Multiplex FISH analysis of a six-species bacterial biofilm

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Abstract

Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 °C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.

Introduction

Biofilms are formed after rapid attachment and growth of microorganisms on a broad range of surfaces in contact with natural fluids. Consisting of single cells, cell aggregates, and microcolonies embedded in an exocellular polymeric matrix gel, biofilms are a most common phenomenon on Earth and have been the subject of intense experimental and theoretical scrutiny over the past decade Costerton et al., 1995, Marsh and Bradshaw, 1995, Wimpenny et al., 2000, Hermanowicz, 2003. Biofilm architecture and the physiological status of the cells contained within these structures are of prime interest for clinical, industrial, and environmental microbiology.

Dental plaques are naturally occurring complex biofilms involved in the development of caries and periodontal diseases. Knowledge of their composition, their members' metabolism and know-how on the successful manipulation of mixed bacterial biofilms may lead to improved preventive measures against these common diseases. Investigations of plaque in vivo are difficult due to the high degree of variability between individuals and since the site-specific microbial composition is reflecting the influences of a multitude of uncontrollable endogeneous and exogeneous dietary factors. In addition, working with humans always raises ethical questions. Therefore, we have developed and applied a six-species in vitro biofilm model of supragingival plaque (Guggenheim et al., 2001a) for the examination of the spatial arrangement and associative behavior of microorganisms, the study of the efficacy of antimicrobial mouthrinses, or the investigation of mass transport of macromolecules Guggenheim et al., 2001b, Shapiro et al., 2002, Thurnheer et al., 2003.

For analyses of the spatial arrangement of bacteria in a multi-species biofilm techniques, which use specific bacterial cell markers and maintain the biofilm's natural architecture are required. The aim of this study was to develop a fluorescent in situ hybridization (FISH) technique using probes to target-specific 16S rRNA sequences and to combine it with confocal laser scanning microscopy (CLSM) for the simultaneous analysis of the spatial distribution of both Gram-positive and Gram-negative bacteria in biofilms. FISH is a recognized tool for the specific and sensitive identification of target organisms within complex microbial communities (Amann et al., 1995). Visualization of FISH labeled cells in biofilms can be carried out by techniques, including fluorescence microscopy and CLSM Lawrence et al., 1991, Davey and O'Toole, 2000, Wimpenny et al., 2000. With biofilms, CLSM is often to be preferred, since it allows a three-dimensional noninvasive visualization of cells and the computational reconstruction of mature biofilms without distortion of their structure. However, the crucial problem was to find experimental conditions that render cell walls of Gram-positive bacteria penetrable to the probes without the simultaneous loss of signal from Gram-negatives that are simultaneously labeled with fluorescent oligonucleotide probes. Moreover, they should allow several consecutive hybridization procedures without harming the biofilm structure.

Section snippets

Preparation of biofilms

Actinomyces naeslundii OMZ 745, Veillonella dispar ATCC 17748T (OMZ 493), Fusobacterium nucleatum KP-F2 (OMZ 596), Streptococcus sobrinus OMZ 176, Streptococcus oralis SK248 (OMZ 607), and Candida albicans OMZ 110 were used as inocula for biofilm formation Guggenheim et al., 2001a, Shapiro et al., 2002. Biofilms were grown in 24-well polystyrene cell culture plates on sintered hydroxyapatite disks as described (Thurnheer et al., 2003). In brief, hydroxyapatite disks (∅ 10.6 mm) that had been

Probe specificity and bacterial permeabilization

Specificity tests under stringent FISH conditions using planktonically cultivated cells showed that all probes displayed the anticipated specificity (data not shown). Successful probe penetration through biofilm grown cells was evaluated with both EUB338 and the specific probes for each target strain. Cell permeabilization of paraformaldehyde-fixed biofilms by exposure to lysozyme proved to be absolutely necessary but the duration of enzyme incubation strongly affected fluorescence intensity

Discussion

In the present study we developed a procedure for the simultaneous spatial analysis of the distribution of both Gram-positive and Gram-negative bacteria in a multi-species biofilm model. The technique combines FISH with oligonucleotide probes to specific 16S rRNA sequences and CLSM to assess fixed but otherwise intact biofilms. Key elements of the procedure are: adequate fixation, optimal permeabilization, and careful selection of the best combination between probe, fluorescence label, and

Acknowledgments

We thank T. Bächi and M. Höchli at the Central Electron Microscopy Laboratory of the University of Zürich for the ability to use the CLSM, A. Meier and V. Osterwalder for excellent technical assistance.

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