A growing body of clinical and scientific evidence points to a potentially significant role of urease in caries development and in oral ecology. In order to determine more definitively this role, we need to first understand some important clinical aspects of oral urease activity. This longitudinal panel study gave us the opportunity to observe the dynamic changes in urease activity in the dental plaque and in the saliva of children over a three-year period, and to associate those changes with some biological, behavioral and socio-demographic caries risk factors.
The trend of plaque urease activity levels was relatively stable in the study panel, however urease activity in saliva showed a trend to increase with time. Part of this trend could be related to loss of urease activity during storage of the samples. The estimated maximum loss of urease activity in saliva samples was up to 60% over 12 months, and in plaque samples up to 25% in 6 months. Samples from the more recent time points were stored for shorter time compared to the samples from the earlier time points, therefore suffered less loss of activity. However, the regression analysis showed a significant positive association between age and urease levels in saliva even when the models were adjusted for length of storage. The mean salivary urease levels in this study were lower than the levels reported in older children 4 to 12 years (18
), and in adults (16
). Although urease activity in these other studies was sometimes measured in plaque and saliva samples that had not been frozen, these observations together suggest that urease levels in saliva may increase with age. This increase could be associated with an increase in the numbers of ureolytic organisms in saliva, such as S. salivarius
, as well as with a general increase in the complexity of the oral flora.
The most important factor affecting urease activity in dental plaque was sugar consumption, as demonstrated by the regression analysis and also by the opposite trends of urease levels and sugar scores during the study period. The negative association between plaque urease levels and sugar consumption observed in this study may reflect lower proportions of ureolytic bacteria in the plaque of subjects who consume high sucrose diets. It has been proposed that frequent sugar consumption can lead not only to an increase in the proportions of cariogenic bacteria, but also to a concomitant decrease in the proportions of beneficial alkali-producing species, including the ureolytic organisms which are usually less aciduric (1
). This hypothesis is supported by the data presented here, and it is currently being more thoroughly evaluated using molecular techniques.
Urease levels in saliva were positively associated with the salivary levels of mutans streptococci, a somewhat unexpected observation based on what was discussed previously. A possible explanation for this finding is that the urease of S. salivarius
, which is the most significant ureolytic organism in saliva, has been shown to be repressed in neutral pH, and becomes de-repressed in acidic environments (27
). It is possible that saliva from subjects with high levels of mutans streptococci may have a more acidic pH than the saliva from subjects with low mutans levels, resulting in stronger induction of the urease genes. A negative correlation was indeed observed between mutans streptococci levels in saliva and salivary pH in the non-fasting samples in this study (Spearman ρ=−0.106, P=0.034); however, this hypothesis needs to be further investigated. The positive relationship between saliva urease and salivary mutans levels could have important clinical applications. Simple, chair-side tests for measuring urease activity in saliva can be easily developed and they could be used as an indicator of salivary mutans infection and possibly caries risk. The advantage of such biochemical tests is that they can give much faster results compared to the currently available microbiological chair-side tests for S. mutans
Salivary urease levels were significantly lower in children who had eaten sugar-containing foods prior to sample collection, compared to children who had not eaten anything since the night before. This observation is likely linked to the previous observation of a positive association between saliva urease activity and salivary mutans levels, because children who had eaten had significantly lower levels of mutans streptococci in their saliva compared to those who were fasting (Kruskal-Wallis P=0.0046). The increased numbers of mutans streptococci in the un-stimulated saliva of fasting children may be related to reduced salivary flow due to lack of stimulation from eating and reduced clearance from the mouth overnight. This could also explain the significant association between saliva urease and plaque levels that was observed in the fasting samples. It could also explain why the association between saliva urease and mutans levels was no longer significant when the analysis was stratified by fasting.
Urease measurements in plaque and in saliva had overall low reproducibility in this study. This low reproducibility can be attributed to the multiple factors that were shown to have a significant association with urease activity in this clinical study, as discussed previously. In vitro
studies have also shown that urease enzymes, including those from oral bacteria, are rarely constitutively expressed. In most cases the expression of urease enzymes in bacteria is highly regulated in response to environmental factors such as substrate availability, nitrogen and carbohydrate availability and pH (2
). In the case of plaque urease, the low reproducibility could also be attributed to the fact that the samples were pooled from multiple teeth and tooth sites. These observations suggest that in order to improve the reproducibility of urease measurements in future studies it will be necessary to control for multiple factors and to use a more site-specific method for plaque collection.
In conclusion, the results of this study reveal some important clinical and epidemiological aspects of oral ureolysis in children and demonstrate interesting and complex interactions between oral urease activity and some important caries risk factors. Of particular clinical interest is the unexpected positive association between urease activity and mutans streptococci in saliva, which suggests that saliva urease could be used as an indicator of mutans infection in children. We are currently finalizing the analysis of the caries data and the microbiological data from this longitudinal study, which will clarify the role of urease in caries development and in the oral ecology of children.