microbial and implant

The International Journal of Oral & Maxillofacial Implants 1521

Osseointegrated oral implants are a major tool in prosthetic dentistry. Although most implants are successful, implant failures occasionally occur. Failing implants are frequently characterized by loss of sup-porting bone, and these implants must be removed. The reasons for failure of implants are multiple (occlu-sion, parafunction, overloading, premature loading, and bacterial infection).1 Above all, two principal rea-sons for implant failure are mechanical stress and/or bacterial infection. Implants that fail after demonstrat-ing osseointegration are considered to be late failures. The factors contributing to late or delayed implant fail-ure such as peri-implantitis are not well-known, and many questions about etiology and treatment remain unanswered. Peri-implantitis is defined as an irrevers-ible inflammatory reaction associated with loss of sup-porting bone around an osseointegrated implant in function.2,3 Two cross-sectional studies reported that peri-implantitis was identified in approximately 28%

1 Assistant Professor, Division of Fixed Prosthodontics and Oral Implantology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, Kanazawa, Hokkaido, Japan.

2 Professor and Chairman, Division of Fixed Prosthodontics and Oral Implantology, Department of Oral Rehabilitation, Health Sciences University of Hokkaido, Kanazawa, Hokkaido, Japan.

3 Assistant Professor, Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, Kanazawa, Hokkaido, Japan.

4 Professor and Chairman, Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, Kanazawa, Hokkaido, Japan.

Correspondence to: Dr Naoki Tamura, Division of Fixed Prosthodontics and Oral Implantology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido; 1757 Kanazawa Ishikari-Tobetsu Hokkaido, 061-0293, Japan. Fax: +81-133-23-2892. Email: [email protected] 2013 by Quintessence Publishing Co Inc.

Analysis of Bacterial Flora Associated with Peri-implantitis Using Obligate Anaerobic

Culture Technique and 16S rDNA Gene SequenceNaoki Tamura, DDS, PhD1/Morio Ochi, DDS, PhD2/Hiroshi Miyakawa, PhD3/Futoshi Nakazawa, PhD4

Purpose: To analyze and characterize the predominant bacterial flora associated with peri-implantitis by

using culture techniques under obligate anaerobic conditions and 16S rDNA gene sequences. Materials and Methods: Subgingival bacterial specimens were taken from 30 patients: control (n = 15), consisting

of patients with only healthy implants; and test (n = 15), consisting of patients with peri-implantitis. In

both groups, subgingival bacterial specimens were taken from the deepest sites. An anaerobic glove box

system was used to cultivate bacterial strains. The bacterial strains were identified by 16S rDNA gene-

based polymerase chain reaction and comparison of the gene sequences. Results: Peri-implantitis sites

had approximately 10-fold higher mean colony forming units (per milliliter) than healthy implant sites.

A total of 69 different bacterial species were identified in the peri-implantitis sites and 53 in the healthy

implant sites. The predominant bacterial species in the peri-implantitis sites were Eubacterium nodatum, E brachy, E saphenum, Filifactor alocis, Slackia exigua, Parascardovia denticolens, Prevotella intermedia, Fusobacterium nucleatum, Porphyromonas gingivalis, Centipeda periodontii, and Parvimonas micra. The predominant bacteria in healthy implant sites apart from Streptococcus were Pseudoramibacter alactolyticus, Veillonella species, Actinomyces israelii, Actinomyces species, Propionibacterium acnes, and Parvimonas micra. Conclusion: These results suggest that the environment in the depths of the sulcus showing peri-implantitis is well suited for growth of obligate anaerobic bacteria. The present study

demonstrated that the sulcus around oral implants with peri-implantitis harbors high levels of asaccharolytic

anaerobic gram-positive rods (AAGPRs) such as E nodatum, E brachy, E saphenum, Filifactor alocis, Slackia exigua, and gram-negative anaerobic rods, suggesting that conventional periodontopathic bacteria are not the only periodontal pathogens active in peri-implantitis, and that AAGPRs may also play an important role.

Int J Oral MaxIIlOfac IMplants 2013;28:15211529. doi: 10.11607/jomi.2570

Key words: 16S rDNA, bacterial flora, obligate anaerobe, peri-implantitis

2013 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

Tamura et al

1522 Volume 28, Number 6, 2013

and 56% of subjects and in 12% and 43% of implant sites, respectively.3 Signs of a failing dental implant are detected both clinically and radiographically, with the diagnosis made in a similar way to periodontitis.4

The initially sterile and clean surface of an implant enters the totally different environment of adult gingival or periodontal tissues and bacterial flora, offering a new surface in the oral cavity for adherence and coloniza-tion of bacteria. Biofilm formation on oral implants can cause inflammation of peri-implant tissue, endangering the long-term success of osseointegrated implants.5 It is known that a large diversity of bacterial species inhabit the peri-implant area. A common finding in previous studies was that similar bacterial flora was found around oral implants and natural teeth.6 A high proportion of coccoid cells and facultative bacterial species and low frequencies of periodontal pathogens including gram-negative bacteria were detected in healthy implant sites. Clinical observations have recently revealed that early colonization patterns differ between implant and tooth surfaces.7 On the other hand, many reports have indicated that the infected lesions in the sulcus around peri-implantitis sites are traditionally associated with chronic periodontopathic bacteria.8,9 It has tradition-ally been thought that peri-implantitis is associated with a group of obligate anaerobic gram-negative rods (OGNRs), in particular black-pigmented and motile rods, and that gram-negative anaerobic cocci such as Veillonella also play an important role.1,6,10 However, these periodontopathic bacteria have also been ob-served in healthy implant sites.11

It has long been known that there are many uncul-tured, undescribed, and unknown bacterial species in the human oral cavity.1214 Some oral bacteria, such as asaccharolytic anaerobic gram-positive rods (AAGPRs), are fastidious and grow poorly on artificial culture me-dia; therefore, some of them are still not well known. Numerous bacterial strains of AAGPRs have been iso-lated from human oral infectious lesions, several of which have been documented as etiologic agents of chronic periodontitis15 based on their frequent isola-tion from diseased periodontal sites.

Previously, new isolates of AAGPRs were classified as members of the genus Eubacterium, which has histori-cally acted as a repository for a large number of diverse organisms16 and contains species and groups that are phylogenetically unrelated.12 Recent studies have de-scribed many oral AAGPR isolates, some of which qual-ify as members of the genus Eubacterium but could not be assigned to any of the established species. In addition, several novel genera have been proposed for some of the AAGPRs, eg, Pseudoramibacter, Slackia, Filifactor, and Mogibacterium, and some species of Eubacterium have been transferred to the novel genera according to their phylogenetic characteristics. It has

been shown that antibody titers against some species of AAGPRs in the sera of patients with periodontitis are higher than those of healthy people, suggesting that these bacteria cause immunologic reactions in periodontal lesions.1,2,17 Nevertheless, AAGPRs are not well-known because of the difficulty in cultivat-ing them, the small size of the colony, and their lack of reactivity in conventional biochemical tests. Thus, further studies are needed to better understand the asaccharolytic microorganisms under the obligate an-aerobic conditions of peri-implantitis.

Recently, the analysis of 16S rDNA gene-based polymerase chain reaction (PCR) has become a useful approach to assess the phylogenetic and taxonomic diversity of bacterial isolates.15,16,18 Culturing under obligate anaerobic conditions is also an important technique to allow the detection of almost every spe-cies in a given sample, revealing the presence of previ-ously uncultivated and unclassified strains.

Peri-implant infection by pathogenic bacteria can-not be understood without a comprehensive knowl-edge of obligate anaerobes. Therefore, it seems appropriate to compare the bacterial flora of peri-implantitis with that of healthy implants by using cul-ture techniques under obligate anaerobic conditions and 16S rDNA gene-based PCR. The aim of this study was to identify and characterize the predominant an-aerobic bacterial flora in the sulcus around progressive peri-implantitis in partially edentulous patients.

MaTerials and MeThods

Patient and sample CollectionThis study was approved by the ethics committee of the Health Sciences University of Hokkaido and the Health Sciences University of Hokkaido Hospital (Kanazawa, Hokkaido, Japan). The examination was performed with the understanding and written con-sent of each patient. The patients were categorized into two groups: the peri-implantitis group and the healthy implant group. Medical and dental histories were ob-tained, and full-mouth periodontal and implant exami-nations were performed. Both groups had one or more dental implants exposed for more than 6 months to the oral environment. The patients in the peri-implantitis group had clinical signs of peri-implantitis around one or more implants. The patients in the healthy implant group had no clinical signs of peri-implantitis. One im-plant was selected from each patient. Patients were ex-cluded if they had severe systemic disease, had taken antibacterial medication, had used mouth rinses dur-ing the previous 6 weeks, or had received peri-implant therapy within the previous 6 months that could inter-fere with the clinical parameters evaluated.


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Peri-implantitis sites were characterized by a prob-ing depth (PD) of 4 mm, suppuration, bleeding on probing (BOP),19 and visible three-thread loss of al-veolar bone clearly extending around the implant as visualized on radiographs.3,20 PD was measured in the deepest pocket of each quadrant between the gingival margin and the base of the peri-implant pocket with a pressure sensitive probe (Click-Probe, KerrHawe).21 BOP was recorded as present or absent. Radiographic bone loss around implants was evaluated from available ra-diographs (periapical or orthopantomography) from the fixture-abutment junction or the shoulder of the implants, or the cemento-enamel junction (Table 1). Based on these data, the diagnosis of peri-implantitis was made.

Microbial samplingSample sites were isolated with cotton rolls, air dried, and supragingival plaque was carefully removed using a pledget of cotton wool to avoid contamination. Sub-gingival bacterial specimens were taken with sterile endodontic paper points (ISO #50), which were gently inserted to the deepest point of each peri-implant or periodontal lesion and kept in place for 30 seconds. The paper points were then removed and placed in an anaerobic sterile transport vial containing 1 mL of semisolid medium (redox potential: 200 to approxi-mately 400 mV, 3.7% brain heart infusion, 0.2% agar). Samples were transferred as soon as possible (within 30 minutes) to an anaerobic glove box containing 80% nitrogen, 10% hydrogen, and 10% carbon dioxide.

Microbiological analysisFor in vitro evaluation, the suspension, dispersion, and dilution of samples and the culture of bacterial strains in the samples were carried out in the anaerobic glove box. The inside of the anaerobic glove box was maintained under 300 mV in redox potential during all experimental procedures. All plates, media, buffer solutions, and experimental instruments were kept in the anaerobic glove box for at least 7 days before use. Anaerobic bacterial isolation was executed by the fol-lowing standard procedures.

Each sample was suspended in 1 mL of sterilized phosphate-buffered saline (PBS) and mechanically dis-persed with a teflon homogenizer and vortex mixer for 30 seconds. Serial 10-fold dilutions (0.1 mL each, from 102 to 106) were spread onto the surface of nonse-lective blood medium (BHI blood agar plate; 3.7% brain heart infusion, 1.5% agar, 5% defibrinated sheep blood, 0.1% hemin, 0.1% menadione) and incubated in the anaerobic glove box at 37C for 7 days. Bacterial strains found on the nonselective blood medium were enumerated, and their percentage of the total number of colony forming units (CFUs) was calculated. When

the number of colonies on a BHI blood agar plate did not exceed 100, all of these colonies were isolated and subcultured for identification.

Subcultured colonies were incubated anaerobically or aerobically for 3 days, and for the purposes of this study, obligate anaerobes were defined as bacteria that grew only in the anaerobic glove box. In addition, these subcultured isolates were examined by gram staining.

Primer selection and 16s rdna Gene-Based PCrFour primer sets were included in individual PCR assays. The following universal primers were used for amplifi-cation of approximately 1,500 bp 16S rDNA22,23: forward primer 16S27F (5-AGAGTTTGATCCTGGCTCAG-3), 16S341F (5-CCTACGGGAGGCAGCAG-3), reverse prim-er 16S1492R (5-TACGGCTACCTTGTTACGACTT-3), and 16S907R (5-CCGTCAATTCCTTTGAGTTT-3). Each DNA amplification technique by colony-directed PCR was performed in a total volume of 50 L in 0.2 mL micro-reaction tubes (MicroAmp, PE Biosystems), contain-ing 1PCR buffer with 23 L deionized water, primers at a concentration of 0.2 mol/L, and 25 L of DNA polymerase (AmpliTaq Gold, PCR Master Mix, Applied Biosystems).

The amplification program was as follows: pre-heating at 95C for 15 minutes, 35 cycles of denaturing at 94C for 1 minute, annealing at 52C for 1 minute, extension at 72C for 5 minutes, and a final exten-sion step for 10 minutes at 72C using a thermocycler (Veriti, Applied Biosystems). The amplification prod-ucts were subjected to electrophoresis on 2.0% aga-rose gel and visualized by ethidium bromide staining.

sequencing of 16s rdna and Phylogenetic analysisThe PCR products obtained above were sequenced at Takara Bio. Then, 16S rDNA sequences of bacterial

Table 1 Patient selection Criteria

Peri-implantitis site

healthy implant site

Bleeding on probing +

Suppuration +

Probing pocket depth (PD)* 4 mm < 4 mm

Radiographic bone loss** +

Periodontal maintenance care

Antibacterial agent None None

Time since implant placement > 6 mo > 6 mo

*Probing pocket depth assessed as greatest distance between gingival margin and base of the peri-implant pocket.**Radiographic bone loss around implants was evaluated from available radiographs from the fixture-abutment junction or the shoulder of the implants, or the cemento-enamel junction.


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strains were compared with 16S rDNA gene sequences from the GenBank database using the BLAST search program through the website of the DNA Data Bank of Japan (DDBJ). A 16S rDNA gene sequence similarity of 98% was used as the cutoff for positive identification of taxa.24,25

statistical analysisThe Mann-Whitney U test (P < .01: average total colony-forming units, P < .05: the proportional distribution of OGNRs and AAGPRs) was used for statistical analyses of the significant differences between the two groups.

resulTs

Clinical features of patients and sites selected for bac-terial sampling are summarized in Table 2. A total of 30 partially edentulous patients involved in this study were selected from systemically healthy patients referred to the Health Sciences University of Hokkaido and Health Sciences University of Hokkaido Hospital. The patients included 12 females and 18 males. Fifteen patients with peri-implantitis (8 females and 7 males) and 15 pa-tients with healthy implants (4 females and 11 males) were characterized as shown in Table 2. The implants of the peri-implantitis patients included three types. One implant was made of single crystal aluminum oxide, 11 were made of commercially pure titanium, and three implants were coated with hydroxyapatite. The healthy implants were made of commercially pure titanium. The mean age was 56.9 years (range, 46 to 76 years) in the peri-implantitis group and 63.4 years (range, 46 to 77 years) in the healthy implant group. The mean PD and CFUs were higher in the peri-implantitis group than in the healthy implant group. The mean PD was 6.8 mm (range, 4 to 10 mm) in the peri-implantitis sites and 1.3 mm (range, 1 to 3 mm) in the healthy implant sites. A high proportion of patients with a diagnosis of peri-implantitis presented with a generalized loss of supporting bone around the implants.

Bacterial strains were detected in all colonies cul-tured on the nonselective blood medium under an-aerobic conditions by serial 10-fold dilutions to a level 1104 per mL. Quantitative bacterial analysis of peri-implantitis sites showed an average total of 6.34 0.52 colony-forming units (logarithm CFUs/mL).

Table 2 Clinical Features of Patients

Clinical featuresPeri-implantitis

sitehealthy implant

site

Patients 15 15

Age (y) 56.9 (range, 4676)

63.4 (range, 4677)

Sex (F/M) 8/7 4/11

Mean probing depths

6.8 (max, 10 mm; min, 4 mm)

1.3 (max, 3 mm; min, 1 mm)

Mean CFUs/mL* 6.34 0.52** 5.16 0.86

CFUs: colony-forming units; max: maximum; min: minimum.*Logarithm CFUs/mL**Statistically different from healthy implant sites, P < .01, Mann-Whitney U test.

Fig 1 Proportional distribution of bacterial genera at peri-im-plantitis sites: orange, OGNRs; blue, AAGPRs. Gram staining characteristics: green, gram-pos-itive cocci; purple, gram-negative cocci; light blue, gram-positive rods; red, gram-negative rods. The bacterial flora of peri-implan-titis sites consisted of bacteria of 31 genera.


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In contrast, the average total CFUs in the healthy im-plant group was 5.16 0.86. Significant differences were seen between the CFUs of the peri-implantitis and healthy implant groups (P < .01) (Table 2).

Figures 1 and 2 show the proportional distribu-tion of bacterial genera in peri-implantitis sites and in healthy implant sites, respectively. Streptococcus was the main genera present at both healthy and peri-implantitis sites. The flora observed at peri-implantitis sites consisted of bacteria of 31 genera, but only 20 genera were detected at healthy implant sites (Figs 1 and 2). The total proportion of OGNRs and AAGPRs

accounted for about 40% of all CFUs at peri-implantitis sites, in contrast to about 10% at healthy implant sites. At peri-implantitis sites, the predominant genera were Streptococcus (34%), Eubacterium (13%), Prevotella (10%), Actinomyces (6%), and Fusobacterium (4%). At healthy implant sites, the predominant genera were Streptococcus (45%), Actinomyces (14%), Veillonella (14%), and Propionibacterium (8%). The proportion of Streptococcus at healthy implant sites was larger than at peri-implantitis sites. At peri-implantitis sites, Eubacterium that were AAGPRs and Prevotella that were OGNRs were the next most predominant genera.

Fig 2 Proportional distribution of bacterial genera at healthy implant sites: orange, OGNRs; blue, AAGPRs. Gram staining characteristics: green, gram-pos-itive cocci; purple, gram-negative cocci; light blue, gram-positive rods; red, gram-negative rods. The bacterial flora of healthy im-plant sites consisted of bacteria of 20 genera.

P < .05

Mea

n pr

opor

tion

of A

AGPR

s (%

)

40

30

20

10

0Peri-implantitis Healthy implants

P < .05

Mea

n pr

opor

tion

of O

GN

Rs

(%)

40

30

20

10

0Peri-implantitis Healthy implants

Fig 3 Mean proportional distribution of AAGPRs. The mean proportional distribution was statistically significantly higher in the peri-implantitis group (P < .05, Mann-Whitney U test).

Fig 4 Mean proportional distribution of OGNRs. The mean pro-portional distribution was statistically significantly higher in the peri-implantitis group (P < .05, Mann-Whitney U test).


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Figures 3 and 4 represent the mean proportional distri-bution of AAGPRs and OGNRs. AAGPRs were detected in the peri-implantitis group (18%) and in the healthy implant group (3%). OGNRs were detected in the peri-implantitis group (20%) and in the healthy implant group (6%). These data reveal statistically significant differences between the two groups. The mean pro-

portional distributions were statistically significantly higher in peri-implantitis (P < .05).

Peri-implantitis sites were colonized by complex bacteria with a large proportion of OGNRs and AAGPRs. A total of 69 different bacterial species were identified in the peri-implantitis sites (Fig 5) and 53 in the healthy implant sites (Fig 6).

Fig 5 Proportional distribution of the 69 different bacterial spe-cies identified at peri-implanti-tis sites: orange, OGNRs; blue, AAGPRs.

Fig 6 Proportional distribution of the 53 different bacterial spe-cies identified at healthy implant sites: orange, OGNRs; blue, AAGPRs.


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Significant differences in bacterial species were observed between healthy implant sites and peri-implantitis sites. The predominant obligate anaero-bic bacterial species at the peri-implantitis sites were E nodatum (7%), P intermedia (5%), F nucleatum (3%), Filifactor alocis (3%), E brachy (3%), Parascardovia denticolens (3%), and Parvimonas micra (3%), while the predominant obligate anaerobic bacterial species at the healthy implant sites were Veillonella species (spp) (14%), Propionibacterium acnes (5%), Pseudoramibacter alactolyticus (3%), and Parvimonas micra (2%).

disCussion

This study aimed to identify the predominant bacteria and characterize the anaerobic bacterial flora in the sulcus around progressive peri-implantitis. Selection of the patients for each group was based on clinical conditions such as BOP, suppuration, PD, and radio-graphic criteria (Table 1).

In this study, various designs of implant were repre-sented in the peri-implantitis group. Previous studies have found no statistically significant difference in the frequency of peri-implantitis related to the type of im-plant, except for a higher frequency of peri-implantitis around implants with rough surfaces than around those with smooth surfaces. There is limited and conflicting evidence to suggest that implant surface and design may be a risk indicator for peri-implantitis. 26 In this study, differences in implant surface were not taken into account, although it may be an important area for future research.

In this study, the bacterial flora of peri-implantitis sites was characterized using anaerobic culture tech-niques and 16S rDNA gene-based PCR for bacterial identification, and compared with that of healthy im-plant sites. All bacterial strains were cultivated un-der obligate anaerobic conditions (redox potential: 400 mV). Culture methods are known to be useful tools for studying the bacterial flora of infectious le-sions by detecting which microorganisms inhabit a given area,15 although the process is labor intensive and time-consuming. However, sensitive and accurate molecular techniques are necessary to characterize the bacterial flora in peri-implantitis in order to deter-mine their association with clinical symptoms and the prognosis of treatment. In the past, a checkerboard DNADNA hybridization technique was the most usual method for identifying bacteria in the field of clinical dentistry.27 Although this technique can be used to detect gene-specific bacteria, it cannot ran-domly detect bacteria. Therefore, culture techniques under obligate anaerobic conditions and 16S rDNA gene-based PCR are important tools for detecting

oral bacterial species in the human oral cavity, and for identifying the predominant bacteria that may be im-portant pathogens for infection.

As a result, the present study has demonstrated sig-nificant differences between the bacterial flora in the two groups, and has revealed characteristics of the bacterial flora of peri-implantitis sites (Figs 5 and 6). In addition, significantly more CFUs were observed in peri-implantitis sites than in healthy implant sites (Table 2).

When the flora of the two groups were compared at the genus level, the present study showed that the pre-dominant genera in the peri-implantitis sites were Strep-toccocus and Eubacterium, while genera Streptoccocus, Veillonella, and Actinomyces were predominant in the healthy implant sites (Figs 1 and 2).

In the literature there has been much speculation surrounding the relationship between implant failures and peri-implantitis, the flora of periodontitis, and the flora of peri-implantitis. Failing oral implants are gener-ally found to harbor bacterial flora traditionally associ-ated with periodontitis such as P gingivalis, T forsythia, T denticola, and F nucleatum. Previous studies have found that the bacterial flora of peri-implantitis and periodontal infections was similar,1,10 and one study demonstrated that in several cases of peri-implantitis, culture techniques identified species that included not only pathogens such as P gingivalis, P intermedius, and A actinomycetemcomitans, but also Escherichia coli and S aureus.28

Although Streptococcus was one of the predomi-nant genera detected in samples from both groups, the species of the genus at the two sites were completely different. S constellatus and S sobrinus were frequently isolated from the peri-implantitis sites, but were not detected at the healthy implant sites (Figs 5 and 6). In contrast, S parasanguinis, S mutans, and S oralis were detected only at healthy implant sites (Figs 5 and 6). A long-term study on colonization of the peri-implant area showed a decrease in the proportion of faculta-tive anaerobic cocci such as Streptococcus species and an increase in the percentage of obligate anaerobic rods, eg, Fusobacterium spp and Prevotella spp.29 These results may indicate the possibility that some species of genus Streptococcus have ecological significance in the flora of peri-implantitis sites.

Veillonella spp were found to be the predominant bac-teria in healthy implant sites in our study. This finding is consistent with the observations of the previous study. 7

OGNRs were detected in both groups. Long-term clinical studies have suggested that the bacterial flora of peri-implantitis sites was characterized by a high proportion of OGNRs belonging to the red and orange complex species.17,27,30,31 In this study, microbiological culture of peri-implantitis sites has detected bacterial species including P gingivalis, P intermedia, F nucleatum,


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Parvimonas micra, and other major pathogenic bacte-ria characteristic of chronic periodontitis. Other studies have reported that implants with peri-implantitis har-bor A actinomycetemcomitans.32,33 However, these find-ings were not confirmed at peri-implantitis sites in this study. Our results showed the presence of P gingivalis, but red complex bacteria (T forsythia, T denticola), A actinomycetemcomitans, S aureus, and Candida spp were not detected. These bacteria may be cultivable but undetectable for a mean CFU threshold level of 1104 per mL; however, these bacteria must certainly be pres-ent at lower levels than other red, orange, green, purple, and yellow complexes.

On the other hand, gram-positive bacilli accounted for 35% of the total proportional distribution at peri-implantitis sites and 27% at healthy implant sites. In addition, AAGPRs were detected in the peri-implan-titis group (18%) but were almost undetectable in the healthy implant group (3%), confirming that the constitution of the bacterial flora differed markedly between the two groups (Figs 3 and 4). A previous study reported that many AAGPRs are significantly as-sociated with periodontitis, periapical infections, and oral abscesses, but are rarely found under healthy oral conditions.13 In the present study, an overwhelming number of AAGPRs (colored green in Fig 3) and OGNRs (colored green in Fig 4) were detected in peri-implantitis sites. These bacteria were obligate anaerobes and were detected only in limited numbers at healthy implant sites. The proportion of AAGPRs was almost the same as the proportion of OGNRs at peri-implantitis sites. However, AAGPRs were outnumbered more than two to one at healthy implant sites. The ratio of AAGPRs to OGNRs and total isolates increased significantly at peri-implantitis sites. Uematsu and Hoshino reported that AAGPRs, such as Eubacterium, Mogibacterium, Slackia, and others, often predominated in periodonti-tis sites.14 Thus, it appears that AAGPR growth and bio-film formation may be influenced by periodontopathic bacteria.

E nodatum, E saphenum, E minutum, and Filifactor alocis, which are species of AAGPRs and were detected in this study, have been known to produce the short-chain fatty acid butyrate as one of the end products.14,15,18,34,35

OGNRs such as Fusobacterium spp and Prevotella spp are also known to produce butyrate.3638 Butyrate has been reported to inhibit cell growth of human gingival fibroblasts39 and human endothelial cell proliferation in vitro,40,41 and to induce apoptosis in T and B cells.37,42,43 Therefore, butyrate may have an important role in the local tissue destructive process in the pathogenesis of peri-implantitis. The presence of AAGPRs in this study suggests that AAGPRs may possibly act in periodontitis to cause tissue destruction at the base of the advanced progressive peri-implantitis sulcus. This suggests that

bacterial flora at peri-implantitis sites reveals that AAGPRs are also predominant in addition to specific bacteria linked with peri-implantitis.

Previously, it was reported that uncultivated bac-terial species were identified in peri-implantitis sites by 16S rRNA gene clone library analysis.44 Since these species might not be detected in the present study with culture techniques, further studies are necessary for considering the role of uncultivated or viable non-culturable bacterial species.

ConClusions

The present study indicates that an obligate anaerobic environment exists at the base of the peri-implantitis sulcus, and is suited for growth of AAGPRs and OGNRs. Although further work is necessary to elucidate the bacterial flora in peri-implantitis, the fact that the sul-cus around oral implants with peri-implantitis showed high levels of AAGPRs and OGNRs suggests that con-ventional periodontopathic bacteria are not the only periodontal pathogens active in peri-implantitis, and that AAGPRs may possibly play an important role.

aCKnowledGMenTs

The Health Sciences University of Hokkaido and the Health Sci-ences University of Hokkaido Hospital supported this work. The authors reported no conflicts of interest related to this study.

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