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The Microbiota of Acute Apical AbscessesDepartment of Endodontics, Faculty of Dentistry, Estácio de Sá University, Av. Alfredo Baltazar da Silveira, 580/Cobertura, Recreio, Rio de Janeiro, RJ, Brazil 22790-701 Correspondence: * corresponding author, jf_siqueira{at}yahoo.com, siqueira{at}estacio.br
As the breadth of bacterial diversity in the oral cavity has been deciphered by molecular studies, several newly identified species/phylotypes have emerged as potential pathogens. We hypothesized that many of these species/phylotypes could also be involved with the etiology of endodontic abscesses. Abscess aspirates from 42 persons were analyzed for the presence of 81 species/phylotypes by means of a reverse-capture checkerboard hybridization assay. Associations between the most frequently detected taxa were calculated. The most prevalent taxa were Fusobacterium nucleatum, Parvimonas micra, and Porphyromonas endodontalis. Other frequently found taxa included Olsenella uli, streptococci, Eikenella corrodens, some as-yet-uncultivated phylotypes (Bacteroidetes clone X083 and Synergistes clone BA121), and newly named species (Prevotella baroniae and Dialister invisus). Several positive bacterial associations were disclosed. Findings not only strengthen the association of many cultivable species with abscesses, but also include some newly named species and uncultivated phylotypes in the set of candidate pathogens associated with this disease.
Key Words: acute apical abscess • endodontic microbiology • 16S rRNA gene • polymerase chain reaction • checkerboard DNA-DNA hybridization
A cute apical abscesses are caused by bacteria that egress the infected root canal and invade the periradicular tissues to establish an extraradicular infection and evoke purulent inflammation. Clinically, the disease leads to pain and/or swelling, and has the potential to diffuse to sinuses and other facial spaces of head and neck, to form cellulitis. The microbiota involved is mixed and dominated by anaerobic bacteria (Siqueira et al., 2004; Sakamoto et al., 2006), with about 12–18 taxa/case, as compared with 7–12 taxa present in chronic cases (Siqueira et al., 2004; Sakamoto et al., 2006). As-yet-uncultivated phylotypes encompass approximately 40% of the taxa found in abscesses and collectively represent more than 30% of the 16S rRNA gene sequences retrieved in clone libraries (Sakamoto et al., 2006). Clone library analysis of 16S rRNA genes has been widely used to unveil the breadth of bacterial diversity in the oral cavity (Siqueira and Rôças, 2005; Paster et al., 2006). Nevertheless, technical demands and high cost can make it difficult for large numbers of samples to be analyzed by this method. Oligonucleotide probes based on sequences revealed by clone libraries can be designed specifically to detect any species/phylotypes. Probes can be used in techniques suitable for large-scale clinical studies, such as the checkerboard hybridization technology, to associate species/phylotypes with disease. This study investigated the presence of 81 bacterial taxa in acute apical abscesses by means of a reverse-capture checkerboard assay. Target taxa for investigation included cultivable species previously linked to endodontic abscesses, as well as newly characterized species and as-yet-uncultivated phylotypes that have been recently detected in clone libraries from periodontal (Paster et al., 2001; Kumar et al., 2005) and endodontic infections (Munson et al., 2002; Sakamoto et al., 2006). Associations between and among the most frequently detected taxa were also calculated.
The study protocol was approved by the Ethics Committee of the Estácio de Sá University, and written informed consent was obtained from all participants. The examined material consisted of purulent aspirates from acute apical abscesses taken from 42 adults who were seeking emergency treatment. Involved teeth showed caries lesions, necrotic pulps, and radiographic evidence of periradicular bone loss. Diagnosis of acute apical abscess was based on the presence of spontaneous pain, exacerbated by mastication, and localized or diffuse swelling, along with fever, lymphadenopathy, or malaise. No fistula connecting the abscess to the oral cavity or the skin surface was observed. No teeth showed significant gingival recession and periodontal pockets deeper than 4 mm. After disinfection of the oral mucosa with chlorhexidine, samples were taken by aspiration of the purulent exudate from the swollen mucosa over each abscess. Sampling procedures and DNA extraction were conducted as outlined previously (Sakamoto et al., 2006). DNA from a panel of oral bacterial species was also prepared to serve as controls (Siqueira et al., 2001).
Probe Design
PCR All PCR amplifications were performed in 50 µ L of reaction mixture containing 1 µ M of each primer, 5 µ L of 10 x PCR buffer, 3 mM MgCl2, 2U of Tth DNA polymerase, and 0.2 mM of each deoxyribonucleoside triphosphate (all reagents from Biotools, Madrid, Spain). Negative controls consisted of sterile ultrapure water instead of sample. Temperature profile for the first PCR reaction with the primers 8f/1492r was: 95°C/1 min, 26 cycles at 94°C/45 sec, 50°C/45 sec, and 72°C/1.5 min, and 72°C/15 min. Cycling conditions for the second round of amplification with the primers digoxigenin-8f/519r or digoxi-genin-515f/1492r included: 95°C/5 min, 28 cycles at 94°C/30 sec, 55°C/1 min, 72°C/1.5 min, and 72°C/20 min. Amplicons were separated by electrophoresis in agarose gels and viewed under ultraviolet transillumination.
Checkerboard The checkerboard assay was performed with the Minislot-30 and Miniblotter-45 system (Immunetics, Cambridge, MA, USA), slightly modified from Paster et al.(1998). First, a 100-pmol quantity of probe in Tris-EDTA buffer was introduced into the horizontal wells of the Minislot apparatus, and cross-linked to the Hybond- N+ nylon membrane (Amersham-Pharmacia Biotech, Buckinghamshire, England) by ultraviolet irradiation by means of a Stratalinker 1800 (Stratagene, La Jolla, CA, USA). The polythymidine tails were preferentially cross-linked to the nylon, leaving the specific probe available for hybridization. The membrane was then pre-hybridized at 55°C/1 hr. Subsequently, a 90-µ L quantity of the labeled PCR products with 50 µ L of 55°C pre-heated hybridization solution was denatured at 95° C/5 min and loaded on the membrane by the Miniblotter apparatus. Hybridization was carried out at 55°C/2 hrs. After hybridization, the membrane was washed and blocked in a buffer with casein. The membrane was sequentially incubated in antidigoxigenin antibody conjugated with alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany) and ultra-sensitive chemiluminescent substrate CDP Star (Roche Molecular Biochemicals). Finally, a square of x-ray film was exposed to the membrane in a cassette for 20 min, so that the hybrids could be detected.
Data Analysis
All sample extracts were positive for PCR amplification with the universal primers. Negative controls yielded no amplicons. Fifty-five taxon-specific probes were reactive with one or more abscess samples. All but one of the 42 samples were positive for at least one taxon-specific probe. The number of bacterial taxa detected per abscess sample ranged from 1 to 24 (mean, 8.3; median, 7). The cases positive for only one of the target species harbored Porphyromonas endodontalis (2 cases), Dialister invisus, or Eikenella corrodens.
Taxa detected more frequently included Fusobacterium nucleatum (27/42 cases-64%), Parvimonas micra (formerly Peptostreptococcus/Micromonas micros) (22/42–52%), P. endodontalis (20/42–48%), Olsenella uli (19/42–45%), streptococci (16/42–38%), E. corrodens (16/42–38%), Bacteroidetes clone X083 (15/42–36%), Prevotella baroniae (15/42–36%), Treponema denticola (15/42–36%), and D. invisus (13/42–31%) (Fig.
Several pairs of bacterial taxa were positively associated (RR > 1). Very strong positive associations occurred among Selenomonas sputigena/streptococci, F. nucleatum/P. micra, T. denticola/streptococci, Filifactor alocis/streptococci, Tannerella forsythia/S. sputigena, F. nucleatum/Synergistes clone BA121, and P. micra/O. uli. Other positive associations are displayed in the Table  . Negative associations were also observed for many pairs (RR < 1).
This study investigated the presence of 81 bacterial taxa in endodontic abscess samples by means of a reverse-capture checkerboard assay. This approach has important advantages over the original checkerboard method, mostly related to the use of oligonucleotide probes instead of whole genomic probes (Paster et al., 1998). Oligonucleotide probes display higher specificity, since the probe sequence is known and consequently permits the calculation of stringent hybridization temperatures. Moreover, mismatches are not tolerated, due to the considerable reduction of bond strength between the short probe and the target (Juretschko et al., 2004). Oligonucleotide probes still have the advantage that they can be designed to detect both cultivable and as-yet-uncultivated bacteria, while in the original checkerboard method, with whole genomic probes, only cultivable species are targeted. Several cultivable species were among the most frequently detected taxa. The most prevalent species were F. nucleatum, P. micra, and P. endodontalis. Previous culture-dependent studies have frequently isolated these species from endodontic abscesses (van Winkelhoff et al., 1985; Sundqvist et al., 1989; de Sousa et al., 2003), but molecular studies have detected them in even higher prevalence (Riggio et al., 2001; Siqueira et al., 2001; Baumgartner et al., 2004; Sakamoto et al., 2006). Other cultivable species often detected in this study included O. uli, streptococci, E. corrodens, T. denticola, T. forsythia, F. alocis, and S. sputigena. O. uli, which has only recently been reported to occur in symptomatic endodontic infections (Rôças and Siqueira, 2005a), was among the most prevalent species, being detected in about one-half of the abscess cases. Previous culture-independent studies have linked T. denticola and T. forsythia to endodontic abscesses (Siqueira et al., 2001; Siqueira and Rôças, 2004; Foschi et al., 2005), which was confirmed by the present findings. Some newly named species, such as P. baroniae and D. invisus, were also found at high frequencies. P. baroniae 16S rRNA gene sequence shares 99.5% similarity with both Prevotella clones PUS9.180 and E9_42-E4, and the possibility exists that they all are the same species. Association of this taxon with endodontic abscesses has been recently suggested by culture-dependent and culture-independent studies (Dymock et al., 1996; Wade et al., 1997; Khemaleelakul et al., 2002; Sakamoto et al., 2006). Indeed, this taxon has been exclusively detected in abscess samples, but not in chronic endodontic infections (Sakamoto et al., 2006). D. invisus was originally found in canals of teeth with chronic apical periodontitis (Munson et al., 2002), but it can also be associated with symptomatic infections (Rôças and Siqueira, 2005b). The present findings confirm its possible involvement with abscesses. It is still worth pointing out that because the Dialister oral clone BS095 shares more than 99.5% 16S rRNA gene sequence similarity with D. invisus, they are likely the same species. If the data from D. invisus were combined with those of BS095 (found in eight cases), then D. invisus would appear in five more cases (18/42–43%). The most prevalent of the 22 as-yet-uncultivated phylotypes targeted in this study were Bacteroidetes X083 (36%), Synergistes BA121 (24%), Dialister BS095 (19%), Lachnospiraceae 55A-34 (12%), and Treponema II:10:D12 (12%). Some phylotypes have been previously reported to be among the most prevalent bacterial taxa in primary endodontic infections, including Lachnospiraceae 55A-34 and Bacteroidetes X083 (Sakamoto et al., 2006). In contrast to the present findings, the latter had been detected only in teeth with chronic apical periodontitis, but not in abscesses (Sakamoto et al., 2006). This difference is possibly related to the larger number of samples surveyed herein. Synergistes bacteria have been recently disclosed in endodontic infections by molecular techniques, with clone BA121 being the most prevalent phylotype (Rôças and Siqueira, 2005b; Siqueira et al., 2005). This was corroborated by this study. Four other as-yet-uncultivated Synergistes clones were also detected, suggesting that this bacterial group may have been overlooked by culture-dependent studies. The occurrence of as-yet-uncultivated phylotypes in endodontic abscess samples suggests that they can be previously unrecognized taxa that participate in the pathogenesis of this disease. Treponema species have been recently linked to endodontic abscesses by studies with highly sensitive PCR approaches (Siqueira and Rôças, 2004). All 10 cultivable and 4 as-yet-un-characterized oral treponemes were targeted in this study. The species/phylotypes detected, in decreasing order of frequency, were T. denticola (36%), T. socranskii (19%), Treponema II:10:D12 (12%), T. pectinovorum (7%), Treponema 6:H:D15A-4 (5%), T. amylovorum (2%), and Treponema I:G:T21/I:W:T040 (2%). These findings confirmed the association of oral treponemes, especially T. denticola and T. socranskii, with apical abscesses and demonstrated that not-yet-characterized treponemes can also take part in the infective microbiota. Except for Veillonella species and Propionibacterium propionicum, there were no significant surprises for the taxa undetected in this study. These taxa may have been actually absent from samples, but the fact that previous, more sensitive, PCR assays have found them in moderate frequencies in abscesses (Siqueira and Rôças, 2003; Rôças and Siqueira, 2006) raises the suspicion that they might have been present, but in levels below the sensitivity of the checkerboard assay. It is also important to point out that although this study has targeted 81 taxa, other taxa not covered in our panel may have been present. Abscesses are typically characterized by a mixed bacterial consortium, the composition of which is largely influenced by positive and negative associations. Enhanced pathogenicity due to additive or synergistic effects is an important feature of mixed infections and relies on positive relationships between and among the community members (Brook, 1986). Interbacterial nutritional interactions are important ecological determinants that result in higher metabolic efficiency of the whole community and increase the probability of certain species being found together in a given habitat. Nutritional interactions are mainly represented by food chains and bacterial cooperation for the breakdown of complex host-derived substrates. In the present study, several positive associations were depicted for the first time for some taxa, particularly the newly named species and as-yet-uncultivated phylotypes. F. nucleatum and P. micra were positively associated with several other taxa. As for the other very prevalent species, strong positive associations were observed for streptococci and T. denticola, T. forsythia, F. alocis, or S. sputigena, and P. endodontalis and O. uli. Several positive associations were also observed for the 2 most prevalent as-yet-uncultivated phylotypes, Bacteroidetes X083 and Synergistes BA121. These findings of bacterial interactions underscore the complex ecological interrelationships that occur among bacteria involved in endodontic infections. The molecular determinants as well as the ecological and pathogenic implications of these various associations remain to be delineated. Molecular methods have refined and redefined the knowledge of the microbial taxa involved in several human infections, including abscesses of endodontic origin. Findings from the present study not only strengthen the association of several cultivable species with abscesses, but also lend support to include several newly named species and as-yet-uncultivated phylotypes in the set of putative pathogens associated with this disease.
This study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), a Brazilian Governmental Institution. The authors are grateful to Dr. Bruce Paster, for providing a detailed protocol of the checkerboard method and for his valuable advice, and to Mr. Marlei Gomes da Silva, for technical assistance.
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. Received for publication May 29, 2007. Revision received . Accepted for publication October 11, 2008.
Journal of Dental Research, Vol. 88, No. 1,
61-65 (2009) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
