Microbiology Laboratory, Dental School, Adelaide University, North Terrace, Adelaide, South Australia 5005, Australia1
Author for correspondence: A. H. Rogers. Tel: +61 8 8303 5104. Fax: +61 8 8303 3444. e-mail: tony.rogers{at}adelaide.edu.au
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ABSTRACT |
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Keywords: oxidative stress, oral bacteria, anaerobes, interactions, continuous culture
Abbreviations: SEM, scanning electron microscopy; SOD, superoxide dismutase
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INTRODUCTION |
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As the oral cavity is an overtly aerobic environment, it is therefore likely that oral anaerobes encounter residual amounts of oxygen both in the early stages of dental plaque development and in established periodontal pockets (Marquis, 1995 ). Indeed, periodontal pockets have been reported to possess residual oxygen at one-tenth the level in air-saturated water (which is 0·021 µmol ml-1) (Mettraux et al., 1984
) and the average Eh (redox potential) in subgingival plaque appears to be only somewhat negative at about -50 mV (Kenny & Ash, 1969
). Moreover, there is evidence in dental plaque of open channels that could deliver oxygen deep into the biofilm (Massol-Deya et al., 1994
). Therefore, the survival of anaerobic bacteria in the mouth might be dependent on the specific tolerance to oxygen of each species and on microbial interactions within the community.
Porphyromonas gingivalis and Fusobacterium nucleatum belong to the group of strictly anaerobic bacteria associated with periodontal diseases (Ximenez-Fyvie et al., 2000 ). Due to its numerous putative virulence factors, P. gingivalis is considered one of the major periodontopathic bacteria (Lamont & Jenkinson, 1998
). F. nucleatum is also regarded as a key organism for dental plaque maturation due to its extensive co-aggregating capacity (Kolenbrander et al., 1989
). Bradshaw et al. (1998)
have suggested that F. nucleatum could be a bridge or mediator of co-aggregation between facultative and obligate anaerobic species and they proposed that this co-aggregation was the mechanism by which strict anaerobes, such as P. gingivalis, survived under aerobic conditions, due to the formation of microenvironments in which the facultative organisms mediated reducing conditions. However, our earlier studies (Diaz et al., 2000
) showed that although F. nucleatum is an anaerobe, its capacity to adapt to and reduce an oxygenated environment is extremely high. Therefore, in the present study we explored the possibility that F. nucleatum might be able to protect P. gingivalis and other anaerobic micro-organisms less tolerant to oxygen. Accordingly, our aims were to determine whether F. nucleatum was able to support the growth of P. gingivalis in a continuous aerated co-culture, comparing this co-culture with the tolerance to oxygen of a monoculture of P. gingivalis grown under the same conditions. The levels of anti-oxidant enzymes in P. gingivalis were also assayed in order to compare them with our previously reported levels of the same enzymes in F. nucleatum (Diaz et al., 2000
). As preliminary experiments showed that F. nucleatum does not require the external addition of CO2 for growth in the chemostat, while CO2 seems to be essential for P. gingivalis, we also explored the possibility that F. nucleatum could supply CO2 for the growth of P. gingivalis.
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METHODS |
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Growth conditions.
For all experiments, if not otherwise indicated, the micro-organisms were grown under continuous culture conditions in BM medium (Shah et al., 1976 ) supplemented with 500 µg haemin l-1. Growth in a 365 ml working-volume chemostat (BioFlo model C30, New Brunswick Scientific) was initiated by inoculating the growth chamber with a 24 h batch culture of the micro-organism(s) grown in the same medium under an atmosphere of N2/H2/CO2 (90:5:5). After 24 h of batch-culture growth in the chemostat vessel, the medium reservoir pump was turned on and the medium flow adjusted to give a dilution rate of D=0·069 h-1 (td=10 h). The temperature was controlled at 36 °C and the pH maintained at 7·4 by the automatic addition of 2 M KOH. The cultures were sparged with the appropriate gas mixture and the Eh was constantly monitored, as was dissolved oxygen. Culture purity was checked daily by Gram staining and cell viability was measured, at steady-state, by viable counts. Vortex mixing of co-culture samples, prior to viable counting, was carried out to disperse co-aggregated cells without affecting cell viability. Blood agar plates, with and without 50 µg kanamycin ml-1, were used for viable counts of P. gingivalis and F. nucleatum, respectively. In the kanamycin-containing medium P. gingivalis was recovered with 100% efficiency. Under all conditions, steady state was achieved after about seven generations, as evidenced by sustained stability of the culture Eh and cell viability. Various culture parameters (see below) were then assayed daily for up to 9 d. Cultures grown under various conditions were also examined by scanning electron microscopy (SEM), as follows. For planktonic-phase analysis, approximately 1 ml of cell culture was removed from the chemostat and 5 µl was placed on a micro glass cover slip and allowed to dry. When formation of biofilms occurred over the chemostat inserts, the chemostat was disassembled and a small amount of the biofilm was mechanically removed with minimal disruption and placed directly onto cover slips. All samples were then placed in fixative solution containing 4% paraformaldehyde, 1·25% glutaraldehyde and 4% sucrose in PBS, followed by post-fixing in 1% OsO4, and dehydration with increasingly concentrated ethanol solutions. Samples were analysed using a Philips XL30 field emission scanning electron microscope.
Initially, P. gingivalis was grown in a gaseous atmosphere of N2/CO2 (95:5) and then under the sequentially increased oxygenated conditions N2/CO2/O2 (85:5:10) and N2/CO2/O2 (75:5:20).
To evaluate the CO2 requirement of the two organisms, each was grown axenically in a N2/CO2 (95:5) atmosphere and when steady state had been achieved, CO2 was excluded from the gas mixture. To determine whether P. gingivalis could survive in a CO2-depleted environment relying only on the amount of CO2 produced by its own metabolism, a closed environment was used, rather than the chemostat, in which the gaseous atmosphere is continually replaced. For these experiments, P. gingivalis was grown in batch culture; two anaerobic jars were used for incubation at 37 °C, one gassed with N2/CO2 (95:5) and the other with N2 alone. P. gingivalis cells growing at steady state in continuous culture under an atmosphere of N2/CO2 (95:5) were used as the initial inoculum. BM medium was used for all experiments and media were inoculated to produce an initial OD560 of 0·45, in a total volume of 50 ml. At late-exponential phase the OD560 was recorded, the pH was measured and the appropriate amount of culture, to produce an initial OD560 of 0·45, was transferred to fresh medium; these broths were incubated under the appropriate gaseous conditions.
The ability of F. nucleatum to support the growth of P. gingivalis under aerated conditions was determined by growing the bacteria in continuous co-culture under the same gaseous conditions as described above for the P. gingivalis continuous monoculture. In a parallel set of experiments CO2 was excluded from the gas mixture.
Enzyme assays.
Since P. gingivalis failed to grow under oxygen concentrations of 10% or more, the activity of anti-oxidant enzymes was assayed in cell-free extracts from the micro-organism grown under anaerobic conditions and under 3% and 6% O2 in the incoming gas mixture. Extracts were prepared as described previously (Diaz et al., 2000 ). Protein content was determined using a Coomassie Plus Protein Assay Reagent Kit (Pierce) with bovine serum albumin as a standard. NADH oxidase and NADH peroxidase activities were assayed at 25 °C following the methods of Higuchi (1992)
. NADH oxidase was assayed by monitoring the oxidation of ß-NADH in the reaction mixture (3 ml) at 340 nm. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7·0), 0·1 mM ß-NADH and cell extract (0·125 mg protein). NADH peroxidase was assayed under anaerobic conditions, achieved in a Thunberg-type cuvette, using the same reaction mixture as for NADH oxidase but with the addition of 0·3 mM H2O2. One unit of activity for both enzymes was defined as the amount of extract that catalysed the oxidation of 1 nmol NADH min-1. SOD activity was measured at 550 nm by competitively inhibiting the reduction of cytochrome c at 25 °C, following the methods of McCord & Fridovich (1969)
. The reaction mixture (3 ml) contained 50 mM potassium phosphate buffer (pH 7·8), 0·1 mM EDTA, 0·01 mM cytochrome c, 0·1 mM xanthine, sufficient volume of xanthine oxidase to produce a constant reduction of cytochrome c at a rate of 0·02 units of absorbance increase min-1 and cell extract (0·125 mg protein). One unit of SOD was defined as the amount of extract that decreased by 50% the rate of reduction of cytochrome c.
Statistical analysis.
Data were expressed as means ± standard deviations. Differences between means were analysed for statistical significance by Students t test.
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RESULTS |
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The co-culture Eh was unaffected by the presence or absence of CO2. Furthermore, the change from anaerobic conditions to 10% O2 resulted in only a small change in Eh values, from about -500 mV to -430 mV. However, the culture Eh increased to about -150 mV under 20% oxygen and only under this condition were large planktonic aggregates and biofilms observed around the growth vessel inserts. Daily Gram stains and SEM analysis of the planktonic phase of the culture showed that F. nucleatum cells increased in length as the culture became more oxygen stressed; cells grown under 20% oxygen were at least five times longer than cells grown under anaerobic conditions (Fig. 2). SEM analysis of the biofilms formed under aeration showed that most obvious on the surface of these biofilms were F. nucleatum cells that appeared to form an intricate network (Fig. 3
). However, viable counts performed on some of these biofilms revealed that P. gingivalis was present (data not shown). It should be noted that both co-aggregating and non-co-aggregating cells of both organisms were observed in the planktonic phase of the culture under all conditions, although the proportion of co-aggregating cells increased at higher oxygen concentrations (Fig. 2
).
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DISCUSSION |
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Socransky et. al. (1998) demonstrated that bacteria in subgingival plaque exist as microbial complexes. Although P. gingivalis and F. nucleatum were identified as forming part of different complexes, a close association was shown to exist between the complexes to which each micro-organism belonged. In fact, the members of the P. gingivalis complex were rarely found in the absence of members of the F. nucleatum complex. Our data provide an explanation for this apparent dependence of P. gingivalis on F. nucleatum, suggesting that the latter might create the necessary reduced conditions and supply CO2 for the survival of the former.
The present study clearly shows that, although F. nucleatum and P. gingivalis are anaerobes, there is a marked difference in their oxygen tolerance. Our previous findings showed that F. nucleatum is able to survive in chemostat cultures sparged with proportions of oxygen even higher than the oxygen content of air (Diaz et al., 2000 ), while the present findings show that P. gingivalis is not able to survive, in the same system, when the gas phase of the culture contains more than 6% oxygen. It should be noted that the ability to metabolize oxygen by a culture of F. nucleatum or P. gingivalis may depend upon the cell numbers (determined by nutrient availability) used in this system, but it reflects the potential of the organisms to survive oxygen tensions that might occur in the oral environment.
Interestingly, if the currently detected levels of the anti-oxidant enzymes in P. gingivalis are compared with our previous report for F. nucleatum (Diaz et al., 2000 ), the major differences occur in NADH oxidase activity. Under anaerobic conditions, F. nucleatum produced 190 units of activity (mg protein)-1, while P. gingivalis activity was only 8·15 units (mg protein)-1, a difference of more than 20-fold. In contrast, SOD activity in F. nucleatum was 10 times lower than in P. gingivalis. This indicates that, in these two micro-organisms, SOD activity does not correlate with tolerance to oxygen, which is at variance with previous suggestions for other species (Park et al., 1992
). NADH oxidase/peroxidase usually exists as a double system, able to metabolize both molecular oxygen and H2O2 (Poole et al., 2000
), while the role of SOD is the detoxification of
with the formation of H2O2 (McCord & Fridovich, 1969
). Therefore, high levels of SOD without correspondingly high levels of NADH oxidase/peroxidase activities could be toxic for anaerobic micro-organisms because of the generation of large amounts of H2O2 that can not be adequately detoxified, as seems to occur in P. gingivalis. The negative aspect of the NADH oxidase/peroxidase systems appears to be that as they are flavin-based enzymes, the formation of
can be promoted (Imlay & Fridovich, 1991
). Therefore, it seems that an efficient radical detoxification system in the absence of catalase, as is the case for F. nucleatum and P. gingivalis, depends on the delicate balance between these two anti-oxidant enzymes, although higher proportions of NADH oxidase/peroxidase seem more beneficial for F. nucleatum.
The present results clearly show that F. nucleatum is able to support the growth of P. gingivalis under aerated conditions in which the latter cannot survive as a monoculture. Indeed, the fact that the populations of F. nucleatum increase under oxygen stress indicates its capacity to survive in natural environments that might be partially oxygenated, in contrast to the low tolerance to oxygen of P. gingivalis. Thus, these results suggest that the capacity of F. nucleatum to protect P. gingivalis from oxidative damage might be one of the reasons why there seems to be a close in vivo association between these two micro-organisms (Socransky et al., 1998 ). Additionally, this study shows that the growth of F. nucleatum does not require a capnophilic environment, whereas CO2 seems to be essential for P. gingivalis. The fact that P. gingivalis survived in co-culture with F. nucleatum without the addition of CO2 indicates that the latter is able to satisfy the CO2 requirement of the former. This might constitute another metabolic interaction that explains the close association between the pair. Other possible interactions between the two micro-organisms include the presence of proteolytic enzymes in P. gingivalis that cleave proteins into peptides and therefore increase the energy sources for F. nucleatum, which does not possess high endopeptidase activity (Grenier, 1994
).
The formation of biofilms, as observed in this study, might be a strategy used by F. nucleatum to overcome high oxidative stress, possibly because of the more reduced microenvironments inside the biofilm, which could in turn benefit P. gingivalis. The intricate networks formed by F. nucleatum in these biofilms are not surprising, considering the shape of the micro-organism, which increases its length in an oxygenated environment.
It is worth noting that haem-limiting conditions were used in this study for the growth of P. gingivalis. This choice was made in order to avoid the possible protective effect of haemin excess under oxidative stress conditions, as it has been suggested that the binding of haemin dimers to the surface of P. gingivalis would serve as a catalase-like buffer system (Smalley et al., 2000 ). The contribution of this mechanism to the ability of P. gingivalis to overcome oxygen stress could be determined by growing the organism under haemin-excess conditions. In this context, the study by Bradshaw et al. (1998)
, showing that P. gingivalis numbers were greatly reduced when F. nucleatum was omitted from a 10-member oral bacterial consortium subjected to aerated conditions, was conducted under haemin-excess conditions.
In conclusion, additional to the known direct potential involvement of F. nucleatum in the disease process (Han et al., 2000 ; Yoshimura et al., 1997
), this study suggests that F. nucleatum could have an important indirect role in the aetiology of periodontal diseases by supporting the growth of P. gingivalis and possibly other oral anaerobes in oxygenated and CO2-depleted environments. Protection against the deleterious effects of oxygen might be important both in the early stages of plaque development and in periodontal pockets, where micro-organisms have to face the constant presence of residual oxygen levels.
From an ecological point of view, the identification of micro-organisms that, like F. nucleatum, support the growth of other periodontopathogens is important because control of such species might radically alter the pathogenic ecosystem.
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ACKNOWLEDGEMENTS |
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Received 9 August 2001;
revised 15 October 2001;
accepted 22 October 2001.