Vinegar inhibits the formation of oral biofilm in situ

Dental problems have been the major reason of Years Lived with disability throughout the world [1]. Oral biofilm has been accepted to be the main reason of oral diseases, such as caries and periodontitis which are caused by dysbiosis and imbalance of the biofilm’s composition [2]. Therefore, biofilm management takes an essential part in prevention and treatment of oral diseases.

Among many other natural antibacterial agents, vinegar is often used for the prevention and treatment of diseases because of its low pH value. Different studies have reported that ear irrigation with diluted vinegar can be effective in the treatment of ear infections, such as chronic suppurative otitis media [3], granular myringitis [4] and otitis externa [5]. Also, the undiluted vinegar can effectively remove the bacteria from dentures, which will not cause oral mucosal damage even if residual vinegar remains on the denture [6]. The explanation for this might be that vinegar affects cell membrane function, leading to transmembrane proton motility destruction [7, 8], as well as other factors such as inhibition of enzyme activity [9], energy competition and inhibition of bacterial protein expression [10].

Although vinegar has such antibacterial effects, there is few research of vinegar applied to the oral biofilm. The impact of vinegar on the initial microflora adherence to enamel and the formation of biofilm in situ have not yet been systematically analyzed. Thus, the present study aims to investigate the effect of commercially available vinegar on in situ formation of the initial pellicle and on 24-h biofilms, as well as on salivary bacteria.

The development of biofilms is a dynamic process [2]. Firstly, after contact with saliva and gingival crevicular fluid, the pellicle is formed on the cleansed tooth surface. The main composition of the pellicle are salivary glycoproteins, carbohydrates and lipids which have been examined by analytical techniques [15]. In the 3-min pellicle experiment, the control group presented the normal process of pellicle formation. The thickness of the globular layer increased upon the 120-min experimental trial (p < 0.001), which proves former research on pellicle formation [16, 17]. In comparison, these particles layers were removed immediately due to vinegar rinsing, and were sparsely formed during the next 120 min after vinegar rinsing, which suggests that the formation of the pellicle was strongly reduced at least for 120 min. Therefore, vinegar presents substantial inhibition in the initial biofilm formation.

There are few investigations dealing with acetic acid applied on the enamel surface for much longer times than 5 s. These studies revealed dental erosion potential of acetic acid; many of them are in vitro studies [18‐21]. According to the daily diet habits, vinegar will not stay in the oral cavity for several minutes due to the swallowing. It has been reported that even the 3-min pellicle could protect the enamel from acid erosion for 60 s [13]. Therefore, short-term application of acid proved to be safe due to the protective effect of the oral biofilm [11]. On the other hand, pure enamel is not exposed intraorally but always covered by biofilms and soaking in saliva in the oral cavity. Thus in situ experiments have advantages in studying erosive processes as compared to in vitro studies [22, 23]. In the 3-min pellicle in situ study, the subsurface pellicle layer below the enamel surface appeared immediately after vinegar rinsing for 5 s with less dense electron appearance as revealed by TEM. This indicated that vinegar could substantially promote the formation of the subsurface pellicle layer, which could prevent further demineralization [11, 15].

After formation of the pellicle, the early bacterial colonizers will adhere to the pellicle surface [2]. In the present experiments, bacteria can already be observed in the control group at 30 min and 120 min after initial 3-min pellicle formation, with many fimbriae firmly fixed on the pellicle surface. Moreover, the quantity of protein particles increased further after the formation of the pellicle for 30 min (p < 0.001) as well as the frequency of single microorganisms adhering to the surface in the control group. However, only the enamel surface covered by a thin pellicle with rarely visible protein particles or microbial attachment was observed after vinegar rinsing. Additionally, the vast majority of microorganism in saliva was strongly affected by vinegar rinsing (p < 0.001), which results in delay of the early microbial colonization process, thus hindering the formation of biofilm. Therefore, vinegar altered the bacteria in both, initial biofilm and saliva, resulting in the inhibition of early biofilm growth.

After colonized by early microbes, biofilms gradually accumulate a wide variety of other species of bacteria, which promotes growth of the biofilm. It has been reported that bacteria embedded in mature biofilms present more tolerance to antibiotic than planktonic cells as well as antiseptics, biocides and other environmental impacts [24, 25]. Therefore, these biofilm are difficult to remove by antibiotics [26]. In the present study, vinegar presented a stronger antibacterial effect to planktonic bacteria in saliva than bacteria embedded in the 24-h biofilm (p < 0.01). Both, TEM as well as SEM images proved that the mature biofilm could functionally protect the microbes better than saliva. Moreover, in the 24-h biofilm experiment, vinegar destroyed the biofilm structure significantly. Especially, 30 min and 120 min after vinegar rinsing, the biofilm was almost wiped out, even the matrix was in part disrupted.

In the oral cavity, saliva as well as the biofilm have buffer capacity for extreme pH values [15, 27, 28]. This buffer capacity is important to maintain the pH value of saliva [29]. In the present experiment, the characteristic of the saliva samples is of great difference immediately after vinegar rinsing (pH = 2.3) compared with the control saliva (pH = 7.0). The control saliva was a very mucus-rich secretion, while the vinegar sample was only a watery fluid essentially devoid of mucus. This suggested that vinegar might change the characteristic of saliva. However, vinegar causes inhibition of biofilm formation as well as biofilm removal, which means that there are some more influencing factors beside the pH value. It has been supposed that salivary flow rate will be increased already before the acid rinsing, which improves the buffer capacity resulting in a protective effect due to the effective dilution of acids [30].

Actually, there are many studies of rinsing solutions used for oral biofilm management and removal, such as chlorhexidine, cetylpyridinium chloride and plant solutions [31‐34]. Among them, chlorhexidine is recognized as the gold standard [35]. However, more and more side effects were reported after wide clinical application of chlorhexidine rinsing, such as tooth staining, taste disturbance and even serious allergic reactions [36, 37]. In addition, more recently, concerns have been raised about an enhanced tolerance or even resistance toward CHX that might lead to an enrichment of resistant strains after widespread use of CHX [38‐40]. In comparison, vinegar present better advantages in clinical experience. Vinegar cannot avoid bacterial resistance in general, but using formulations containing vinegar instead of antiseptics or antibiotics as oral antibacterial agents may be worthwhile in reducing risk of development of resistance in oral bacteria. As demonstrated in the present study, vinegar reveals a significant efficacy to manage and inhibit short-term biofilm formation. However, further experiments on mature biofilms are needed. These experiments should focus on quantification of the proliferation capacity of bacteria after vinegar treatment, using for example colony-forming unit counts. Furthermore, the effects of vinegar on oral biofilm formation in comparison to gold standard antimicrobials (like CHX) should also be investigated.

To sum up, this in situ study has demonstrated the potential of the vinegar rinsing approach for inhibition of biofilm formation in the oral cavity, as well as strong removal effect towards the outer globular particles of the initial pellicle on the enamel surface. Although the results of vinegar were significant in this study, the long-term clinical efficacy required further studies.