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Red fluorescent dental plaque: An indicator of oral disease?

Volgenant, C.M.C.

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Citation for published version (APA):Volgenant, C. M. C. (2016). Red fluorescent dental plaque: An indicator of oral disease?.

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Download date: 29 Apr 2020

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2Effect of metalloporphyrins on red

autofluorescence from oral bacteria

C.M.C. Volgenant

M.H. van der Veen

J.J. de Soet

J.M. ten Cate

Published in the European Journal of Oral Sciences (2013)

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Abstract

AimThe aim of this study was to assess the red autofluorescence from bacterial species related to dental caries and periodontitis in the presence of different nutrients in the growth medium.

MethodsBacteria were grown anaerobically on tryptic soy agar (TSA) supplemented with nutrients, including magnesium-porphyrins from spinach and iron-porphyrins from heme. The autofluorescence was then assessed at 405 nm excitation.

ResultsOn the TSA without additives, no autofluorescence was observed from any of the species tested. On the TSA containing sheep blood, red autofluorescence was observed only from Parvimonas micra. When the TSA was supplemented with blood, hemin and vitamin K, red autofluorescence was observed from Actinomyces naeslundii, Bifidobacterium dentium and Streptococcus mutans. Finally, on the TSA supplemented with spinach extract, red autofluorescence was observed from strains of Aggregatibacter actinomycetemcomitans, A. naeslundii, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus salivarius, S. mutans and Veillonella parvula.

ConclusionsWe conclude that the bacteria related to dental caries and periodontal disease exhibit red autofluorescence. The autofluorescence characteristics of the tested strains depended on the nutrients present, such as metalloporphyrins, suggesting that the metabolic products of the oral biofilm could be responsible for red autofluorescence.

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Autofluorescence from oral bacteria

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IntroductionMicroorganisms are associated with oral diseases, such as caries and inflammation of the periodontal tissues. The principal means of preventing these diseases is to remove, or at least disturb, the microbial biofilm on the oral surfaces. This is achieved by instructing the patient to maintain strict daily oral hygiene. The early detection of matured plaque allows for more focused preventive treatment by both the dental team and the patient.

It is assumed that mature plaque produces red autofluorescence when illuminated with blue light at 405 nm (Coulthwaite et al., 2006; Thomas et al., 2008). Such red autofluorescence has been observed in the fluorescence images of teeth coated with plaque or calculus (Heinrich-Weltzien et al., 2003). This extrinsic red autofluorescence is formed in the dental plaque (König et al., 1993; Pretty et al., 2005; Van der Veen et al., 2006) and is particularly found on active caries (Lennon et al., 2002; Shigetani et al., 2008). Little is known about the exact origin of this autofluorescence, but the red autofluorescence is assumed to originate from specific bacterial metabolites formed in the oral biofilm, such as protoporphyrin IX (König et al., 1993). If red autofluorescence is correlates with disease-associated plaque, this autofluorescence could serve as an indicator of plaque-induced diseases. A validated method to quantify this plaque would be a useful tool in the general dental practice for early risk assessment and to support longitudinal preventive treatment.

To identify the origin of the red autofluorescence observed in dental plaque, recent research has focused on isolated bacteria typically grown in batch cultures or single-species biofilms. Not all microorganisms fluoresce when grown on agar plates (König et al., 1993; König, 1994b) and previous studies also did not demonstrate red autofluorescence from Streptococcus mutans, a species strongly associated with caries (König, 1994b; Lennon et al., 2006b).

No research has yet focused on the influence of species interactions on red autofluorescence, except for a report mentioning red autofluorescence from Parvimonas micra only when grown in close proximity to Porphyromonas gingivalis (Van der Veen et al., 2006).

Hemin and vitamin K are required for bacteria to produce metal-free porphyrin (Dolowy et al., 1995). Buchalla et al. (2009) have suggested that bacteria can generate red autofluorescence when spinach extract is added to the agar. Chlorophyll and hemin molecules have the same ring structure as the porphyrin molecule except that there is a magnesium cation in the centre of the chlorophyll ring and an iron ion at this position in the hemin molecule. We hypothesise that molecules containing this ring structure influence the ability of bacteria to generate red autofluorescence.

Therefore, the first aim of this study was to investigate the influence in tryptic soy agar (TSA) of the metalloporphyrin-containing chlorophyll from spinach

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extract, hemoglobin from sheep blood and a combination of hemoglobin, hemin and vitamin K on the autofluorescence of 11 different bacterial species related to caries and periodontal disease. This study also sought to investigate the effects of different growth media on the autofluorescence from these bacteria when grown in the presence of Porphyromonas gingivalis.

Material and MethodsMediaTo assess the ability of 11 different bacterial strains to develop red autofluorescence, we used four types of media consisting of TSA and different supplements, poured into six-well cell-culture plates (CELLSTAR; Greiner Bio-One, Alphen a/d Rijn, the Netherlands). Specifically, the media used included TSA (BD Difco, Becton, Dickinson, Le Pont de Claix, France); TSA supplemented with aseptically collected, defibrinated sheep blood (50 mL/L; bioTRADING Benelux B.V., Mijdrecht, the Netherlands); TSA supplemented with sheep blood, bovine hemin (5 mg/L; Sigma-Aldrich, St. Louis, MO, USA) and vitamin K (0.5 mg/L; Sigma-Aldrich, Steinheim, Germany) and TSA supplemented with spinach extract (50 mL/L).

The spinach extract was obtained from fresh spinach leaves washed with ice-cold water. The spinach leaves were then cut into small pieces, mashed using a hand-held blender and centrifuged at room temperature for 5 min at 600 g. The supernatant was collected and further centrifuged for 10 min at 4,000 g. The resultant supernatant was again collected and centrifuged for 10 min at 16,100 g. The supernatant was then filtered (0.2 mm pore size cellulose acetate filter unit; Whatman, Dassel, Germany) to obtain a sterile spinach extract, of which 50 mL was added to 1 L of TSA. The concentration of chlorophyll in the spinach extract was determined to be 13 ng/mL, as described by Porra et al. (1989) using 80% aqueous acetone as the solvent.

MicroorganismsThe following bacterial strains were studied: Aggregatibacter actinomycetemcomitans (ATCC 29522), Actinomyces naeslundii (DSM 43013), Bifidobacterium dentium (DSM 20436), Enterococcus faecalis V583 (a clinical isolate), Fusobacterium nucleatum subsp. polymorphum (DSM 20482), Lactobacillus salivarius (ATCC 11741), P. micra (DSM 20468), P. gingivalis W50 (a clinical isolate), S. mutans UA 159 (ATCC 700610), Streptococcus sobrinus (DSM 20742) and Veillonella parvula (DSM 2008). The bacteria were cultured separately on each of the four media, described earlier at 37°C under anaerobic conditions (80% N2, 10% H2 and 10% CO2) for 4 -7 days, depending on the growth rate of the respective strains. When inoculating the bacteria, we tried to distribute a dense layer of evenly spread colonies on the agar. To check for interspecies



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interactions, P. gingivalis W50 was cocultured with each of the other species on the four media under the above-mentioned conditions.

Fluorescence assessmentThe ability of the bacterial strains to fluoresce at an excitation of 405 nm was visually assessed using fluorescence photographs and spectroscopic analysis. White-light and fluorescence photographs of each six-well agar plate were generated using a QLF-D biluminator camera system (QLF, Inspektor Research Systems., Amsterdam, the Netherlands). The QLF-D biluminator system consists of an illumination tube (Biluminator; Inspektor Research Systems) fitted onto an SLR camera (Canon model 450D, fitted with a 60 mm macro lens; Canon, Tokyo, Japan). The illumination tube is composed of a ring mounted with eight violet-blue light-emitting diodes (LEDs; 405 ± 20 nm) and four white LEDs (broad spectrum, 6500K) with filtering optics in front of the camera lens. The camera system was controlled using dedicated software to capture both fluorescence photographs and white-light photographs (C2 version 14; Inspektor Research Systems). The photographs were taken in a dark room with fixed camera settings (white light photographs: shutter speed 1/50 s; aperture value 13; ISO 1600; QLF-photographs: shutter speed 1/20 s; aperture value 5; ISO 1600). The biluminator tube was placed directly on the 6 well cell culture plates. To confirm the colours observed in the photographs, a spectrophotometer (SPECTRAmax M2; Molecular Devices, Sunnyvale, CA, USA) was used to quantitate the emission spectrum after direct excitation. The excitation wavelength of the monochromator was set at 405 nm and the emission was measured in the range of 500 - 750 nm with a step size of 2 nm. Measurements were performed in each sextant of a well, and the results were averaged. The entire experiment was performed in duplicate.

ResultsVisual observations of the autofluorescence and the autofluorescence peaks determined by spectroscopic assessment for all tested strain and agar combinations are presented in Table 2.1.

All the strains were found to grow on each of the four media, with two exceptions: P. micra did not grow on the agar plates supplemented with spinach extract, and P. gingivalis (solitary) did not grow on the two media lacking sheep blood.

On the TSA without additives, no autofluorescence was observed from any of the tested species. On the TSA supplemented with blood, we observed red autofluorescence from P. micra when grown either alone or in combination with P. gingivalis. In contrast, green autofluorescence was found from F. nucleatum, and no autofluorescence was observed from the other tested strains.

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On the TSA supplemented with blood, hemin and vitamin K, red autofluorescence was observed from A. naeslundii, B. dentium and S. mutans, whereas green autofluorescence was noted from F. nucleatum. The autofluorescence from these strains was not influenced by the presence of P. gingivalis. P. micra showed no autofluorescence when grown alone but exhibited intense red autofluorescence when co-cultured with P. gingivalis (Figure 2.1).

Table 2.1 Overview of the autofluorescence properties of four different media.TSA TSA with

spinach extractTSA with blood

TSA with blood, vitamin K and hemin

Peak and visual aspect

Peak (nm)

Visual aspect

Peak (nm)

Visual aspect

Peak (nm)

Visual aspect

Aggregatibacter actinomycetemcomitans

- 676 Red - - - -

A. actinomycetemcomitans / Porphyromonas gingivalis

- 676 Red - - - -

Actinomyces naeslundii - 672 Red - - 620 Red A. naeslundii / P. gingivalis - 674 Red - - 634 Red Bifidobacterium dentium - - - - - 634, 700 Red ++B. dentium / P. gingivalis - 674 Red - - 634, 696 Red ++Enterococcus faecalis - 678 Red 620 - 630 - E. faecalis / P. gingivalis - 672 Red 636 - - - Fusobacterium nucleatum - 674 Red - * Green - * Green F. nucleatum / P. gingivalis - 674 Red - * Green - * Green Lactobacillus salivarius - 676 Dark red - - - - L. salivarius / P. gingivalis - 672 Dark red - - - - P. gingivalis - - - - - - - Parvimonas micra - - - 624 Red - - P. micra / P. gingivalis - - - 628 Red 632 Red ++ Streptococcus mutans - 674 Red - - 636 RedS. mutans / P. gingivalis - 672 Red - - 634 RedStreptococcus sobrinus - - - - - - - S. sobrinus / P. gingivalis - - - - - - - Veillonella parvula - 678 Red - - - - V. parvula / P. gingivalis - 674 Red - - - -

“Red ++” is defined as intense red autofluorescence (as shown in Figures 2.1B and 2.3B) compared with average red autofluorescence (as shown in Figure 2.2D).* Fusobacterium nucleatum fluoresced green in the fluorescence photographs. The spectra demonstrated an increase in and a shift of the blue/green background peak toward the green part of the spectrum.



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Figure 2.1 Parvimonas micra increased its red autofluorescence when grown together with Porphyromonas gingivalis. P. gingivalis and P. micra were grown together on a blood agar plate supplemented with vitamin K and hemin. A comparison of the white-light photograph (A) with the fluorescence photograph (B) shows the ability of the separate P. micra colonies to fluoresce red when they are in close proximity to P. gingivalis. A distinct peak in the fluorescence intensity was observed at approximately 632 nm when P. micra and P. gingivalis were grown together on an agar plate containing blood, vitamin K and hemin. (C) This increase in intensity (measured in arbitrary units) was not observed when the two species were grown separately on the same type of growth medium. Pg: Porphyromonas gingivalis; Pm: Parvimonas micra.

On the TSA containing spinach extract, red autofluorescence was observed from A. actinomycetemcomitans, A. naeslundii, E. faecalis, F. nucleatum, L. salivarius, S. mutans and V. parvula. Coculture with P. gingivalis did not influence the red autofluorescence from these strains. In contrast, B. dentium showed red autofluorescence when grown on the TSA supplemented with spinach extract only when grown together with P. gingivalis. The fluorescence photographs of the A. actinomycetemcomitans colonies and corresponding emission spectra are shown in Figure 2.2.

The visual autofluorescence observations were confirmed by spectrophoto-metric analysis in all cases except three (Table 2.1). Enterococcus faecalis on the TSA containing blood, grown either alone or with P. gingivalis, exhibited a peak in the red part of the spectrum that was not observed during visual observation. A peak

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Figure 2.2 Red autofluorescence from Aggregatibacter actinomycetemcomitans. A. actinomycetemcomitans has the ability to fluoresce red. A tryptic soy agar (TSA) plate was observed via a white-light photograph (A) and a fluorescence photograph (B). Photograph C exhibits a white-light image of the agar plate with added spinach extract, whereas D is a fluorescence photograph. A. actinomycetemcomitans did not fluoresce on the blood agar plate or on the blood agar plate enriched with vitamin K and hemin. The graph (E) shows the red autofluorescence as a peak at approximately 676 nm (intensity is measured in arbitrary units).

in this part of the spectrum was also measured for E. faecalis grown on the TSA supplemented with blood, vitamin K and hemin. In the case of F. nucleatum, the bacteria fluoresced green on the agar plates containing blood with or without vitamin K and hemin.

Maximum emission peaks were found at different wavelengths for the bacteria and conditions studied. The emission wavelength peaks measured on the TSA containing spinach extract ranged from 672 - 678 nm. On the TSA supplemented with blood, these peaks appeared between 620 nm and 636 nm, and on the TSA containing blood, vitamin K and hemin, these peaks ranged from 620 - 700 nm. For B. dentium, bright red autofluorescence was observed on the TSA containing blood, vitamin K and hemin, and the measured emission spectrum showed peaks at 634 nm and 696 nm, in the red part of the spectrum (Figure 2.3).



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Figure 2.3 Bright-red autofluorescence from Bifidobacterium dentium. B. dentium colonies were grown on a blood agar plate supplemented with vitamin K and hemin. A tryptic soy agar (TSA) plate was observed in a white-light photograph (A) and a fluorescence photograph (B). The red autofluorescence from B. dentium grown on this medium was very distinct and presented two emission peaks (C) at approximately 634 nm and 700 nm. * The average of the results from the TSA containing blood, or blood, hemin and vitamin K, is represented on the secondary y-axis of the graph (in arbitrary units).

DiscussionIn this study, we investigated the ability of 11 bacterial species associated with caries or periodontal disease to fluoresce red when grown on agar plates either with or without added metalloporphyrins. From our experimental observations, we conclude that both bacterial interactions and growth conditions, specifically the availability of metalloporphyrins, determine red autofluorescence from oral bacteria. In this study, the intensity and the location of the emission peaks of the red autofluorescence varied among the tested bacteria. We selected strains of 11 bacterial species that have been linked to either caries or periodontitis, the majority of which have been genetically sequenced, which allows for future studies of their metabolic pathways at a genetic level. Previous research on the red autofluorescence observed in dental plaque has mainly focused on a single species grown on blood- or hemin-containing agar (Coulthwaite et al., 2006; Van der Veen et al., 2006). Research on the effects of hemin, hemoglobin or chlorophyll, which are all molecules with porphyrin

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ring structures, on the ability of bacteria to induce red autofluorescence has only been reported in an abstract (Buchalla et al., 2009).

On the TSA plates supplemented with spinach extract, the majority of the tested strains showed red autofluorescence, in contrast to those strains grown on the TSA plates without this extract. Spinach contains chlorophyll, which has a ring structure similar to that of porphyrin; thus, the increased number of strains showing red autofluorescence in the presence of spinach extract could be attributed to the chlorophyll. Chlorophyll itself (at room temperature) has an emission peak of around 685 nm and a broad shoulder of around 740 nm (Krause, 1991). The supplied metalloporphyrins are metabolized and could contribute to the red autofluorescence. Unlike the known effect of P. gingivalis on the autofluorescence from P. micra, P. gingivalis only had a small effect on B. dentium when grown on the TSA containing spinach extract, where autofluorescence was not visible without P. gingivalis. However, P. gingivalis did not affect the autofluorescence from the other tested strains. It should be noted that P. gingivalis alone did not grow on the TSA without blood as a result of the lack of iron, which is critical for the growth of P. gingivalis (Bramanti and Holt, 1991) and is typically supplied in nature by heme.

In all experiments, the fluorescence spectra were measured per sextant of a well and then averaged to one output per well. As a consequence, the fluorescence signals are also obtained from areas without bacterial colonies grown on it. When inoculating the bacteria, we tried to distribute a dense layer of evenly spread colonies on the agar. However, we could not avoid the possibility that the bacteria and hence the fluorescence signal, were not evenly distributed over the well, which might have influenced the spectral output.

Enterococcus faecalis grown on the agar plates revealed emission peaks when examined using a spectrophotometer. However, autofluorescence was not observed in the fluorescence photographs of the agar plates containing blood alone or blood, vitamin K, and hemin. The intensity of the spectroscopic peak indicated that the red autofluorescence was relatively low, which could explain the absence of visible red autofluorescence.

König et al. (1994) did not observe red autofluorescence from S. mutans, whereas Lennon et al. (2006b) reported green autofluorescence following the visual assessment of S. mutans. In the current study, we noted red autofluorescence from S. mutans when grown on the TSA supplemented with spinach extract or on the TSA containing blood, hemin and vitamin K. Mutans streptococci constitute approximately 30% of the total flora in cavitated lesions in dentin (Takahashi and Nyvad, 2011), so the ability of S. mutans to fluoresce red suggests that red autofluorescence measurements could be attributed to the reported fluorescence on carious tissues.



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Previous research indicated that Bifidobacterium species are frequently present in caries (Becker et al., 2002; Modesto et al., 2006). Bifidobacterium dentium was found in the majority of active carious lesions but was significantly less frequently present on carious-free surfaces (Mantzourani et al., 2009a; Mantzourani et al., 2009b). Given the highly intense red autofluorescence produced by B. dentium and the clinical relationship between Bifidobacterium species and caries, follow-up research could focus on the detailed mechanism of red autofluorescence from Bifidobacteria as well as the connection between red fluorescent plaque and the risk of caries in patients.

We assume that the observed double-emission peak of B. dentium is attributable to the different types of porphyrins produced by the bacteria. The emission maxima of the different solutions of porphyrins, excited at 407 nm, are around 633 nm and 700 nm for protoporphyrin IX, 623 nm and 690 nm for coproporphyrin and 593 nm and 646 nm for zinc protoporphyrin (König et al., 1993; Seo, 2009). A double-emission peak has been previously described for the emission spectrum of calculus (Dolowy et al., 1995). However, the reported emission maxima do not explain the emission wavelengths observed in the spinach extract-supplemented TSA group (672 - 678 nm).

The observation that the red autofluorescence emission peaks of the bacteria were not of the same wavelength may be attributed to potential different fluorophores, the pH, the buffer system, the medium used and/or the solvent (Polo et al., 1988). In the present study, we did not measure the pH of the agar after the bacterial growth because of the high buffering capacity of the agar. Any influence of the pH on the ability of the microorganisms to fluoresce red or on the peak wavelength thus remains unknown. In the current study, it is not possible to further specify the type(s) of porphyrin that produced the red autofluorescence.

Our findings confirm the influence of the environment on the ability of bacteria to produce red-fluorescing metabolic products. To identify the source of the clinically observed red autofluorescence, the rapidly changing conditions within the mouth must be considered. More information regarding the origin of the underlying process is needed to achieve a full understanding of the circumstances leading to the autofluorescence from the bacteria in the oral biofilm. More specifically, a study of the metabolic pathways of specific bacteria could clarify the role of the fluorescent molecule(s) in the functions of the cell.

We conclude that the autofluorescence characteristics of the strains examined here vary extensively under different circumstances, specifically with the composition of the growth medium used. This finding suggests that the intrinsic metabolic products of the oral biofilm, and not the presence of specific bacteria in the dental plaque, are responsible for the red autofluorescence. The ability of S. mutans and B. dentium to fluoresce red could be of further interest due to the potential use of these autofluorescence properties in assessing the risk of caries in patients.

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