Anaerobic Bacteria in Implants and Homologous Teeth 2-14 Years after Implantation.

OBJECTIVE: The objective of the study was to establish whether there is a difference in the presence of potentially pathogenic anaerobic microorganisms around the implant and the homologous tooth in implant-prosthetic patients who received individual information about maintaining their oral hygiene.
MATERIAL AND METHODS: The study included 30 subjects with dental implants and metal-ceramic crowns. A periodontal probe was used to record the approximal plaque index (API), the papilla bleeding index (PBI), the periodontal pocket probing depth (PD) and the gingival recession. The fluid around the implant and the gingival sulcus fluid around the homologous tooth on the opposite lateral side were sampled.
RESULTS: The results have shown a positive API and PBI on 30% of the implants and a negative one on 70% of the implants. The average mucosal retraction measured around the implants was 0.15 mm, and the average probing depth was 2.25 mm. The API and PBI were positive on 78.3% of the homologous teeth. The average gingival retraction measured was 1.06 mm, and the average probing depth was 1.85 mm. Anaerobic bacteria were found in 12 out of 30 subjects (40%). Anaerobic bacteria were isolated only on the implant in 7 subjects, only on the homologous tooth in 3 subjects and both on the implant and the homologous tooth in 2 subjects.
CONCLUSIONS: Anaerobic bacteria were more abundantly present on implants than on homologous teeth.

Introduction

It has been almost five years since Albrektson et al. () concluded that peri-implantitis is “an infection with suppuration associated with clinically significant progressive crestal bone loss after the adaptive phase”, but it appears that the number of patients with peri-implant infections is constantly rising (). The etiology of peri-implantitis is complex, and the series of risk factors that influence its emergence and progression can only be explained by the multicausality model. Nevertheless, the organization and formation of biofilm on dental implants foster the host's reaction, which leads to the development of peri-implant mucositis, and soon also to peri-implantitis (). Biofilm can be defined as an aggregation of one or more groups of different microorganisms, embedded in a self-produced matrix and adhering to a firm surface ().

The initial phases of biofilm formation on teeth and on implants can be considered identical. The pellicle on the surface of implants/suprastructures is very similar to the pellicle on natural teeth. In the initial phase of biofilm formation, Streptococcus mutans makes up for 60%-80% of all early colonizers, with different bacterial adhesives responsible for the adhesion to the pellicle. Even though the growth and diversification of biofilm are somewhat different on implants than on natural teeth, certain elements remain identical. For example, the collective consciousness that the bacteria develop is enhanced by the stimulating peptides that are released after the exposure to low pH ().

The surface of implants is preferably uneven, but that is precisely what favors the formation of biofilm, the organization that is nowadays considered to be a primitive multicellular organism (). There are four elements that are favorable for the growth and formation of biofilm on the surface of dental implants: (a) random transport of bacteria to the surface of the implant through saliva, (b) initial (reversible) adhesion, (c) colonization of the surface and (d) strong adhesion to the surface ().

Controlling the biofilm is one of the main prerequisites for keeping the peri-implant tissue, as well as the periodontal tissue, healthy. However, due to morphological and anatomical differences, peri-implant tissue is more prone to developing inflammation than periodontal tissue, and it appears that the inflammation around dental implants progresses faster (). There is a series of factors, primarily bacteria-related, that affects the extent to which the biofilm will constitute a challenge for the host. The emergence of inflammation leads directly to significant changes in the composition of biofilm (), primarily in terms of increasing and decreasing the proportion of certain species (). These differences are especially noticeable when it comes to gingivitis (), but the roles of certain species within biofilm in developing peri-implant mucositis and peri-implantitis are unknown. Since periimplantitis is a serious condition that can lead to progressive destruction of the supporting alveolar bone and adjacent tissues, it is of general interest to identify the local parameters which significantly influence the initiation and progression of this disease ().

Precisely in view of that, in this study we have attempted to establish whether there is a difference in the presence of potentially so-called periodontopathogenic bacteria (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola) around the implant and homologous tooth in patients who, after having the dental implants placed, received information about an individual approach to maintaining oral hygiene.

Materials and methods

Subjects

The study included 30 subjects (10 males and 20 females), whose average age was 49.6 years (ranging from 22 to 78 years). Aside from having implant-prosthetic interventions done, the chosen participants were required to have natural teeth as well. On top of the participant’s implants, dental prostheses in the form of metal-ceramic crowns had been fixed, whose average age before the examination was 5.26 years (ranging from 2 to 14 years). Four implants were placed in the area of front teeth (incisors), and 26 were placed in the back (premolars and molars). The subjects were healthy and did not show clinical signs of periodontal disease. All the participants had signed an informed consent form for participating in a scientific study, approved by the Ethics Committee of the School of Dental Medicine, University of Zagreb.

Methods

The condition of the participant’s tooth-supporting apparatus and of the tissue surrounding the implant was established during an examination. A periodontal probe (Tekno-medical Optik-Chirurgie, Tuttlingen Germany) was used to record the following indexes and measurements: the approximal plaque index (API), the papilla bleeding index (PBI), the periodontal pocket probing depth (PD) and the gingival recession.

After disinfecting with the 3% hydrogen peroxide (H2O2), drying with compressed air and placing dental cotton rolls, paper points () (Absorbent Paper Point Pearl Endopia, Pearl Dent, Kyunggi-Do, Korea) were used to vestibularly sample the fluid around the implant and the gingival sulcus fluid around the homologous tooth on the contra lateral side. The paper points were kept in the sub gingival area of the implant for 90 seconds, after which they were stored in the anaerobic transport medium (Thioglycollate Medium G, Biolab, Budapest, Hungary) (Figure 1) until they were transported to the microbiological laboratory, where they were immediately incubated in the same medium at 37°C for three days. The samples were then cultivated on the Columbia anaerobically agar base with 5% horse blood (Columbia Agar Base, Biolife, Milano, Italy) and the bacteria were identified using the protein mass spectrometry method (matrix-assisted laser desorption/ionization time-of-flight mass spectrometer, MALDI-TOF-MS).

Figure 1

Paper Point () (Absorbent Paper Point Pearl Endopia, Pearl Dent, Kyunggi-Do, Korea), anaerobic transport medium (Thioglycollate Medium G, Biolab, Budapest, Hungary)

Statistical Analysis

The results obtained were analyzed by the statistical program SPSS 21 (IBM, Armnok, USA). The probability of the correlation of anaerobic bacterial findings with the depth of probing was determined by multiple linear regression model (p <0.05), while estimates of statistically significant differences when comparing the homologous tooth and implant were analyzed by t-test on the difference between the two populations. The remaining results describing the characteristics of the sample were processed with the help of descriptive statistics, regarding measures of central tendency, measures of variability and measures of asymmetry (Table 1-7).

Table 1

Absolute and relative frequencies of APIs and PBIs expressed in the number of positive and negative findings

PLAQUE VESTIBULAR (H)	Apsolutefrequencies	Percentfrequences	CumulativePercentage
Negative	6	20,0	20,0
Positive	24	80,0	100,0
PLAQUE ORAL (H)
Negative	7	23,3	23,3
Positive	23	76,7	100,0
BLEEDING VESTIBULAR (H)
Negative	6	20,0	20,0
Positive	24	80,0	100,0
BLEEDING ORAL (H)
Negative	7	23,3	23,3
Positive	23	76,7	100,0
PLAQUE VESTIBULAR (I)
Negative	21	70,0	70,0
Positive	9	30,0	100,0
PLAQUE ORAL (I)
Negative	21	70,0	70,0
Positive	9	30,0	100,0
BLEEDING VESTIBULAR (I)
Negative	21	70,0	70,0
Positive	9	30,0	100,0
BLEEDING ORAL (I)
Negative	21	70,0	70,0
Positive	9	30,0	100,0

Table 2 T

-test for estimating the depth of probe depth around the implant and the homologous tooth (mm).

Var/indices	N	SD	AS	MD	t	p	Lower 95%	Upper95%
Probing depth (H)	30	2,43	7,50	-1,47	-2,74	<0,05	-2,56	-0,37
Probing depth (I)	30	3,29	8,97

Table 3 T

-test for estimating the difference of mucosa recession around the implant and gingiva around the homologous tooth (mm).

Var/indices	N	SD	AS	MD	t	p	Lower95%	Upper 95%
Recession (H)	30	2,76	2,13	1,83	4,38	<0,05	0,98	2,69
Recession (I)	30	1,06	0,30

Table 4

Descriptive statistics of depth probes around the homologous tooth by API

API (H)		Depth probes distal (mm)	Depth probes mesial (mm)	Depth probes vestibular (mm)	Depth probesoral (mm)
Negative	N	6	6	6	6
	A. mean	1,83	1,83	1,67	1,33
	Median	2,00	2,00	2,00	1,00
	Std. Deviation	0,75	0,75	0,52	0,52
	Asymmetry coefficient	0,31	0,31	-0,97	0,97
	Curvature coefficient	-0,10	-0,10	-1,88	-1,88
	Data range	2	2	1	1
	Minimum	1	1	1	1
	Maximum	3	3	2	2
Positive	N	24	24	24	24
	A. mean	2,38	2,33	1,21	1,79
	Median	2,00	2,00	1,00	2,00
	Std. Deviation	0,92	1,17	0,41	0,66
	Asymmetry coefficient	2,75	1,60	1,53	0,24
	Curvature coefficient	10,33	3,17	0,38	-0,55
	Data range	5	5	1	2
	Minimum	1	1	1	1
	Maximum	6	6	2	3

Table 5

Descriptive gingival recession statistics according to the API

API (H)		Recession vestibular (mm)	Recession oral (mm)
Negative	N	6	6
	A. mean	1,50	0,83
	Median	0,50	0,00
	Std. Deviation	2,07	2,04
	Asymmetry coefficient	1,21	2,45
	Curvature coefficient	0,20	6,00
	Data range	5	5
	Minimum	0	0
	Maximum	5	5
Positive	N	24	24
	A. mean	1,42	0,67
	Median	1,00	0,00
	Std. Deviation	1,64	1,17
	Asymmetry coefficient	1,32	2,51
	Curvature coefficient	1,72	7,67
	Data range	6	5
	Minimum	0	0
	Maximum	6	5

Table 6

Descriptive statistics of probe depth around the implant to the API

API (I)		Depth probes distal (mm)	Depth probes mesial (mm)	Depth probes vestibular(mm)	Depth probes oral(mm)
Negative	N	21	21	21	21
	A. mean	2,52	2,71	1,57	2,29
	Median	2,00	3,00	1,00	2,00
	Std. Deviation	1,03	1,42	1,03	0,96
	Asymmetry coefficient	0,54	0,56	1,92	0,50
	Curvature coefficient	0,34	-0,19	5,55	-0,44
	Data range	4	5	5	3
	Minimum	1	1	0	1
	Maximum	5	6	5	4
Positive	N	9	9	9	9
	A. mean	2,11	2,11	2,22	2,22
	Median	2,00	2,00	2,00	2,00
	Std. Deviation	0,78	0,78	1,20	0,83
	Asymmetry coefficient	-0,22	-0,22	1,68	-0,50
	Curvature coefficient	-1,04	-1,04	3,69	-1,28
	Data range	2	2	4	2
	Minimum	1	1	1	1
	Maximum	3	3	5	3

Table 7

Descriptive recession of mucose around the implant to the API

API (I)		Recession vestibular (mm)	Recession oral (mm)
Negative	N	21	21
	A. mean	,24	,00
	Median	,00	,00
	Std. Deviation	1,09	,00
	Asymmetry coefficient	4,58
	Curvature coefficient	21,00
	Data range	5	0
	Minimum	0	0
	Maximum	5	0
Positive	N	9	9
	A. mean	,33	,11
	Median	,00	,00
	Std. Deviation	1,00	,33
	Asymmetry coefficient	3,00	3,00
	Curvature coefficient	9,00	9,00
	Data range	3	1
	Minimum	0	0
	Maximum	3	1

Results

The results of our study have shown a positive API on 30% of the implants and a negative one on 70% of the implants. The PBI values were identical to the API values. The average mucosal retraction measured around the implants was 0.15 mm (ranging from 0 to 5 mm), and the average probing depth was 2.25 mm (ranging from 0 to 6 mm). As regards the homologous teeth, the API and PBI were positive on 78.3% of the teeth. The average gingival retraction measured was 1.06 mm (ranging from 0 to 6 mm), and the average probing depth was 1.85 mm (ranging from 1 to 6 mm).

Furthermore, the results of the analysis indicate the presence of a statistically significant difference between the probe depth around the implant and about the homologous tooth (MD = -1.47; t = -2.74; p <0.05) (Table 2). In the previous study, a statistically significant difference was observed when retraction results were measured, with a higher retraction in the homologous tooth compared to the implant (MD = 1.83; t = 4.38; p <0.05) (Table 3).

Anaerobic bacteria were found in 12 out of 30 participants (40%), while no potentially pathogenic anaerobic bacteria were found in the remaining 18 participants (60%). Out of 12 participants, in 7 of them the anaerobic bacteria were present only on the implant, in 3 of them only they were present on the homologous tooth, while in 2 participants the anaerobic bacteria were present on both the implant and the homologous tooth. In those subjects, 13 anaerobic bacteria were found, including Streptoccocus anginosus on 2 implants, Propionibacterium acnes on 1 implant, Lactobacillus fermentum on 2 implants and 2 homologous teeth, Lactobacillus spp. on 1 implant and 1 homologous tooth, Bifidobacterium dentium on 1 implant, Veilonella spp. on 1 homologous tooth, and Veilonella parvula on 1 homologous tooth. In one subject, Veilonella parvula and Prevotela denticola were identified on the implant, while in one subject 5 anaerobic bacteria were isolated on the implant (Prevotella nigrescens, Fusobacterium nucleatum, Selenomonas infelix, Capnocytophagia spp., Parvimonas micra). A connection between the type of bacteria and the time of placing the implant has not been found.

The model of multiple linear regressions was used to investigate the existence of statistically significant correlation between probing depth and anaerobic bacterium findings. The results of the homologous tooth analysis indicate the absence of statistically significant correlation, with the model comparing 11.40% of the total variance. No depth of probing in the homologous tooth has a statistically significant association with the presence of anaerobic bacteria. The results of the implants were also analyzed, showing a partial statistically significant correlation between the depth of the probe and the findings of anaerobic bacteria. Specifically, 31.1% of the total variance was interpreted in the model, and in one of the four sites for the measurement of the depth of the probes, they have statistically significant correlation with anaerobic bacterial findings (b2 = 0.637, t = 2.82, p <0.05). Additionally, with the increasing depth of probe, the probability of finding anaerobic bacteria increases.

Discussion

The results of the study have shown that despite the expected potentially pathogenic bacteria, the so-called periodontal pathogens () (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola), potentially pathogenic anaerobic bacteria from the red complex (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) have not been isolated in any of the 30 subjects, neither on the implant nor on the homologous tooth.

In 40% of the subjects (), other anaerobic bacteria have been isolated, including the bacteria from the orange complex. Cortelli et al. () demonstrated the trend of a more frequent presence of anaerobic bacteria on natural teeth than on implants, while the results of our study have shown that more anaerobic bacteria have been isolated on implants compared to homologous teeth.

According to the available literature on the subject, in view of the biofilm formation, it is expected that a larger number of anaerobic bacteria will be isolated on natural teeth or that in the subjects in which anaerobic bacteria have been isolated on natural teeth, they will be isolated on the implant as well (, ). Some authors believe that bacteria on natural teeth are the primary source of pathogens and that they directly affect the outcome of the newly-placed implants (). However, Schierano et al. () analyzed the biofilm in relation to periodontal pathogens around clinically healthy teeth and around implants and they have not found any substantial differences in terms of the number and type of bacteria considering the two sampling locations. Botero et al. () stated that there was a significant connection between the sub gingival bacteria in implants and in the neighboring teeth. The results of our study show the presence of sub gingival bacteria on the implant and on the neighboring tooth in two subjects.

Koyanagi et al. () found that the microbial flora in peri-implantitis is more varied than in periodontitis and that Fusobacterium spp. and Streptococcus spp. are the dominant pathogens in both conditions. However, they found that Parvimonas micra had been isolated only in the patients with peri-implantitis, which is consistent with the results of our study.

Some authors () believe that periodontal disease is related to peri-implantitis and that Porphyromonas gingivalis, Aggregatibacter actinomicetemcommitans, Prevotella intermedia; Tannerella forsythia and Treponema denticola were isolated in healthy tissue, but also in individuals with peri-implant mucositis and peri-implantitis.

Sumida et al. () found that Porphyromonas gingivalis and Prevotella intermedia are transported from periodontal pockets of healthy teeth onto the area around the implant. Stingu et al. () isolated Prevotella intermedia and Prevotella nigrescens both in healthy individuals and in patients with the periodontal disease. Prevotella intermedia species belongs to the orange complex of bacteria, together with Fusobacterium nucleatum, Fusobacterium periodonticum, Parvimonas micra, Streptococcus constellatus, Eubacterium nodatum and Campylobacter rectus (, ). It is difficult to identify Prevotella intermedia and distinguish it from Prevotella nigrescens using ordinary laboratory methods, including gas chromatography (, ), but that can be achieved using the protein mass spectrometry method (MALDI-TOF-MS method). Prevotella nigrescens has recently been accepted as a possible periodontal pathogen. It is believed that it fosters the production of mediators of inflammation and that its lipopolysaccharide may cause alveolar bone resorption (). In some more recent studies Prevotella nigrescens was isolated in a substantially larger proportion from the places with a clinically more prominent inflammation associated with deeper periodontal pockets, which was demonstrated in one of the subjects of our study, whose probing depth was 5 mm vestibularly (). A higher percentage of Prevotella nigrescens was also detected in patients with localized and generalized forms of periodontitis, as well as in patients with generalized aggressive and chronic periodontitis (, ). In accordance with this study, we have also isolated Prevotella nigrescens in one subject with a clinically prominent inflammation around the implant, with the probing depth of 5 mm.

Using meta-analysis, Heitz-Mayfield () determined that poor oral hygiene, history of periodontitis and smoking are the most important risk factors for the development of periimplantitis.

It is known that the rough surface of implants has an effect on the colonization of biofilm. A titanium surface with the average roughness of Ra < 0.088 µm inhibits the colonization and maturation of biofilm (). Conversely, the surface roughness of Ra > 0.2 µm leads to increased biofilm formation and favors the emergence of peri-implantitis (). Ra < 0.2 µm does not have an influence on the formation of supragingival and sub gingival plaque (); consequently, some scholars have concluded () that Ra < 0.2 µm does not affect micro flora. A study carried out in 2016 demonstrated that the microorganisms present in peri-implant lesions are similar to those in periodontal lesions, but that correlation between the conducted studies is quite unlikely because of different sampling methods; it was concluded that new metagenomic sampling techniques should be the method of choice for future studies ().

Conclusion

In a group of 30 subjects no potentially pathogenic anaerobic bacteria from the red complex have been isolated (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola).

The study has shown that in 12 out of 30 subjects (40%) other types of anaerobic bacteria have been isolated, including bacteria from the orange complex, either on a tooth or on an implant.

Anaerobic bacteria were more abundantly present on implants than on homologous teeth. The presence of anaerobic bacteria both on a tooth and on an implant was found in few samples.

In view of the biofilm formation, it was expected that a larger number of anaerobic bacteria would be isolated on natural teeth or that in the subjects in which anaerobic bacteria were isolated on natural teeth, they would be isolated on the implant as well (, ).

Some authors believe that the microorganisms related to the development of periodontal diseases differ in different parts of the world and that they can vary due to a series of factors. In light of that presumption, every country should establish its own dental microbiological profile in order to develop guidelines for the implementation of the corresponding preventive measures and in order to take targeted therapeutic measures accordingly ().

In order to obtain more relevant results, further studies with larger samples are needed.