In this letter we report a fast and easy method which could be used for initial screening of multispecies-biofilm development on putative new dental implant materials. Most staining methods require numerous washing steps that can result in detachment of loosely bound biofilms and therefore falsify the results. Thus, we used glutaraldehyde fixation, which induces autofluorescence through bacterial membrane protein cross-linking and concurrently stabilizes the biofilm structure. We analyzed the biofilms with nonlinear laser scanning microscopy and were able to (I) evaluate the multispecies-biofilm growth and (II) distinguish between bacterial species based on different two-photon autofluorescence intensities.
Multispecies-biofilms play an important role in the development and progression of periimplantitis, which is considered as one of the major complications in dental implantology. If left untreated, the bacterial induced inflammation leads to bone loss and in the end to implant failure [1, 2]. To improve the clinical situation, current research efforts focus on the development of new implant materials with antimicrobial properties. However, these candidate materials have to be tested for their antimicrobial efficacy in sophisticated screening procedures that mostly include microscopic examination. For increased specificity and sensitivity, microorganisms are usually stained with fluorescent dyes or probes. However, these staining procedures often require numerous washing steps whereby loosely bound biofilms can be easily washed away and thus impeding data analysis and interpretation. Furthermore, DNA-intercalating dyes are often carcinogen or at least toxic, making their handling and disposal more demanding. The aim of this study was therefore the establishment of a fast and easy method without complex staining procedures which can be used for initial screens. For this purpose we made use of a well-known property of aldehyde fixatives: the generation of fixative-induced fluorescence. This normally can be a problem if using specific probes or dyes because it complicates the distinction of real signals from the fixative-induced fluorescence in histological tissues . During the fixation with glutaraldehyde, which was first described as a fixative for electron microscopy in 1963 , the aldehyde reacts with amino groups of proteins in the tissue via covalent cross-linking [5, 6]. Bacteria contain a large number of membrane proteins which can react with the aldehydes during the fixation process. Their product, a Schiff base, leads to a high autofluorescence of the cells while microscopic examination [7, 8]. The fixation procedure is easy to perform, cost efficient and increases the mechanical stability of biofilms [9–11]. We expected that a species-specific membrane protein composition would result in different fluorescence intensities. Initially, we performed a multiprobe fluorescence in situ hybridization assay (urea-NaCl-FISH; modified after ). Due to probe labeling with different fluorescent dyes, this method allowed the simultaneous localization of three individual bacterial species within a biofilm. To analyze the biofilm with species-specific labels, confocal laser scanning microscopy (CLSM; C2si, NIKON, Tokio, Japan) was used. The urea-NaCl-FISH experiment (Figure 1) showed Streptococcus oralis as the most prevalent species (red). Actinomyces naeslundii occurred in bigger aggregates (green) whereas Veillonella dispar formed occasional spots or little microcolonies (yellow).
With this background knowledge, we started NSLM analysis of autofluorescence signals. Therefore, the laser scanning microscope TriM Scope II (LaVision Biotec GmbH, Bielefeld, Germany) equipped with the titanium:sapphire femtosecond laser system Chameleon Ultra II (Coherent Inc., Santa Clara, CA, USA) was used. With such a microscope short laser pulses from the near infrared are focused into the sample. Simultaneous absorption of two-photons within the focal volume excites fluorescent molecules inside the biofilm. After internal molecular conversion processes a transition from the excited state into the ground state finally leads to fluorescence. Since the excitation is restricted to the focal volume no pinhole (as in confocal microscopy) is necessary in the detection beam paths. Thus, spatially localized excitation takes place and defined imaging planes can be scanned. In the experiments excitation of two-photon fluorescence was realized with a central laser pulse wavelength of 780 nm. In all cases, two-photon autofluorescence signals could be observed over a broad wavelength range (435–485 nm and 500–550 nm; detectable wavelength regimes defined by bandpass filters). The images from the two wavelength ranges were comparable and showed no difference in structural information. Therefore, only NLSM images on two-photon fluorescence signals in the range of 500–550 nm are displayed in this letter.
In a first step, single-species biofilms of S. oralis, A. naeslundii and V. dispar were investigated for their fixative-induced two-photon fluorescence patterns and are displayed in Figure 2. The 48 h old biofilms of S. oralis (Figure 2A) and A. naeslundii (Figure 2B) showed very dense and spongy structures compared to the thinner biofilm of V. dispar (Figure 2C). Inside the single species biofilms, no differences regarding the fluorescence intensities could be detected. In contrast, brighter fluorescing aggregations of bacteria could be detected within a dual-species biofilm in Figure 3A and in a three-species biofilm in Figure 3C (in both exemplarily marked with red arrows). The aggregates can also be seen in the images which were modified with a 16-color lookup table as green, yellow and red cluster (Figure 3B, D). In the three-species biofilm the third bacterial species occurs occasionally as brighter spots between the two others (Figure 3C; exemplarily marked with yellow arrows). We were able to correlate these two-photone autofluorescence images from NLSM with the results from urea-NaCl-FISH (Figure 1) and demonstrated the similarities in Figure 4: S. oralis dominates the biofilm (exemplarily marked with a white arrow). The brighter fluorescing aggregates could be identified as A.naeslundii (exemplarily marked with a striped arrow) and the spots or microcolonies could be identified as V. dispar (exemplarily marked with a pointed arrow). We were able to distinguish between the three bacterial species based on different two-photon autofluorescence intensities.
In situ hybridization labeling is more laborious and costly than the glutaraldehyde fixation method. The latter is therefore more suited for standardized medium to high volume screening applications where cost-effectiveness and ease of use are given priority. A further advantage is the direct fixation of the biofilms without previous staining and washing procedures. We think that this method, once a biofilm-model is established, can be applied for the initial microscopic screening of new implant materials to get a first impression of their antimicrobial efficacy.
This work was carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative, which is financially supported by the ministry of Lower Saxony and the VolkswagenStiftung. NIFE is the Lower Saxony Center for Biomedical Engineering, Implant Research and Development, a joint translational research centre of the Hannover Medical School, the Leibniz University Hannover, the University of Veterinary Medicine Hannover and the Laser Zentrum Hannover e. V.
Funding: Ministry of Lower Saxony and VolkswagenStiftung (both BIOFABRICATION FOR NIFE: VWZN2860).
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