Dental plaque biofilm is part of the oral microbiome that co-evolves in symbiosis with the human host (Marsh, 2012). Recently the importance and beneficial role of the oral microbiome in maintaining oral and general health has been brought forward (Marsh, 2012; Hezel and Weitzberg, 2013). On the other hand, undisturbed dental plaque accumulation is associated with an enhanced host inflammatory response and gingival inflammation (gingivitis) (Lee et al., 2012). Gingivitis is known to be associated with the onset of periodontitis (Schatzle et al., 2003), therefore the importance of maintaining gingival health is well understood.

Although regular mechanical plaque removal is recommended for prevention of periodontal diseases, the quality of self-performed mechanical plaque removal may not always be sufficient (Hioe and van der Weijden, 2005). When this fails or cannot be optimally maintained, for instance in physically or mentally disabled populations, a chemical approach, such as the use of an antimicrobial mouthwash, can be an alternative or an adjunct.

Anti-plaque agents should not eradicate the oral microbiota. Instead, they should maintain the microbiota of the mouth at the level and composition that is compatible with oral health, this way preserving the beneficial functions of resident microbes (Marsh, 2012; ten Cate and Zaura, 2012). This requirement is not met by so-called broad spectrum antimicrobial agents such as chlorhexidine (CHX). Interestingly, oxygenating mouthwashes containing peroxoborate are able to reduce the dental plaque amount and retard the colonization and growth of anaerobes (Wennstrom and Lindhe, 1979; Binney et al., 1992; Moran et al., 1995) and Gram-negative bacteria (Hernandez et al., 2013). Gram-negative anaerobes are generally associated with oral infections (e.g., periodontitis, peri-implantitis, endodontic infections).

Among oxygenating agents, boron-derived compounds such as sodium perborate (peroxoborate) generate active oxygen in aqueous solutions. This characteristic is the basis for their use as bleaching agents in detergents, cleaning products and cosmetic preparations, as well as a preservative in eye drops (Safety, 2010). In clinical dentistry, boron-derived compounds are used as a bleaching agent for teeth and as an adjunct to CHX to counteract extrinsic staining of the tongue and tooth surfaces (Dona et al., 1998; Grundemann et al., 2000; van Maanen-Schakel et al., 2012; Feiz et al., 2014).

Ardox-X® technology (AX) was introduced to the market and promoted as a teeth whitening, anti-microbial, anti-fungal and anti-inflammatory compound (NGen Oral Pharma, www.ngenpharma.com)¹. According to the manufacturer, the AX compound is formed by chemical complexation of peroxoborate with specific carriers such as glycerol and cellulose. This produces sodiumperborate-1,2-diol-glycerol/cellulose-ester adducts, i.e., single-reaction products containing all the atoms of all components. The manufacturer considers this to be a distinct molecular compound that provides controlled release of active oxygen without generating hydroxyl radicals. However, the scientific evidence for the antimicrobial efficacy of this compound is scarce. So far, only one in vitro study has been published which showed that AX has an antimicrobial effect against polymicrobial biofilm (microcosm) grown on titanium surfaces (Ntrouka et al., 2011).

The aims of the current study were: first, to determine the antimicrobial effect of AX against oral bacteria in vitro; second, to evaluate in vivo the effect of AX containing mouthwash on the composition of undisturbed plaque accumulation in a one-week non-brushing model in healthy adults.

The in vitro experiments of this study indicated that oxygen-releasing compound Ardox-X® technology (AX) selectively inhibits oral bacteria, with anaerobe Gram-negative species being the most sensitive. These promising findings were further tested in vivo, during a pilot clinical study with experimental period without any oral hygiene measures but twice-daily rinse with mouthwash containing AX. After a week of non-brushing, the plaque scores increased, while the microbial composition showed a shift toward compositionally less diverse plaque, dominated by primary colonizing genera Streptococcus and Veillonella compared to the Corynebacterium dominated plaque at the baseline.

It has been proposed that in order to study the effects of a mouthwash, a population with a high amount of plaque should be studied (Wennstrom, 1988). The study population therefore included individuals that proved to be good plaque formers at the screening visit. Moreover, it is known that the periodontal condition affects the rate of supragingival plaque forming (Hillam and Hull, 1977; Rowshani et al., 2004). Participants showed to have moderate gingivitis at baseline whereby 51% of the sampled sites were bleeding on marginal probing. The one-month interval between the pre-experiment assessment and baseline did not result in significant changes in plaque scores or in plaque composition of the study population. This is in line with previous studies, which have demonstrated the stability of the oral microbiome (Zhou et al., 2013). Microbial composition of the supragingival plaque in this gingivitis population 1 month before the experiment and at the baseline resembled mature plaque (Haffajee et al., 2009) and plaque associated with gingivitis (Huang et al., 2011). The major taxon in these samples was identified as Corynebacterium, a Gram-positive, facultatively anaerobe bacterium that resembles Gram-negatives with respect to the lipid layer in the cell membrane (John, 1984). Corynebacterium is associated with mature plaque and is found in dental calculus (Moorer et al., 1993).

The participants of the classical experimental gingivitis model (Loe et al., 1965; Theilade et al., 1966) received a prophylaxis and subsequently refrained from oral hygiene for 21 days inducing an acute stage of inflammation in otherwise healthy subjects. For the present study a seven-day model was chosen since the purpose was not to change the level of gingival health but to assess the effects on the microbial composition of undisturbed plaque. In the absence of oral hygiene, bacterial re-colonization increases after professional oral hygiene reaching or exceeding pre-prophylaxis levels at 2 days (Uzel et al., 2011) and de novo plaque formation reaches a stable microbial community between 4 and 7 days (Uzel et al., 2011). Higher diversity of mature supragingival plaque compared to younger plaque has been found in a recent experimental gingivitis study (Kistler et al., 2013). In the present study after the baseline assessment, the participants received a thorough professional oral hygiene and were not allowed to brush their teeth for 1 week. Instead, they were asked to perform a twice daily rinsing with AX-containing mouthwash. As expected, the plaque amount increased during the experimental period. The composition of the sampled plaque also changed impressively, whereby microbial diversity had decreased significantly, when compared to the pre-experimental and baseline visits. Genus Corynebacterium was considerably reduced, while streptococci, Veillonella and Haemophilus—all health-associated primary colonizers (Colombo et al., 2009; Simon-Soro et al., 2013)—dominated the post-experimental plaque.

In vitro diffusion and susceptibility tests showed that AX is highly selective in inhibiting oral bacteria. The Gram-negative anaerobes such as prevotellas, but also Veillonella, Tannarella, Campylobacter, Fusobacterium, and Porphyromonas were highly sensitive, while streptococci and lactobacilli, facultative anaerobe Gram-positive bacteria, were not inhibited even by the highest dose of AX tested in the diffusion test. In the clinical samples genus Prevotella were found at very low levels throughout the study and no effect of AX was discernible. However, other Gram-negative taxa such as genera Derxia and Leptotrichia, as well as OTUs classified as Cardiobacterium (OTU#245) and Capnocytophaga (OTU#251) were significantly reduced during the experimental period. Unfortunately, in vitro tests did not include any genus Corynebacterium species which allows only for speculation whether this Gram-positive bacterium with the characteristics of Gram-negatives (John, 1984) was also highly susceptible to exposure to AX. Alternatively its nearly complete elimination from the post-experimental plaque samples could have other reasons for instance its growth could have been inhibited due to ecological shifts in the entire community (Bradshaw et al., 1989). Another intriguing bacteria was Veillonella—anaerobe Gram-negative bacteria, associated with early supragingival plaque (Li et al., 2004; Haffajee et al., 2009). Although Veillonella was found to be susceptible to AX in in vitro testing, this genus showed a significant increase after the experimental period with twice-daily exposure to the AX mouthwash. This could be attributed to the “pioneering” function of this bacteria; it is found in healthy individuals, in young supragingival plaque, in similar proportions with streptococci (Keijser et al., 2008; Haffajee et al., 2009). Veillonellae are secondary fermenters—they consume lactic acid produced during glucose fermentation by streptococci (Keller and Surette, 2006; Periasamy and Kolenbrander, 2010) and produce other, weaker acids such as acetic and propionic acid. By doing so, the environmental conditions are created that promote growth of both of these genera (Bradshaw et al., 1989). The most likely explanation of the increase of genus Veillonella during the experimental period, although sensitive to direct exposure to AX in vitro, could be related to this ecologically beneficial relationship with streptococci.

The selective inhibition of oral bacteria by AX is of interest with respect to gingival and periodontal diseases, since infections associated with Gram-negatives would be selectively suppressed whereas the microorganisms regarded as more beneficial for periodontal health such as streptococci and lactobacilli would not. Several in vitro studies (Teughels et al., 2007; van Essche et al., 2012) and recent clinical studies (Iniesta et al., 2012; Teughels et al., 2013; Yanine et al., 2013) have suggested that these allegedly beneficial bacteria can cause antagonism toward Gram-negative bacteria.

In the agar diffusion assay, AX had significantly lower inhibitory effect than CHX. The activity of AX could have been limited to a short period right after its administration until the active component is broken down and oxygen is released. CHX is known to retain its activity for a longer time period after a single application in vitro (Carrilho et al., 2010). The only other study that has assessed the antimicrobial effect of AX, showed that the AX containing product had higher antimicrobial capacity than chlorhexidine toward monospecies bacterial biofilm and microcosm obtained from pooled saliva (Ntrouka et al., 2011).

So far, chlorhexidine (CHX) has proven to be the most effective antimicrobial agent in clinical dentistry and is considered as the “gold standard” disinfectant in dental research (Jones, 1997; Arweiler et al., 2001). Although it is widely used in periodontics and is among the most effective compounds preventing plaque formation (Addy, 1986), it has several side effects (Keijser et al., 2003; Gurgan et al., 2006) and therefore may result in poor rinsing compliance by patients (Addy and Moran, 1985; Cortellini et al., 2008; Van Strydonck et al., 2012). Optimizing anti-plaque agents, reducing their side effects while at the same time taking care that the oral microbiota are kept in balance with oral health has initiated interest in the development of other chemotherapeutical agents. Interestingly, AX showed selective inhibition of oral bacteria that may contribute to this demanding balance and deserves further investigation.

The lack of controls is a major limitation of this study that could have potential bias on the interpretation of this study outcomes. This pilot however indicates a potential rationale for more elaborate studies with a randomized clinical trial protocol that would include both a positive control such as CHX, and a negative control without any antimicrobial effects. The potential effect of AX on reductions in both the clinical manifestations of gingivitis and the inhibition of or reduction of plaque or plaque pathogenicity still needs to be demonstrated. For that purpose a 21 days experimental gingivitis model could be used or alternatively a 4-week trial among gingivitis subject as proposed by the American Dental Association in their Acceptance Guidelines of Chemotherapeutic Products for Control of Gingivitis (ADA, Acceptance Guidelines, 2008).

In conclusion, a mouthwash containing the oxygenating agent Ardox-X® technology showed potential for selective inhibition of oral bacteria. Twice-daily exposure for 1 week to this mouthwash resulted in a shift in the microbial composition toward a less diverse and less mature plaque. The clinical consequences of this shift in the oral microbiota need to be established.

ACTA Research BV received financial support from NGen Oral Pharma N. V., Curacao, for the role of the Departments of Periodontology and Preventive Dentistry of ACTA in this project. NGen Oral Pharma N. V., Curacao, provided study products. The authors report that this company had no influence on the design, content, results and publication of this study.