo). Quantitative features extracted from the retraction curve involved the maximal adhesive force, Fmax, and the adhesive energy, Eadhesion, as obtained from the area under the retraction curve. The maximal adhesive force between co-aggregating actinomyces and streptococci is three-to four-fold higher (p < 0.001, one-sided Student t test) than for the non-co-aggregating pair at all temperatures. Similarly, the adhesive energy is significantly (p < 0.001, one-sided Student t test) higher for the co-aggregating than for the non-co-aggregating pair at all temperatures. Note that the adhesive interactions between the two members constituting the pair are significantly (p < 0.02, one-sided Student t test) smaller at 40°C than at lower temperatures.


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  		Table. Force-Distance Curves between a Co-aggregating and a Non-co-aggregating Bacterial Pair in Buffer at 3 Temperatures
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Interactive forces between a co-aggregating and a non-co-aggregating oral bacterial pair were compared by AFM. Qualitatively, consistent features could be obtained from the force-distance curves between co-aggregating and non-co-aggregating pairs, but quantitatively, large standard deviations occurred, believed to be due to biological variations between individual bacteria that could have been exacerbated even more by the use of batch cultures. Moreover, due to the nature of the technique, the numbers of individual bacteria that can be realistically studied in an experiment are small. Yet it is interesting that, in the absence of specific forces between A. naeslundii 147 and S. sanguis PK1889, an adhesive force upon retraction of around 1 nN was measured, while for the co-aggregating pair, a three- to four-fold-higher force was found. In a similar study on enterococci with or without the ability to aggregate (Waar et al., 2005), adhesive forces of 3 and 1 nN were observed, respectively. This suggests that the ubiquitously present non-specific interactive force between 2 bacteria amounts is approximately 1 nN, and that larger specific contributions add 3 to 4 nN to the interactive force, and therewith mediate (co-)aggregation.

During AFM, contact is forced upon the interacting surfaces, and a repulsive energy barrier has to be overcome for contact to be established. The range of these repulsive forces is considerably longer than expected for electrostatic interactions, suggesting an electro-steric nature (Heinz and Hoh, 1999). At close approach, contact requires removal of water molecules from between hydrophobic actinomyces (water contact angle, 53 degrees) and the more hydrophilic streptococci (S. sanguis PK1889 and S. oralis J22 water contact angles of 28 and 24 degrees, respectively) (Bos et al., 1996b), which yields an additional source of repulsion. Interestingly, the repulsive interaction to be overcome for the organisms to come together effectively is not convincingly higher for the non-co-aggregating pair than for the co-aggregating pair. Therefore, it must be concluded that contact between the bacterial cell surfaces will occur, regardless of whether the organisms have the ability to co-aggregate or not. Co-aggregation, however, becomes manifest only if contact can be maintained under prevailing shear conditions, as occurring either during vortexing in a test tube (Kolenbrander, 1989) or in the oral cavity (Palmer et al., 2003).

This conclusion is supported by the adhesive forces measured in the retraction curves, i.e., the forced disruption of the contact between partner organisms. Upon retraction, bonds induced by contact are stretched until detachment, yielding distinct adhesive forces extending over a long distance for the co-aggregating pair only. Similar adhesive events between bacteria and inert surfaces have been sensed by AFM until a separation distance of 1000 nm, and have been attributed to extracellular polysaccharides (Frank and Belfort, 1997) or proteinaceous fibrils (Van der Mei et al., 2000; Vadillo-Rodriguez et al., 2003).

The adhesive interactions upon retraction for the co-aggregating pair are smaller at 40°C than at lower temperatures, suggesting involvement of entropic effects (Leckband, 2000) in co-aggregation. Earlier, it was demonstrated that co-adhesion between an already-adhering actinomyces and planktonic streptococci was favored at temperatures between 22°C and 35°C, as compared with experiments done at 37°C and 40°C (Bos et al., 1996a). Since relevant oral surface temperatures range from 30°C to 35°C (Spiering et al., 1984) up to 37°C, the influence of temperature upon the adhesive interactions measured here indicates that co-aggregation and co-adhesion may be reversible processes under the dynamic conditions of the human oral cavity.


   ACKNOWLEDGMENTS
 
This study was funded by the University Medical Center Groningen, Groningen, The Netherlands.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received February 8, 2005; Last revision September 27, 2005; Accepted October 13, 2005


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Benoit M, Gabriel D, Gerisch G, Gaub HE (2000). Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat Cell Biol 2:313–317.[ISI][Medline]

Bos R, van der Mei HC, Busscher HJ (1996a). Influence of temperature on the co-adhesion of oral microbial pairs in saliva. Eur J Oral Sci 104(4 Pt 1):372–377.[Medline]

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Handley PS, Harty DW, Wyatt JE, Brown CR, Doran JP, Gibbs AC (1987). A comparison of the adhesion, co-aggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius. J Gen Microbiol 133:3207–3217.[Medline]

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