Study of oral microorganisms contributing to non‐carious cervical lesions via bacterial interaction and pH regulation

Abstract There is a lack of evidence about the relationship between microorganisms and non‐carious cervical lesions (NCCLs) due to limited technologies. A group of 78 patients was enrolled for microbial 16S rRNA sequencing of dental plaques on normal and defective cervical surfaces. Parallel data from 39 patients were analysed with paired t tests, and Fusobacteriales exhibited significantly less distribution on NCCLs than on normal surfaces. As a result, Fusobacterium nucleatum, the most common oral bacterial strain belonging to the order Fusobacteriales, was selected for further research. From a scanning electron microscopy (SEM) scan, the tooth surface with Fusobacterium nucleatum and Streptococcus mutans culture was more intact than that without Fusobacterium nucleatum. Furthermore, the calcium contents in groups with Fusobacterium nucleatum were significantly higher than that without it. In further mechanistic research, Fusobacterium nucleatum was demonstrated to adhere to and disturb other organisms as well as producing alkaline secretions to neutralize the deleterious acidic environment, protecting the tooth structure. In conclusion, microorganisms and NCCLs were confirmed directly related through adherent bacterial interactions and pH regulation. The research provides a new perspective and experimental evidence for the relation between microorganisms and NCCLs, which guides clinical treatment and preventive dentistry in the future.


| INTRODUC TI ON
A NCCL is defined as loss of dental hard tissue at the cementoenamel junction (CEJ), which is commonly encountered in clinical practice. 1,2 The prevalence of NCCLs has not been well documented because of varying investigation methods and samples; thus, it is difficult to compare the results obtained from diverse scientific studies. 3,4 According to a study in 1985, non-carious destruction contributed to nearly 25% of the pathological processes of hard dental tissue. 5 However, it is well recognized that their prevalence and severity increase with age, and the first premolars are affected most frequently. 6,7 Dental lesions at the neck produce a series of unpleasant symptoms, such as sensitivity, pain and unaesthetic appearance. The problems draw increasing attention as the life expectancy of humankind increases and awareness of the importance of oral health increases. 8 The most commonly acknowledged aetiology causing the progression of NCCLs on the neck is multifactorial, including erosion, abrasion and occlusal trauma. 3,4 Evidences suggest that abrasion and erosion from long-term physical-chemical effects could lead to the destruction of the dental crystalline structure of hydroxyapatite and fluorapatite. Lee et al proposed the occlusal stress and tooth flexion theory, noting that traumatic occlusion results in tooth tissue deformation to create microcracks under the magnitude and direction of masticatory forces. 9 A comprehensive understanding of the impact of the oral microbiota on host health and disease is considered a holistic view of intra-and cross-kingdom interactions among members of the oral microbiome, and imbalance of the oral flora leads to caries, periodontal disease and other oral pathologies. 10,11 Due to the special oral environment of the cervical teeth, anatomical structure changes the wedge-shaped defects, residual food and unqualified cleaning can bring about corresponding changes in the local microecology.
In recent years, most surveys involving wedge-shaped defects have focused mainly on the occlusion and comparison of different filling materials and methods.
In fact, there is a lack of definite evidence to demonstrate that microorganisms do not lead to NCCLs or that no microflora variation occurs in NCCLs. Considering that the official definition of NCCLs denies the function of caries, few researchers are interested in the relationship between oral microorganisms and defect sites. 2,12 Moreover, previous microflora studies were limited mainly by the inefficiency of technologies because of the extremely minimal flora existing on the smooth surface of tooth defects for most people without caries. The microbial community on the surface of teeth is diverse, so we are interested in conducting a systematic study on the correlation between defects and microorganisms. The rapid development of high-throughput sequencing technologies in the past decade provides scientists with new perspectives as well as opportunities to explore the composition of the microbial world and the unknown area of life science. 13,14 In this study, microorganism specimens from the facial surface of the cervical tooth in subjects with healthy tooth surfaces and those with wedge-shaped defects were collected, measured and analysed. Furthermore, some functional experiments were conducted in vitro to investigate the correlation of dental wedge-shaped defects and oral microbes, especially whether oral microbes could affect the occurrence and development of wedgeshaped defects. The aim of this study was to explore the role of oral microbes in NCCLs and provide an experimental basis for NCCLs prevention and treatment.  Upon receipt, these intact teeth first underwent professional periodontal curettage to remove dental calculus, pigmentation and attached soft tissue remnants. Subsequently, the teeth were kept in a 0.5% sodium hypochlorite solution for surface disinfection overnight and cleaned in a phosphate-buffered saline (PBS) solution (pH 7.4).

Fusobacterium nucleatum is an obligate anaerobe, while
Streptococcus mutans is a facultative anaerobe. Both microorganisms used in this study were cultured under anaerobic conditions with a biobag (Thermo AnaeroGen, USA) at 37°C.

| Specimen treatment
Either the tooth crown or root was transversely and partly cut off using a rotating highly concentrated diamond saw (Buehler, USA) under water cooling. The retained tooth tissue portion was also sectioned in half parallel to the long axis of the teeth, from buccal to lingual surfaces, by the cutting wheel. Finally, the tooth slices were stored in sterile artificial saliva (Chuangfeng Technology, China) at 4℃. The two equivalent tooth slices obtained from each tooth were sorted into two groups with differently appointed numbers. For example, half of the tooth specimen was named 1A, 2A…10A in the control group, while the other half was named 1B, 2B…10B in the treatment group.
The specimens of 1B, 2B…5B were placed in five conical flasks containing only Streptococcus mutans suspensions, while the specimens 6B, 7B…10B were placed in another five conical flasks containing Streptococcus mutans following Fusobacterium nucleatum culture, where they remained separately for 6 days at 37℃ under gentle agitation. Afterwards, the abovementioned dentinal cylinders were removed from the flasks and cleaned with PBS.

| pH value variation
After incubation, the pH values of Fusobacterium nucleatum and Streptococcus mutans cultures were detected by a pH meter after 2, 4 and 6 days (Mettler, Switzerland). The culture conditions were the same as those above.

| SEM and outcome measure
After inoculation for up to 6 days, the treated specimens were prepared for SEM examination. Dentinal cylinders were fixed in 35% glutaraldehyde and dried with ascending ethanol concentrations. 18 Afterward, they were dehydrated to their critical point with a drying apparatus (Bio-Rad, E3000, Watford, UK), fixed on microscope slides and gold-coated using a sputter coater (Bio-Rad). The surface morphology of the tooth specimens was captured by a tungsten filament SEM (Quanta 250*, FEI, USA). EDX spectroscopy mapping was performed by using an XFlash 6130 microscope (Bruker, Germany) to identify the chemical compositions of the surface of crystal phases.

| Bioinformatics and statistical analysis
Raw reads were demultiplexed and quality filtered by QIIME 2, a next-generation microbiome bioinformatics platform, to determine the alpha and beta diversity indexes. 16 The alpha and beta diversity data were performed using Wilcoxon rank-sum test.
Quantitative data are shown as the mean ± standard deviation (SD), median or interquartile range, while qualitative data are presented as frequencies. Normally distributed continuous variables were processed through paired and 2 independent samples t tests. 19 Univariate and multivariate linear regression models were applied to analyse the association between given factors and NCCL risk. A two-tailed P < 0.05 was defined as statistically significant. All data analyses were performed with R software (version 3.6.0). Models were built using forward variable selection. Variables explored included stable demographic factors and modifiable lifestyle factors. 20

| Paired t test analysis
Eliminating unpaired cases of healthy and defective samples in the same patient, the available data were ultimately obtained from wedge-defect samples and normal samples from 39 patients.
Through sequencing, the relative abundance of all strains in each sample was obtained. The relative proportion of each order was calculated and compared with a paired t test. As shown in Table 2, Bacteroidales and Firmicutes seemed to display an association with NCCL risk, although the association was not statistically significant (Bacteroidales, P = 0.056; Firmicutes, P = 0.066), while Actinomycetales, Clostridiales and Fusobacteriales exhibited statistically significant differences (Actinomycetales, P = 0.023; Clostridiales, P = 0.011). Among these, Fusobacteriales showed an extremely significant difference (P = 0.000345, t = 3.932) between the normal group and the NCCL group. 23

| Correlation analysis of NCCL and possible factors
In addition to the traditional aetiology, additional possible factors associated with oral organisms were speculated to explore the relationship among Fusobacterium nucleatum, lifestyle factors and clinical variables by univariate and multivariate linear regression. The related statistical analysis with coef., P values and 95% CIs of 39 survey respondents is described in Tables 3 and 4. As shown in Table 3, using univariate linear regression, there was no significant difference revealed between

| SEM and element mapping data of tooth surfaces after Fusobacterium nucleatum and Streptococcus mutans culturing
Relative to other destructive bacteria, Fusobacterium nucleatum was suspected to have protective effects on dental hard tissues.
To simulate the oral microenvironment, we selected the most common damaging bacterial strain, Streptococcus mutans, and cultured it separately or followed with Fusobacterium nucleatum to explore and compare their effects.
Oral multispecies communities were formed on dentin specimens by culturing Fusobacterium nucleatum and Streptococcus mutans. At the end of the incubation period, images were obtained with a SEM (magnification: ×1000). In comparison with that of the tooth slices from the other half of the same tooth or those incubated with Streptococcus mutans culture alone, the surface morphology of the tooth specimens with only Streptococcus mutans exhibited the most distinct cracks (Figure 3A-D). The most interesting appearance we detected from the specimens immersed in the Fusobacterium nucleatum and Streptococcus mutans culture revealed nearly similar morphology to that of untreated control slices ( Figure 3E-H). Since the specimens we selected were teeth isolated from oral cavities of different humans, the tooth surface itself was greatly affected by individual differences.
Some teeth surfaces were relatively smooth, homogenous and shiny, while others originally suffered potential structural damage. Through random allocation, we selected both relatively smooth visual fields ( Figure 3A,B,E,F) and relatively rough visual fields (Figure 3C,D,G,H) to objectively illustrate the possible influence of bacteria on the tooth surface. It was observed that Fusobacterium nucleatum, on both the relatively intact and relatively rough tooth surfaces, produced no significant changes in morphology compared with that of the control.
In the Streptococcus mutans alone group, the relatively smooth tooth surface exhibited significant defects, while the originally rough surface showed widened and deepened destruction.
According to the elemental mapping data ( Figure

TA B L E 3 Factors associated with
Fusobacterium sp in a univariate linear regression model prevent structural integration and counteract the damage induced by Streptococcus mutans. Every group contained 5 specimens, and every test was performed 3 times.

| Fusobacterium nucleatum adheres to other bacteria and affects the pH value
To further explore the mechanism of tooth tissue damage by Streptococcus mutans and protection by Fusobacterium nucleatum, SEM was utilized to visibly record the morphology and growth statuses of bacteria. As shown in Figure 4A, pure Streptococcus mutans was distinctively biojoint-spherical shaped, while pure Fusobacterium nucleatum was long and rod shaped. When the two kinds of bacteria were cultured, we found that Fusobacterium nucleatum could adhere to Streptococcus mutans, which exhibited significant transformation and stretching at the contact region, as if a tentacle was extended to capture other bacteria.
Also, we conducted pH test experiments under different conditions. The BHI and sheep blood culture medium was slightly alkaline, and its pH values were approximately 7.2 to 7.4 ( Figure 4B). With the growth of Fusobacterium nucleatum, the pH value of the bacterial suspension slowly increased but still appeared alkaline. In contrast, the pH value in the Streptococcus mutans suspension decreased to nearly 5.34, changing from alkaline to acidic.  In this study, a novel nucleic acid extraction method was first adopted to extract extremely small traces of microbiological DNA at the cervical dentin surface, and metagenomic sequencing was Fusobacterium nucleatum, an obligate anaerobic gram-negative rod, has long been found to cause opportunistic infections in a wide spectrum of human diseases, such as oral infections 24 and colorectal cancer. 25 High content of Fusobacterium colonizes mainly the human oral cavity and has a symbiotic relationship with its hosts. 26 To conclude, the wedges were confirmed to have a certain correlation with the distribution of microorganisms in our research.

TA B L E 4 Factors associated with
Although there was no significant difference between the distribution and diversity of the microbial population at the defect site and that on normal teeth, statistical analysis of each bacteria using a paired t test revealed significant differences in Actinomycetales, Clostridiales and Fusobacteriales, among which the difference in Fusobacteriales was the most significant. The most common oral flora constituent Fusobacterium nucleatum, a Fusobacteriales species, was selected to further explore its interaction with cervical defects, and a hypothesis suggesting protection of the tooth neck by Fusobacterium nucleatum was proposed. It is speculated that Fusobacterium nucleatum may pro-

CO N FLI C T O F I NTE R E S T
The authors declare no competing interest.

E TH I C A L A PPROVA L
This study was approved by the Institutional Review Board of School and Hospital of Stomatology, Fujian Medical University, and written informed consent was obtained from all subjects before interviews.

DATA AVA I L A B I L I T Y S TAT E M E N T
The sequencing raw data reported in this article have been uploaded to the NCBI-SRA Database under the accession number of PRJNA589106.