Discussion

The main finding of this study was that the oral microflora appeared basically unchanged following a supplementary semi-annual varnish program in a group of vulnerable preschool children. Thus, the null hypothesis could not be rejected. It is likely that the negative results were a consequence of, and mirrored the absent benefits of the fluoride varnish program on caries increment as reported from the main project [10] and from similar studies in young children [16, 17]. A second explanation could be that the semi-annual schedule of varnish applications was too infrequent to make a difference. It is suggested that fluoride in concentrations found in dental plaque may act as a metabolic inhibitor and lower the bacterial acid production in the oral biofilm but the clinical implications are still unclear [7]_. Many bacteria are highly susceptible to fluoride in planktonic stages or simple biofilm models but this may not be the case in complex biofilms communities in vivo [9, 18]_. Chau and co-workers [19] have recently shown that fluoride varnish applications can affect biofilm formation and acidogenicity but these effects were strongly reduced by time and biofilm age. Notably, the present samplings were performed approximately 6 months after the latest varnish application. A third but less plausible explanation could be that the daily exposure of fluoride from tooth paste in both groups may have obscured the results. Therefore, further clinical research to elucidate the impact of fluoride on bacterial physiology and adaption seems warranted.

Recent findings from molecular-based studies have confirmed the importance of mutans streptococci, Actinomyces and Veillonella for the development of early and severe childhood caries [20–22]_. We were able to verify a frequent recovery of V. parvula but found no differences in the S. mutans or A. odontolyticus counts between caries-free children and those with initial, moderate and extensive lesions. Interestingly, we noted a higher prevalence of Lactobacillus, Neisseria and Bifidobacterium species among children free from caries. These findings support the concept that caries is more due to absence or under-abundance of beneficial bacteria rather than linked to specific pathogens [23, 24]. Thus, future research should focus on functional rather than phylogenetic diversity in order to fully understand host-microbiome interactions. An illustration of this complexity is L. rhamnosus that can be linked to both mineral loss [25] and caries prevention [26]. The relatively frequent detection of Neisseria and Haemophilus in our samples was likely a reflection of the low age of the present subjects and the oral sampling technique representing more oral structures than just dental plaque.

The obtained results must however be regarded with caution. First of all, the study groups constituted a convenience sample from a major project and the groups were unfortunately not fully balanced concerning socio-economy and caries. A previous study has established a relationship between social deprivation and the isolation frequencies of caries-associated microorganisms, such as lactobacilli and mutans streptococci, in 3- and 4-year-old-children [27]. However, when the data was adjusted for the imbalance between the groups concerning immigrant status, parental smoking, sweet drinks and caries, the bacterial outcome remained unchanged. Secondly, the checkerboard DNA-DNA hybridization technique has its strengths and shortcomings; the main advantage is that the method permits enumeration of large numbers of species in a large numbers of samples and hence considered as a useful tool for the enumeration of bacterial species in complex microbial systems [28]. Among the limitations, possible cross-reactions and varying reproducibility for different strains has been discussed [29]. In fact, the probes used were not particularly specific and did not differentiate between genotypes of the same species. For example, among some Gram-positive genera (e. g. Actinomyces, Lactobacillus and Streptococcus) there was a risk for cross-reactions between closely related species. Such cross-reactions between the probes were checked prior to the study and the selection of the present twelve DNA probes was done in order to get as few cross-reactions as possible. Furthermore, the selected species represented early colonizers of the oral cavity (mucosal membranes and teeth) and therefore considered suitable for this low age group. The methodological limitations made us however to present data according as proportions over the thresholds 10⁴ and 10⁵ cells respectively [29]. We used the swab-technique to collect oral samples rather than dental samples for practical reasons; because of the low age of the subjects, it was not possible to collect enough plaque and saliva for separate analysis. One should also keep in mind that the present assay only mirrored 12 selected species out of the over 600 prevalent taxa at species level that are reported from the oral cavity [30].

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