Received: 19 October 2023

| Accepted: 30 March 2024

DOI: 10.1111/iej.14071

REVIEW ARTICLE

Apical root canal microbiome associated with primary and

posttreatment apical periodontitis: A systematic review

José F. Siqueira Jr1,2 | Warley O. Silva1 | Kaline Romeiro1 | Luciana F. Gominho3 |

Flávio R. F. Alves1,2 | Isabela N. Rôças1,2
1
Postgraduate Program in Abstract
Dentistry, University of Grande Rio
Background: Microorganisms colonizing the apical root canal system are con-
(UNIGRANRIO), Rio de Janeiro, RJ,
Brazil ceivably the ones directly involved with the causation and maintenance of apical
2
Department of Endodontics, Faculty periodontitis.
of Dentistry, Iguaçu University (UNIG), Objectives: This article systematically reviews the reports on the microbiome oc-
Nova Iguaçu, RJ, Brazil
3
curring exclusively at the apical root canal of teeth with primary and posttreatment
Department of Restorative Dentistry,
Federal University of Paraíba (UFPB), apical periodontitis.
João Pessoa, PB, Brazil Methods: The electronic databases PubMed, Embase, Web of Science, Science
Direct, and Proquest were searched up to August 2023. Clinical studies using culture
Correspondence
Flávio R. F. Alves, University of Grande and molecular microbiology methods to identify the microbial taxa present exclu-
Rio, Postgraduate Program in Dentistry, sively in the apical root canal segment of infected teeth with apical periodontitis
Rua Professor José de Souza Herdy,
1160, Duque de Caxias 25071-­202, RJ,
were included. Studies were critically assessed using the Joanna Briggs Institute
Brazil. Critical Prevalence Assessment Checklist.
Email: [email protected] Results: From 2277 articles initially detected, 52 were selected for full reading and
Funding information 21 were eventually included in this review. Of these, molecular methods were used
CNPq; FAPERJ in 19 and culture in 2 studies. Ten studies evaluated primary infections, 8 evalu-
ated posttreatment infections, and 3 included both. Cryopulverization of the apical
root specimens was conducted in 11 studies. All studies evaluated the prevalence
and diversity of bacteria, and only one also reported on fungi. Overall, the most fre-
quent/abundant bacterial taxa found in the apical canal of primary infections were
Pseudoramibacter alactolyticus, Olsenella uli, Fusobacterium species, Streptococcus
species, Porphyromonas endodontalis, Prevotella species, Actinomyces species,
Parvimonas micra, Treponema denticola, Synergistetes species, and an as-­yet unchar-
acterized taxon. In posttreatment infections, the most prevalent/abundant bacterial
taxa included species of Streptococcus, Enterococcus, Fusobacterium, Actinomyces,
Pseudoramibacter, Pseudomonas, and Propionibacterium. At the phylum level,
Firmicutes was the most represented. The average apical bacterial load ranged from
105 to 106 in primary infections and from 103 to 104 in posttreatment infections.
Discussion: Microbial diversity in the apical part of the root canal system was
examined encompassing data from both primary and posttreatment infections.
Heterogeneity amongst the studies, especially in sample collection and microbial
identification methods, is an important limitation that prevented a meta-­analysis.

© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd

Int Endod J. 2024;00:1–16.  wileyonlinelibrary.com/journal/iej | 1

 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2    APICAL ROOT CANAL MICROBIOME

Conclusions: There is a pronounced bacterial diversity in the infected apical canal,

with a high interindividual variability. Different microbiome compositions at the
species/genus level are observed according to the infection type.
Registration: PROSPERO CRD42021275886.

KEYWORDS
apical root canal system, microbiome, posttreatment apical periodontitis, primary apical
periodontitis

I N T RO DU CT ION the pathogenesis and maintenance of apical periodon-

titis, it becomes imperative to determine the main taxa
Apical periodontitis is an inflammatory disease in re- involved. Therefore, this study aimed to systematically
sponse to microbial infection of the root canal system review the reports on the microbiome present in the api-
(Ørstavik, 2020). Given the proximity and direct access cal root canal segment of infected teeth with primary and
to the periradicular tissues, bacteria located in the apical posttreatment apical periodontitis.
segment of the root canal can be regarded as the main
culprits of apical periodontitis (Siqueira & Rôças, 2022a).
Bacteria have been reported to build biofilm communities METHOD
in the apical canal segment in the large majority of cases
of primary and posttreatment apical periodontitis, with Protocol and registration
higher prevalences in teeth with large lesions (>5 mm in
diameter on radiographs) and those diagnosed as apical This systematic review followed the PRISMA (Preferred
cysts (Ricucci & Siqueira, 2010). In teeth with posttreat- Reporting Items for Systematic Reviews and Meta-­
ment apical periodontitis, persistent bacterial infection Analyses) guidelines (Moher et al., 2010). The protocol
has been found in the apical canal in virtually all cases was registered in the International Prospective Register of
(Ricucci et al., 2009). This strongly suggests a role in the Systematic Reviews (PROSPERO: CRD42021275886).
failure of the root canal treatment.
As a consequence, the apical region of the root
canal system can be considered as a critical territory Focused question
(Simon, 1994). Microorganisms in this area can derive
nutrients from the tissue fluids and inflammatory ex- The question that guided this study was formulated using
udate that seep into the canal via apical foramen and the PECO (population, exposure, comparison, outcome)
ramifications (Siqueira & Rôças, 2022). In addition, strategy: what is the microbiome present in the apical root
they establish a close interaction with the host cells and canal segment of teeth with primary or posttreatment api-
tissues that will invariably result in inflammation and cal periodontitis? Thus, the outcome of the research ques-
accumulation of the host defences around the portal tion (O) was to identify the microbial taxa of the apical
of exit of microorganisms to the periradicular tissues. root canal in infected teeth. The sample (P) was apical
Given microbial association with persistent disease, the root segments (derived from periradicular surgery or ex-
apical canal system is also a critical area for disinfection, traction) from adult patients, and the exposition (E) was
cleaning, shaping, and filling. primary or posttreatment apical periodontitis. The com-
Since the introduction of sophisticated molecular parative group (C) was not applicable.
methods in endodontic microbiology studies, there has
been a significant refinement and expansion in the list
of candidate pathogens associated with apical periodon- Eligibility criteria
titis. Curiously, despite these advances, only a limited
number of studies have focused on microbial identifi- Cross-­sectional clinical studies were included, which in-
cation exclusively in the apical part of the canal system. vestigated the microbiome of the apical segment of the
This may be mostly related to technological difficulties root canal in teeth with primary or posttreatment infec-
in effectively sampling this specific area (Siqueira & tions using molecular and/or culture methods. Samples
Rôças, 2022b). consisted of resected root apexes from extracted teeth
Because microorganisms located in the apical canal or obtained by periradicular surgery. The exclusion cri-
system are allegedly the most important associated with teria included studies that did not specify the sampled
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SIQUEIRA et al.    3

root canal segment and those evaluating the extraradic- Reviews, available in https://​amstar.​ca/​Amstar_​Check​
ular microbiome associated with pus samples or lesion list.​php).
specimens.

RESULTS
Search strategy and selection of articles
Type of infection and sampling method
This review was conducted in the following data-
bases: Medline via PubMed (www.​pubmed.​gov), Web Initially, 2277 articles were identified, of which 727 were
of Sciences (http://​www.​isikn​owled​ge.​com), Science duplicates and were removed. Of the 1550 articles that
Direct (www.​scien​cedir​ect.​com), Embase (https://​www.​ went through the stage of title and abstract selection, 52
embase.​com), and Proquest (proqu​est.​com; Table S1). remained for complete reading and analysis. The final
Searches included studies published up to 22 August sample consisted of 21 studies (Figure 1). Ten studies
2023. investigated only primary infections (Baumgartner &
Three independent reviewers performed the electronic Falkler, 1991; Dougherty et al., 1998; Ozok et al., 2012;
searches. Articles found in the databases were exported to Persoon et al., 2017; Rôças et al., 2010; Siqueira et al., 2009;
Rayyan (https://​www.​rayyan.​ai) for management and to Siqueira et al., 2011; Siqueira, Rôças, Alves, et al., 2004;
eliminate duplicates. Before the article selection process, Takahama et al., 2018; Tatikonda et al., 2017), 8 only
calibration of the reviewers was performed with a sam- posttreatment infections (Antunes et al., 2015; Pereira
ple of 20 articles retrieved from the databases using the et al., 2017; Perez-­Carrasco et al., 2023; Ping et al., 2015;
search strategy. Titles and abstracts were analysed for the Siqueira et al., 2016; Siqueira et al., 2020; Subramanian
screening of articles based on the eligibility criteria. The & Mickel, 2009; Wang et al., 2012), and 3 included both
pre-­selected articles from the screening process under- types (Bouillaguet et al., 2018; Chugal et al., 2011; Qian
went full-­text analysis. et al., 2019). The search for grey literature using the
Proquest database did not return any document related to
the objective of the present study.
Data extraction Regarding the methods used for microbial detection
and identification, 2 studies used culture, whereas the
The following data were extracted from the articles other 19 studies used culture-­independent molecular mi-
included in the present review: author(s), year of pub- crobiology methods. The characteristics of these studies
lication, country of origin, sample type (obtained by are listed in Tables 1, 2, and 3. The countries with more
periradicular surgery or extraction), infection/lesion publications were Brazil, China, and the United States
type (primary or posttreatment), identification method (Figure 2).
(culture or molecular), identified microorganisms, and All included studies evaluated root apex specimens
limitations. obtained either from extracted teeth or after root-­end re-
section during periradicular surgery. Extracted teeth were
used in 13 studies, with the length of the apical segment
Quality assessment varying from 2 to 7 mm. Apical segments obtained by per-
iradicular surgery were used in 8 studies, with the api-
Two reviewers performed independent analyses of the cal root segment ranging from 2 to 5 mm in length. For
methodological quality of the eligible studies. A third sample processing, cryopulverization was employed in
reviewer decided on the discordant cases. The Joanna 11 studies (Antunes et al., 2015; Ozok et al., 2012; Perez-­
Briggs Institute (JBI) checklist for cross-­sectional stud- Carrasco et al., 2023; Persoon et al., 2017; Ping et al., 2015;
ies was applied to evaluate the quality assessment (Moola Qian et al., 2019; Rôças et al., 2010; Siqueira et al., 2011;
et al., 2020). Each of the eight JBI questions was answered Siqueira et al., 2016; Siqueira et al., 2020; Takahama
with ‘Yes’, ‘No’, ‘Unclear’, or ‘Not applicable’. A score et al., 2018). In one study, the root apex specimens were
of one point was given for each ‘Yes’ answer, whereas a triturated using orthodontic pliers (Pereira et al., 2017). As
score of zero was assigned for ‘No’ or ‘Unclear’ responses. for the other studies, 5 used endodontic files and paper
Overall scores for each paper were calculated as percent- points (Baumgartner & Falkler, 1991; Chugal et al., 2011;
ages, and the quality was rated as high (80%–100%), fair Siqueira et al., 2009; Siqueira, Rôças, Alves, et al., 2004;
(50%–79%), or low (<50%). Finally, the quality of the pre- Tatikonda et al., 2017) and 2 studies used only endodon-
sent review was evaluated using the AMSTAR Checklist tic files (Bouillaguet et al., 2018; Dougherty et al., 1998)
(Assessing the Methodological Quality of Systematic to take samples from the root canal in the resected apical
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4    APICAL ROOT CANAL MICROBIOME

F I G U R E 1 Flowchart illustrating
the screening and article selection
approaches.

root segment; in the other 2 studies, samples were ob- et al., 2009; Siqueira, Rôças, Alves, et al., 2004; Takahama
tained by agitation and centrifugation of the root speci- et al., 2018; Tatikonda et al., 2017). Olsenella uli was found
mens (Subramanian & Mickel, 2009; Wang et al., 2012). in primary infections in 3 studies, with prevalence varying
from 10.5% to 76.5% (Rôças et al., 2010; Siqueira et al., 2009;
Tatikonda et al., 2017). Fusobacterium species, especially
Closed-­ended molecular methods F. nucleatum, were found in 7 closed-­ended studies, 5
from primary (range, 8%–53%) and 2 from posttreatment
Closed-­ended methods are intended to identify targeted infections (range, 14%–73.3%; Antunes et al., 2015; Pereira
microbial species or groups. Table 1 shows the studies that et al., 2017; Rôças et al., 2010; Siqueira et al., 2009; Siqueira,
used closed-­ended molecular methods for the analyses of Rôças, Alves, et al., 2004; Takahama et al., 2018; Tatikonda
the apical microbiome, 3 in posttreatment and 6 in pri- et al., 2017). Streptococcus species were detected in 5 stud-
mary infections. The number of samples that were posi- ies, 3 in primary (range, 21%–35%) and 2 in posttreatment
tive for bacteria ranged from 10 to 38 in primary infections infections (range, 76%-­91.6; Antunes et al., 2015; Siqueira
and from 21 to 36 in posttreatment infections. Amongst et al., 2009; Takahama et al., 2018; Tatikonda et al., 2017;
the studies using molecular methods, the reverse-­capture Siqueira et al., 2020). Actinobacteria species were al-
checkerboard DNA–DNA hybridization was used in 3 and ways detected in high prevalences: 2 studies of posttreat-
the nested polymerase chain reaction (PCR) in 1 study ment infection (52%–97%; Antunes et al., 2015; Siqueira
evaluating primary infections; real-­time PCR was used in et al., 2020) and in 1 of primary infection (53%; Takahama
3 studies of posttreatment infection and in 1 study of pri- et al., 2018). Other relatively frequent taxa included
mary infection (Figure 3). Porphyromonas endodontalis, black-­pigmented Prevotella
In primary infections, Pseudoramibacter alactolyti- species, Treponema denticola, and an as-­yet uncultivated/
cus was one of the most prevalent bacterial species-­level uncharacterized taxon Bacteroidaceae (G-­ 1) bacterium
taxon, with frequencies ranging from 6% to 44% (Siqueira HMT 272 (also Bacteroidetes oral clone X083).
 TABLE 1 Closed-­ended studies to evaluate the apical microbiota.

Mean no.
N (samples Apical species-­level
positive for fragment Identification taxa/canal
SIQUEIRA et al.

Reference N (sample) bacteria) Sample type length method Infection type Most prevalent taxa (range)

Dougherty et al. (1998) 18 10 Extracted teeth 7 mm Culture Primary infection Black-­pigmented bacteria (55.6%) Not reported
Prevotella nigrescens (50%)
Prevotella melaninogenica (16.6%)
Porphyromonas gingivalis (5.5%)
Prevotella intermedia (5.5%)
Siqueira, Rôças, Alves, 23 17 Extracted teeth 5 mm Nested PCR Primary infection Pseudoramibacter alactolyticus (44%) Not reported
et al. (2004) Treponema denticola (26%)
Fusobacterium nucleatum (26%)
Porphyromonas endodontalis (17%)
Filifactor alocis (9%)
Dialister pneumosintes (4%)
Porphyromonas gingivalis (4%)
Tannerella forsythensis (4%)
Siqueira et al. (2009) 20 19 Extracted teeth 5 mm Checkerboard Primary infection Pseudoramibacter alactolyticus (32%) 1.4 (1–6)
Bacteroidetes clone oral X083 (26%)
Streptococcus species (21%)
Olsenella uli (10.5%)
Synergistetes oral clone BA121 (10.5%)
Fusobacterium nucleatum (10.5%)
Porphyromonas endodontalis (10.5%)
Dialister clone oral BS016 (5%)
Parvimonas micra (5%)
Treponema denticola (5%)
Filifactor alocis (5%)
Rôças et al. (2010) 17 17 Extracted teeth 4–6 mm Checkerboard Primary infection Olsenella uli (76.5%) 8 (1–25)
Prevotella baroniae (71%)
Porphyromonas endodontalis (65%)
Fusobacterium nucleatum (53%)
Tannerella forsythia (47%)
Antunes et al. (2015) 27 21 Root-­end resection 3–5 mm Real-­time PCR Posttreatment Streptococcus species (76%) Not reported
infection Actinobacteria species (52%)
Pseudoramibacter alactolyticus (19%)
Enterococcus faecalis (14%)
Fusobacterium species (14%)
Parvimonas micra (14%)

(Continues)
|   
5

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 | 6
  

TABLE 1 Continued

Mean no.
N (samples Apical species-­level
positive for fragment Identification taxa/canal
Reference N (sample) bacteria) Sample type length method Infection type Most prevalent taxa (range)

Pereira et al. (2017) 30 30 Root-­end resection 3 mm Real-­time PCR Posttreatment Dialister pneumosintes (73.3%) 4 (1–8)
infection Fusobacterium nucleatum (73.3%)
Tannerella forsythia (53.3%)
Aggregatibacter actinomycetemcomitans
(23.3%)
Porphyromonas gingivalis (16.6%)
Prevotella intermedia (13.3%)
Treponema denticola (13.3%)
Porphyromonas endodontalis (10%)
Prevotella nigrescens (3.3%)
Tatikonda et al. (2017) 40 38 Extracted teeth 5–6 mm Checkboard Primary infection Pseudoramibacter alactolyticus (32%) Not reported
Bacteroidetes clone X083 (26%)
Streptococcus species (21%)
Olsenella uli (10.5%)
Synergistetes clone BA121 (8%)
Fusobacterium nucleatum (8%)
Porphyromonas endodontalis (8%)
Takahama et al. (2018) 17 17 Extracted teeth 5 mm Real-­time PCR Primary infection Actinobacteria species (53%) Not reported
Streptococcus species (35%)
Fusobacterium species (18%)
Parvimonas micra (18%)
Porphyromonas endodontalis (12%)
Dialister species (12%)
Pseudoramibacter alactolyticus (6%)
Filifactor alocis (6%)
Siqueira et al. (2020) 36 36 Root-­end resection 3–5 mm Real-­time PCR Posttreatment Actinobacteria species (97%) Not reported
infection Streptococcus species (91.6%)
Enterococcus faecalis (19%)
APICAL ROOT CANAL MICROBIOME

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 TABLE 2 Open-­ended studies from the first (culture) and third (molecular methods) generations to evaluate the apical microbiota.
SIQUEIRA et al.

N (samples Apical Mean no. species-­

positive for fragment Identification level taxa/canal
Reference N (sample) bacteria) Sample type length method Infection type Most prevalent taxa (range)

Baumgartner and 10 10 Extracted teeth 5 mm apical Culture Primary infection Prevotella intermedia (50%) 5 (4–6)
Falkler (1991) Prevotella buccae (50%)
Peptostreptococcus anaerobius (50%)
Veilonella parvula (50%)
Lactobacillus species (40%)
Enterococcus faecalis (40%)
Streptococcus mutans (30%)
Fusobacterium nucleatum (30%)
Actinomyces species (20%)
Actinomyces naeslundii (20%)
Subramanian and 34 33 Root-­end resection 2–3 mm PCR, cloning and Posttreatment Enterococcus faecalisa (11.5%) Not reported
Mickel (2009) sequencing infection Burkholderia cepaciaa (8.8%)
Bifidobacterium dentiuma (4.8%)
Streptococcus gordonia (4.1%)
Campylobacter gracilisa (3.8%)
Firmicutes oral clone CK057a (3.3%)
Achromobacter xylosoxidansa (3.0%)
Chugal et al. (2011) 26 26 Extracted teeth 5 mm PCR-­DGGE Primary infection Primary infection Primary
(n = 18); Fusobacterium nucleatum ssp. animalis (68%) infection
Posttreatment Fusobacterium nucleatum ssp. nucleatum (68%) 33 (16–50)
infection (n = 8) Porphyromonas endodontalis (57.8%) Posttreatment
Synergistetes species (57.8%) infection
Actinomyces species (57.8%) 16 (9–26)
Anaeroglobus geminatus (47%)
Posttreatment infection
Fusobacterium nucleatum ssp. nucleatum (80%)
Fusobacterium nucleatum ssp.animalis (70%)
Anaeroglobus geminatus (50%)
Pseudomonas species (50%)
Burkholderiales species (50%)
Actinomyces species (50%)
Enterococcus faecalis (40%)
Propionibacterium species (40%)
Comamonadaceae (30%)

(Continues)
|   
7

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8    APICAL ROOT CANAL MICROBIOME

Open-­ended molecular methods from

Mean no. species-­

level taxa/canal
1st and 3rd generations

Not reported
(range)
Open-­ended methods are intended to identify all micro-
bial taxa, or at least the most dominant ones, in a given
sample. These methods comprise the 1st (culture), 3rd
(PCR-­cloning-­Sanger sequencing, PCR-­DGGE) and 5th
Propionibacterium oral clone HE018 (61.5%) (NGS, see next section) generations of endodontic micro-
Uncultured clone ncd2794c06c1 (38.5%)
biology studies (Siqueira & Rôças, 2014). Table 2 shows

Pseudoramibacter alactolyticus (23.1%)

Propionibacterium propionicus (46.2%)
Actinomyces oral clone EL030 (84.6%)

Porphyromonas endodontalis (30.8%) studies that used open-­ended methods, 1 that evaluated

Firmicutes oral clone CK057 (7.7%)

Capnocytophaga gingivalis (15.4%) only primary infections by culture and 3 others using mo-

Bilophila wadsworthia (15.4%)

lecular methods to evaluate posttreatment infections (2
studies) and both primary and posttreatment infections (1
Dialister invisus (23.1%)
Most prevalent taxa

study). The number of samples ranged from 10 to 18 per

study of primary infections and from 8 to 34 in posttreat-
ment infections.
In primary infections, the most prevalent bacte-
rial taxa were F. nucleatum, P. endodontalis, Prevotella
species, Peptostreptococcus anaerobius, Synergistetes
and Actinomyces species, and Veillonella parvula (all
Infection type

Posttreatment

above 50% of the cases) (Baumgartner & Falkler, 1991;

infection

Chugal et al., 2011). In posttreatment infections, the

most prevalent taxa were F. nucleatum, Anaeroglobus
geminatus, Pseudomonas species, Actinomyces species,
Propionibacterium species, and Burkholderiales spe-
Identification

cies, each being found in more than 50% of the cases

PCR-­DGGE

(Chugal et al., 2011; Subramanian & Mickel, 2009; Wang

method

et al., 2012).
Abbreviations: DGGE, denaturing gradient gel electrophoresis; PCR, polymerase chain reaction.

Next-­generation sequencing (NGS)

fragment
length
Apical

3 mm

The studies using massively parallel NGS technologies

are grouped in Table 3. The number of samples evaluated
Root-­end resection

ranged from 10 to 23 in cases of primary infection and

Sample type

from 8 to 22 in posttreatment infection.

In primary infections, the most abundant bacterial
phyla detected in the apical canal included Firmicutes
(25% – 48%; Bouillaguet et al., 2018; Ozok et al., 2012;
identification

Qian et al., 2019; Siqueira et al., 2011), Proteobacteria

subjected to
positive for
N (samples

(2.4%–43%; Bouillaguet et al., 2018; Qian et al., 2019;

13 samples
bacteria)

Siqueira et al., 2011), Actinobacteria (5%–30%; Bouillaguet

et al., 2018; Ozok et al., 2012; Qian et al., 2019; Siqueira
Data provided as abundance (% clones).

et al., 2011), Bacteroidetes (9%–26.6%; Bouillaguet

et al., 2018; Ozok et al., 2012; Qian et al., 2019; Siqueira
N (sample)

et al., 2011), Fusobacteria (4.2%–16%; Bouillaguet

Continued

et al., 2018; Qian et al., 2019; Siqueira et al., 2011), and

23

Synergistetes (9.9%; Bouillaguet et al., 2018). One study

did not report on phyla abundance (Persoon et al., 2017).
Wang et al. (2012)

In posttreatment infections, the most abundant phyla were

TABLE 2

Reference

Firmicutes (18%–48.4%; Bouillaguet et al., 2018; Perez-­

Carrasco et al., 2023; Ping et al., 2015; Qian et al., 2019;
Siqueira et al., 2016), Proteobacteria (4.8%–46%; Bouillaguet
a
 TABLE 3 Next-­Generation Sequencing (NGS) studies to evaluate the apical microbiota.
SIQUEIRA et al.

N (samples Mean no. species-­

positive for level taxa/canal No. different
Reference N (samples) bacteria) NGS platform Infection Type (range) phyla Phylum (abundance) Phylum (richness) Genus (abundance) Genus (richness)

Siqueira 10 extracted 10 Pyrosequencing Primary infection 37 (13–80) 10 Proteobacteria (43%) Firmicutes (65) Fusobacterium (15%) Pseudomonas (5)
et al. (2011) teeth Firmicutes (25%) Proteobacteria Pseudoramibacter (8%) Bacteroidetes (4)
(4–6 mm) Fusobacteria (15%) (57) Novosphingobium (8%) Bacillus (4)
Bacteroidetes (9%) Bacteroidetes (20) Ralstonia (6%) Atopobium (3)
Actinobacteria (5%) Bacteroides (5%) Eubacterium (3)
Fusobacterium (3)
Mogibacterium (3)
Streptococcus (3)
Ozok et al. (2012) 23 extracted 23 Pyrosequencing Primary infection 44 (13–111) 24 Firmicutes (48%) Not defined Lactobacillus (14.3%) Not reported
teeth Actinobacteria (30%) Actinomyces (11.9%)
(1/2 root) Bacteroidetes (12%) Streptococcus (8.4%)
Actinobacteria 6.9%
Prevotella (6.1%) Parvimonas
(3.4%)
Pseudoramibacter(3%)
Bacteroidales (2.7%)
Veillonella (2.5%)
Fusobacterium (2.0%)
Peptostreptococcus (2.0%)
Ping et al. (2015) 20 root-­end 20 Pyrosequencing Posttreatment 418 20 Firmicutes (31%) Firmicutes (564) Streptococcus (12%) Prevotella (564)
resection infection (235–654) Proteobacteria (23%) Proteobacteria (428) Burkholderia (8%) Streptococcus (104)
(3 mm) Bacteroidetes (19%) Bacteroidetes (359) Prevotella (7%) Actinomyces (102)
Fusobacteria (12%) Actinobacteria (241) Fusobacterium (7%) Capnocytophaga (76)
Actinobacteria (10%) Fusobacteria (158) Veillonella (5%) Burkholderia (71)
Synergistetes (2%) Leptotrichia (5%) Fusobacterium (59)
Spirochaetes (2%) Capnocytophaga (4%) Leptotrichia (55)
Actinomyces (4%) Veillonella (40)
Siqueira 10 root-­end 10 Illumina MiSeq Posttreatment 116 (86–146) 11 Proteobacteria (46%) Proteobacteria (192) Fusobacterium (15.3%) Not reported
et al. (2016) resection infection Firmicutes (18%) Firmicutes (137) Unclassified (14.4%)
(3–5 mm) Fusobacteria (15%) Actinobacteria (53) Pseudomonas (11%)
Actinobacteria (8%) Fusobacteria (51) Stenotrophomonas (5.2%)
Unclassified (41) Rhodococcus (4.8%)
Bacteroidetes (26) Alcanivorax (4.2%)
Synergistetes (21) Rhizobium (4.0%)
Spirochaetes (9) Pseudoramibacter (2.8%)
Chloroflexi (4) Alcaligenes (2.7%)
Pyramidobacter (2.5%)
Enterococcus (2.4%)
|   
9

(Continues)

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 | 10
  

TABLE 2 Continued

N (samples Mean no. species-­

positive for level taxa/canal No. different
Reference N (samples) bacteria) NGS platform Infection Type (range) phyla Phylum (abundance) Phylum (richness) Genus (abundance) Genus (richness)

Persoon 26 extracted 23 Illumina MiSeq Primary infection 87 (46–184) Not reported Not reported Not reported Bacteriome Not reported
et al. (2017) teeth Prevotella (12.7%)
(1/2 root) Lactobacillus (11.2%)
Actinomyces (7.5%)
Fusobacterium (7.2%)
Atopobium (6.9%)
Streptococcus (4.4%)
Leptotrichia (4.3%)
Phocaeicola (3.5%)
Pyramidobacter (2.9%)
Porphyromonas (2.7%)
Mycobiome
Candida (90.7%)
Malassezia (7.9%)
Bouillaguet 43 extracted 43 Illumina MiSeq Primary infection Not reported 18 Primary infection Firmicutes (703) Primary infection Prevotella (112)
et al. (2018) teeth (n = 21) Firmicutes (36%) Bacteroidetes (434) Fusobacterium (15.9%) Treponema (50)
(5 mm) Posttreatment Bacteroidetes (23.8%) Proteobacteria (227) Parvimonas (8.0%) Lachnospiraceae_
infection Fusobacteria (16%) Actinobacteria (158) Pyramidobacter (6.3%) Unclassified (26)
(n = 22) Synergistetes (9.9%) Spirochaetes (55) Porphyromonas (5.6%) Bacteroides (22)
Actinobacteria (6.4%) Tenericutes (44) Prevotella (5.6%) Alloprevotella (18)
Proteobacteria (2.4%) Lentisphaerae (34) Bacteroides (5.0%) Actinomyces (16)
Posttreatment Saccharibacteria (20) Fretibacterium (3.5%) Lactobacillus (16)
infection Cyanobacteria (17) Dialister (3.4%) Catonella (15)
Firmicutes (48.4%) Fusobacteria (15) Alloprevotella (3.0%) Corynebacterium (14)
Actinobacteria (23.4%) Synergistetes (9) Treponema (3.2%) Mitsuokella (12)
Bacteroidetes (9.5%) Chloroflexi (7) Posttreatment infection Leptotrichia (9)
Fusobacteria (5.6%) (Average) Porphyromonas (9)
Proteobacteria (4.8%) Enterococcus (18.8%) Capnocytophaga (8)
Synergistetes (4.5%) Propionibacterium (9.9%) Olsenella (8)
Fusobacterium (5.2%) Streptococcus (8)
Bacteroides (4%) Olsenella Bifidobacterium (7)
(3.7%) Bulleidia (7)
Pseudoramibacter (3.5%) Dialister (7)
Schwartzia (3.5%) Pseudomonas (7)
Streptococcus (3.4%) Megasphaera (6)
Parvimonas (2.6%) Campylobacter (3)
Actinomyces (2.3%)
APICAL ROOT CANAL MICROBIOME

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SIQUEIRA et al.    11

et al., 2018; Perez-­Carrasco et al., 2023; Ping et al., 2015;

Qian et al., 2019; Siqueira et al., 2016), Actinobacteria
Genus (richness)
(8%–23.4%; Bouillaguet et al., 2018; Ping et al., 2015; Qian

Not reported

Not reported
et al., 2019; Siqueira et al., 2016), Bacteroidetes (9.5%–19%;
Bouillaguet et al., 2018; Perez-­Carrasco et al., 2023; Ping
et al., 2015; Qian et al., 2019), and Fusobacteria (5.6%–
15%; Bouillaguet et al., 2018; Perez-­Carrasco et al., 2023;
Ping et al., 2015; Qian et al., 2019; Siqueira et al., 2016).

Porphyromonas (5.9%)
Genus (abundance)

Fusobacterium (16%)

Pseudomonas (4.7%)
Streptococcus (7.6%)

Enterococcus (4.8%)
Richness findings for the detected phyla are shown in
Table 3.
Not reported

At the genus level, the following were more

abundant in the apical canal of primary infections:
Fusobacterium (2%–15.9%; Bouillaguet et al., 2018; Ozok
et al., 2012; Persoon et al., 2017; Siqueira et al., 2011),
Phylum (richness)

Lactobacillus (11.2%–14.3%; Ozok et al., 2012; Persoon

Not reported

Not reported

et al., 2017), Prevotella (5.6%–12.7%; Ozok et al., 2012;

Persoon et al., 2017), Actinomyces (7.5%–11.9%; Ozok
et al., 2012; Persoon et al., 2017), Streptococcus (4.4%–
8.4%; Ozok et al., 2012; Persoon et al., 2017), Parvimonas
Phylum (abundance)

Actinobacteria (13.6%)
Proteobacteria (25.3%)

Proteobacteria (28.7%)
Bacteroidetes (26.6%)

Bacteroidetes (17.3%)
Actinobacteria (11%)

Proteobacteria (10%)

(3.4%–8%; Bouillaguet et al., 2018; Ozok et al., 2012),

Primary infection

Fusobacteria (4.2%)

Fusobacteria (8.6%)

Bacteroidetes (19%)
Fusobacteria (14%)
Firmicutes (25.6%)

Firmicutes (26.2%)

Synergistetes (7%)
Firmicutes (38%)
Posttreatment

Pseudoramibacter (3%–8%; Ozok et al., 2012; Siqueira

infection

et al., 2011), Pyramidobacter (2.9%–6.3%; Bouillaguet

et al., 2018; Persoon et al., 2017), and Porphyromonas
(2.7%–5.6%; Bouillaguet et al., 2018; Persoon
et al., 2017). As for posttreatment infections, the fol-
No. different

lowing were the most abundant genera: Enterococcus

phyla

(2.4%–18.8%; Bouillaguet et al., 2018; Perez-­Carrasco

26

21

et al., 2023; Siqueira et al., 2016), Fusobacterium

Mean no. species-­

diseased teeth

(5.2%–16%; Bouillaguet et al., 2018; Perez-­Carrasco

level taxa/canal

specifically for

et al., 2023; Ping et al., 2015; Siqueira et al., 2016),

Not reported

Not reported

Streptococcus (3.4%–12%; Bouillaguet et al., 2018; Perez-­

(range)

Carrasco et al., 2023; Ping et al., 2015), Actinomyces

(2.3%–4%; Bouillaguet et al., 2018; Ping et al., 2015), and
Primary infection

Pseudoramibacter (2.8%–3.5%; Bouillaguet et al., 2018;

infection (8)
Infection Type

Posttreatment

Posttreatment
infection

Siqueira et al., 2016). Genus richness is depicted in

(23)

Table 3.
Fungi were targeted and detected in the apical canal
segment with primary endodontic infection in one study
Illumina Mi-­Seq

Illumina Mi-­Seq
NGS platform

(Persoon et al., 2017), containing 5 ± 2 OTUs (range 2–8)

per sample. Candida and Malassezia were the fungal taxa
found.
positive for
N (samples

bacteria)

Number of taxa per apical canal

50

21

Data from closed-­ended analysis showed a range of 1–25

length not
reported)
(segment
N (samples)

resection
Qian et al. (2019) 31 extracted

38 root-­end
Continued

distinct bacterial taxa per apical canal. However, this in-

teeth
(2 mm)

formation is biased and limited to the number of taxa tar-

geted in each study. Open-­ended analysis in turn using
culture revealed 4–6 species per canal, while PCR-­DGGE
et al. (2023)
Perez-­Carrasco
TABLE 2

showed 9–50, and NGS studies found 13–184 species-­level

Reference

taxa, excluding a study with too discrepant findings (235–

654 taxa/canal) (Ping et al., 2015).
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
12    APICAL ROOT CANAL MICROBIOME

FIGURE 2 Publications by country.

F I G U R E 3 Methods used to identify

microorganisms according to the study.

Bacterial load in the apical canal most of the checklist requirements; however, parameters
related to conducting a meta-­analysis were not fulfilled
Only 4 studies quantified the total bacterial cells present (Data S1).
in the apical canal segment. Of these, 2 used culture to
evaluate primary infections: Dougherty et al. (1998) re-
ported a mean 3.89 × 105 bacterial cells per canal (range, DISC USSION
2 × 101–4.2 × 106), whereas Baumgartner and Falkler
(Baumgartner & Falkler, 1991) found 1.08 × 106 cells/ Knowledge about the microbial populations colonizing
canal (range, 5.4 × 104–4.3 × 106). The other 2 studies the apical portion of necrotic root canals of teeth with api-
used molecular methods and evaluated posttreatment in- cal periodontitis is of utmost importance because this is
fections: Siqueira et al. (Siqueira et al., 2020) reported a regarded as a critical zone for infection control. The mi-
mean 1.82 × 103 cell equivalents/canal (range, 1.53 × 102– crobial taxa located in the apical segment of the canal
1.18 × 104) and Antunes et al. (2015) found 5.7 × 104 (range, system are conceivably the ones directly involved with
4 × 101–3.7 × 105). disease pathogenesis and those remaining in this area
after treatment have the potential to sustain periapical
inflammation. The apical canal third contains the most
Quality assessment significant anatomical challenges compared to the mid-
dle and coronal portions, including a large number of
Regarding the quality assessment, the included studies ramifications. The present systematic review evaluated
were rated as fair quality (score 50%–70%), and amongst the microbial diversity in the apical portion of the root
the 21 studies, all were rated as ‘moderate quality’. A canal system, compiling data from primary and posttreat-
summary of the quality appraisal ratings of each study is ment infections. The study emphasizes the importance
presented in (Table S2). According to AMSTAR 2, this sys- of understanding the microbiome in the apical segment
tematic review demonstrated moderate quality, meeting of infected root canals, which plays a critical role in the
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SIQUEIRA et al.    13

pathogenesis and persistence of apical periodontitis. This most dominant taxa in the sample and provide informa-
knowledge can guide effective infection control strategies. tion on the overall community members, closed-­ended ap-
According to this review, there are not many studies proaches are generally more sensitive to detect the target
that have specifically focused on investigating the apical taxa, thus offering valuable information about their preva-
microbiome of the root canal system. This is highly likely lence (Siqueira & Rôças, 2005, 2022b).
to be due to the methodological difficulties in obtaining While only one study reported on detection of fungi
samples exclusively from the apical segment of the canal in the apical canal of teeth with primary infection, most
during treatment (Siqueira & Rôças, 2022b). Because the studies evaluated the prevalence and diversity of bac-
methods used for taking samples from the root canals of teria. The occurrence of archaea in the apical canal was
teeth undergoing treatment or retreatment cannot dis- not reported in these studies. A high interindividual vari-
tinguish the exact region sampled, the most feasible ap- ability in the composition of the apical microbiome was
proach is to resect the apical segment of extracted teeth or reported in all studies using bacterial community profil-
teeth subjected to periradicular surgery. While the former ing methods such as DGGE and NGS (Alves et al., 2009;
permits the study of the apical microbiome in primary Chugal et al., 2011; Ozok et al., 2012; Siqueira et al., 2011,
and posttreatment apical periodontitis, the latter is typi- 2016; Wang et al., 2012). This agrees with other studies
cally used to evaluate cases of posttreatment disease. All evaluating the microbiome of the entire canal using the
the studies included in this review used one or the other conventional paper point sampling (Kruly et al., 2022;
sample source. The length of the apical segments ranged Rôças et al., 2004; Siqueira et al., 2008; Siqueira, Rôças, &
from 2 to 7 mm, which precludes us to accurately refer to Rosado, 2004; Zandi et al., 2018), and supports the concept
them as the “apical third.” that apical periodontitis is a disease with heterogeneous
Once the root apex is resected, samples from the main aetiology (Siqueira & Rôças, 2009a). Only a few studies
root canal can be obtained by burs, paper points and/ compared the composition of the apical microbiome with
or files or agitation and/or centrifugation (Siqueira & that found in the most coronal segments of the canal,
Rôças, 2022b). However, these methods cannot predict- with the results showing a significant difference (Alves
ably retrieve microbial cells entrapped in the intricate et al., 2009; Ozok et al., 2012; Rôças et al., 2010).
anatomy of the root canal system (Siqueira et al., 2009). To Data evaluating the apical microbiome revealed that
address these limitations, Alves et al. (Alves et al., 2009) Firmicutes was the most represented/abundant bacte-
proposed the use of cryogenic grinding (cryopulveriza- rial phylum followed by Proteobacteria, Actinobacteria,
tion) as an alternative approach. Since then, several stud- Bacteroidetes, and Fusobacteria. At lower taxonomic hi-
ies have adopted this methodology (Antunes et al., 2015; erarchies, the most frequent/abundant taxa in teeth with
Ozok et al., 2012; Perez-­Carrasco et al., 2023; Persoon primary infections were Pseudoramibacter alactolyticus,
et al., 2017; Ping et al., 2015; Qian et al., 2019; Rôças Olsenella uli, Fusobacterium species, Streptococcus spe-
et al., 2010; Siqueira et al., 2011, 2016, 2020; Takahama cies, Porphyromonas endodontalis, Prevotella species,
et al., 2018). In 11 of the selected studies, cryopulveriza- Actinomyces species, Parvimonas micra, Treponema denti-
tion was used to process the root apexes. An important cola, Synergistetes species, and an as-­yet uncharacterized
limitation of cryopulverization, however, is that it is a taxon Bacteroidaceae (G-­1) bacterium HMT 272 (clone
destructive approach, which does not allow for longitu- X083). As for species-­level taxa, those present in more
dinal analyses of samples taken in different time periods than 20% of the cases of primary infections included
(Siqueira & Rôças, 2022b). Also, because of the risk of F. nucleatum (4 studies), P. alactolyticus (3 studies), clone
contamination of the root during surgical (especially ex- HMT 272 and P. endodontalis (2 studies). Other 12 species
traction) procedures, attention should be given to prop- were found in >20% prevalence in 1 study each. In teeth
erly disinfecting the outer root surface and taking sterility with posttreatment infections, the most prevalent/abun-
control samples therefrom (which should yield negative dant taxa occurring in the apical canal included species of
results for bacteria; Alves et al., 2009). Streptococcus, Enterococcus, Fusobacterium, Actinomyces,
Of the studies included in this review, 19 used molecu- Pseudoramibacter, Pseudomonas, and Propionibacterium.
lar methods in either closed-­or open-­ended strategies for The species-­level taxa found in more than 20% of the teeth
microbial identification, while 2 studies relied on culture, with posttreatment disease included F. nucleatum (2 stud-
one for closed-­ended and the other for open-­ended iden- ies), and 7 other species (1 study). Enterococcus faecalis
tification. Molecular methods are usually more sensitive was present in more than 20% of the specimens in one
and some can be even more specific than culture, allow- study of primary and another one of posttreatment infec-
ing the detection of difficult-­to-­grow and even uncultiva- tion. All these most prevalent/abundant taxa found in the
ble microorganisms (Siqueira & Rôças, 2005). Although apical canal have also been reported to be amongst the
the use of open-­ended analyses is essential to detect the common members of the endodontic microbiome when
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
14    APICAL ROOT CANAL MICROBIOME

samples from the entire length of the main canal were Development of an infectious disease is related not
evaluated (Manoil et al., 2020, Siqueira & Rôças, 2009b). only to the type and number of species, but also the counts
The present results strengthen their association with api- of individual bacterial cells present (infectious load).
cal periodontitis and reinforce their status as candidate Obviously, the bacterial counts in the apical canal repre-
endodontic pathogens. sent only a fraction of the overall counts in the full canal,
Another critical aspect is how many species-­level bac- which may range from 103 to 108 in primary infections and
teria compose the apical microbiome (species richness). from 103 to 107 in posttreatment infections (Siqueira &
For this, data from open-­ended studies are the most accu- Rôças, 2022c). Only a few studies have quantified the total
rate. In primary infections, a culture-­based study reported bacterial cells in the apical root canal segment and they
a mean of 5 species/apical canal (range, 4–6; Baumgartner reported an average apical bacterial load ranging from 105
& Falkler, 1991). Two PCR-­DGGE analyses of apical speci- to 106 in primary infections and from 103 to 104 in post-
mens were published: one revealed a mean 28 species/api- treatment infections.
cal canal (range, 18–48; Alves et al., 2009), while the other Rigorous inclusion criteria were applied in the study
found a mean 33 species (range, 16–50; Chugal et al., 2011). selection of the present review, and the quality assessment
Open-­ended studies from the 5th generation (NGS) are par- was consistently rated as ‘moderate’ for all the included
ticularly effective in detecting low abundance species-­level studies. In general, the manuscripts were penalized in re-
bacteria, resulting in increased richness values. Three stud- lation to the strategies to deal with confounding factors
ies evaluated primary infections and reported the follow- in the methodology. Also, the majority of studies did not
ing mean numbers of species-­level taxa in the apical canal: perform sample size calculation. The limited number of
37 (range, 13–80; Siqueira et al., 2011); 44 (range, 13–111; studies and the heterogeneity among them, particularly in
Ozok et al., 2012); and 87 (46–184; Persoon et al., 2017). terms of sample taking and microbial identification pro-
Only two studies, both using NGS, reported on the species cedures, can be regarded as limitations of this review, pre-
richness in the apical canal of teeth with posttreatment cluding a meta-­analysis.
disease; one reported a mean 116 species-­level taxa/ canal In conclusion, findings from this systematic review
(range, 86–146; Siqueira et al., 2016) and the other found revealed a significant diversity of bacterial taxa in the
418 species-­level taxa (range, 235–654; Ping et al., 2015). It apical segment of infected root canals. There is a high
is worth pointing out that the latter study reported excep- interindividual variability in the apical microbiome
tionally discrepant findings, which could potentially be an composition. Differences in the prevalence order of the
outlier and may require further investigation. detected taxa were observed among the studies and are
The environmental conditions within the root canal possibly related to the specificity/sensitivity of the de-
system play a pivotal role in shaping the microbiome spe- tection methods for both primary and posttreatment
cies composition. Factors such as oxygen tension, nutrient infections and/or geographical influences. While no sig-
type and availability, pH, host defences, type of adhesin nificant variability at the phylum level was observed in
receptors, and microbial interactions are ecological deter- both infection types, differences were evident at the spe-
minants that exert selective pressure on the type of bac- cies/genus level. Further studies are required to provide
terial species that will survive, establish themselves and more details of the microbiome present in the apical
prevail (Siqueira & Rôças, 2022a; Sundqvist, 1992). In the canal of teeth with different types of apical periodon-
apical portion, the reduced oxygen tension along with the titis, its association with clinical and radiographic fea-
main source of nutrients in the form of proteins and gly- tures, as well as its response to antimicrobial intracanal
coproteins present in the inflammatory exudate that seep procedures.
into the canal, favour the establishment of obligate an-
aerobic bacteria that are inflammophilic and utilize pro- AUTHOR CONTRIBUTIONS
teins as the main source of energy (Fabricius et al., 1982; Warley O. Silva: formal analysis (lead). Kaline Romeiro
Hajishengallis, 2014; Sundqvist, 1994). Conversely, in the and Luciana F. Gominho: formal analysis (supporting).
most coronal parts of the canal, the oxygen tension is Isabela N. Rôças, José F. Siqueira Jr, and Flávio R. F.
higher and the main source of nutrients is allegedly car- Alves: writing – review and editing (supporting). José F.
bohydrates from diet that seep into the canal via pulpal ex- Siqueira Jr: Conceptualization (lead).
posure, favouring the higher dominance of saccharolytic
species and lower dominance of obligate anaerobes. These ACKNOWLEDGEMENTS
conditions evolve with time, as anaerobic conditions in This study was supported by grants from Fundação
the canal intensify with pulp necrosis, pioneer species Carlos Chagas Filho de Amparo à Pesquisa do Estado
change the environment, and periapical inflammation de- do Rio de Janeiro (FAPERJ) and Conselho Nacional de
velops (Fabricius et al., 1982; Sundqvist, 1994). Desenvolvimento Científico e Tecnológico (CNPq).
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SIQUEIRA et al.    15

Moher, D., Liberati, A., Tetzlaff, J., Altman, D.G. & Group P. (2010)
CONFLICT OF INTEREST STATEMENT Preferred reporting items for systematic reviews and meta-­
The authors state no conflict of interest. analyses: the PRISMA statement. International Journal of
Surgery, 8, 336–341.
DATA AVAILABILITY STATEMENT Moola, S., Munn, Z., Tufanaru, C., Aromataris, E.C., Sears, K., Sfetc,
The data that support the findings of this study are avail- R. et al. (2020) Systematic reviews of etiology and risk. In:
able from the corresponding author upon reasonable Aromataris, E. & Munn, Z. (Eds.) JBI manual for evidence syn-
request. thesis. JBI. Available from: https://​jbi-­​globa​l-­​wiki.​refin​ed.​site/​
space/​​MANUAL [Accessed 5th November 2024].
Ørstavik, D. (2020) Apical periodontitis: microbial infection and host
ETHICS STATE MENT responses. In: Ørstavik, D. (Ed.) Essential endodontology, 2nd
Not applicable. Not applicable since is a Systematic edition. Oxford: Wiley Blackwell, pp. 1–10.
Review. Ozok, A.R., Persoon, I.F., Huse, S.M., Keijser, B.J., Wesselink, P.R.,
Crielaard, W. et al. (2012) Ecology of the microbiome of the in-
ORCID fected root canal system: a comparison between apical and coro-
Warley O. Silva https://orcid.org/0000-0002-2326-2367 nal root segments. International Endodontic Journal, 45, 530–541.
Pereira, R.S., Rodrigues, V.A.A., Furtado, W.T., Gueiros, S., Pereira,
Flávio R. F. Alves https://orcid.
G.S. & Avila-­Campos, M.J. (2017) Microbial analysis of root
org/0000-0002-9922-8202
canal and periradicular lesion associated to teeth with end-
odontic failure. Anaerobe, 48, 12–18.
REFERENCES Perez-­Carrasco, V., Uroz-­ Torres, D., Soriano, M., Crielaard, W.,
Alves, F.R., Siqueira, J.F., Jr., Carmo, F.L., Santos, A.L., Peixoto, Krom, B.P., Zaura, E. et al. (2023) Microbiome in paired root
R.S., Rôças, I.N. et al. (2009) Bacterial community profiling apices and periapical lesions and its association with clinical
of cryogenically ground samples from the apical and coronal signs in persistent apical periodontitis using next-­generation
root segments of teeth with apical periodontitis. Journal of sequencing. International Endodontic Journal, 56, 622–636.
Endodontics, 35, 486–492. Persoon, I.F., Buijs, M.J., Ozok, A.R., Solana, C., Ruiz-­ Linares,
Antunes, H.S., Rôças, I.N., Alves, F.R. & Siqueira, J.F., Jr. (2015) M., Garcia-­Salcedo, J.A. et al. (2017) The mycobiome of root
Total and specific bacterial levels in the apical root canal sys- canal infections is correlated to the bacteriome. Clinical Oral
tem of teeth with post-­treatment apical periodontitis. Journal Investigations, 21, 1871–1881.
of Endodontics, 41, 1037–1042. Ping, Y., Wang, J. & Liang, J. (2015) Pyrosequencing analysis of api-
Baumgartner, J.C. & Falkler, W.A., Jr. (1991) Bacteria in the api- cal microbiota of teeth with persistent apical periodontitis.
cal 5 mm of infected root canals. Journal of Endodontics, 17, Journal of Dental Sciences, 10, 6–371.
380–383. Qian, W., Ma, T., Ye, M., Li, Z., Liu, Y. & Hao, P. (2019) Microbiota in
Bouillaguet, S., Manoil, D., Girard, M., Louis, J., Gaïa, N., Leo, S. the apical root canal system of tooth with apical periodontitis.
et al. (2018) Root microbiota in primary and secondary apical BMC Genomics, 20, 189.
periodontitis. Frontiers in Microbiology, 9, 2374. Ricucci, D. & Siqueira, J.F., Jr. (2010) Biofilms and apical peri-
Chugal, N., Wang, J.K., Wang, R., He, X., Kang, M., Li, J. et al. (2011) odontitis: study of prevalence and association with clinical
Molecular characterization of the microbial flora residing at the and histopathologic findings. Journal of Endodontics, 36,
apical portion of infected root canals of human teeth. Journal 1277–1288.
of Endodontics, 37, 1359–1364. Ricucci, D., Siqueira, J.F., Jr., Bate, A.L. & Pitt Ford, T.R. (2009)
Dougherty, W.J., Bae, K.S., Watkins, B.J. & Baumgartner, J.C. Histologic investigation of root canal-­treated teeth with apical
(1998) Black-­pigmented bacteria in coronal and apical seg- periodontitis: a retrospective study from twenty-­four patients.
ments of infected root canals. Journal of Endodontics, 24, Journal of Endodontics, 35, 493–502.
356–358. Rôças, I.N., Alves, F.R., Santos, A.L., Rosado, A.S. & Siqueira,
Fabricius, L., Dahlen, G., Ohman, A.E. & Moller, A.J. (1982) J.F., Jr. (2010) Apical root canal microbiota as determined by
Predominant indigenous oral bacteria isolated from infected reverse-­capture checkerboard analysis of cryogenically ground
root canals after varied times of closure. Scandinavian Journal root samples from teeth with apical periodontitis. Journal of
of Dental Research, 90, 134–144. Endodontics, 36, 1617–1621.
Hajishengallis, G. (2014) The inflammophilic character of Rôças, I.N., Siqueira, J.F., Jr., Aboim, M.C. & Rosado, A.S. (2004)
the periodontitis-­ associated microbiota. Molecular Oral Denaturing gradient gel electrophoresis analysis of bacterial
Microbiology, 29, 248–257. communities associated with failed endodontic treatment. Oral
Kruly, P.C., Alenezi, H., Manogue, M., Devine, D.A., Dame-­Teixeira, Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and
N., Garcia, F.C.P. et al. (2022) Residual bacteriome after chemo- Endodontology, 98, 741–749.
mechanical preparation of root canals in primary and secondary Simon, J.H. (1994) The apex: how critical is it? General Dentistry, 42,
infections. Journal of Endodontics, 48, 855–863. 330–334.
Manoil, D., Al-­Manei, K. & Belibasakis, G.N. (2020) A systematic Siqueira, J.F., Jr. & Rôças, I.N. (2005) Exploiting molecular methods
review of the root canal microbiota associated with apical peri- to explore endodontic infections: part 1-­current molecular tech-
odontitis: lessons from next-­generation sequencing. Proteomics. nologies for microbiological diagnosis. Journal of Endodontics,
Clinical Applications, 14, e1900060. 31, 411–423.
 |

13652591, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/iej.14071 by Jenny Guerrero - Readcube (Labtiva Inc.) , Wiley Online Library on [23/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
16    APICAL ROOT CANAL MICROBIOME

Siqueira, J.F., Jr. & Rôças, I.N. (2009a) Community as the unit of Brazilian and Norwegian subjects. Journal of Endodontics, 34,
pathogenicity: an emerging concept as to the microbial patho- 1457–1461.
genesis of apical periodontitis. Oral Surgery, Oral Medicine, Subramanian, K. & Mickel, A.K. (2009) Molecular analysis of per-
Oral Pathology, Oral Radiology, and Endodontology, 107, sistent periradicular lesions and root ends reveals a diverse mi-
870–878. crobial profile. Journal of Endodontics, 35, 950–957.
Siqueira, J.F., Jr. & Rôças, I.N. (2009b) Diversity of endodontic mi- Sundqvist, G. (1992) Ecology of the root canal flora. Journal of
crobiota revisited. Journal of Dental Research, 88, 969–981. Endodontics, 18, 427–430.
Siqueira, J.F., Jr. & Rôças, I.N. (2014) Present status and future direc- Sundqvist, G. (1994) Taxonomy, ecology, and pathogenicity of the
tions in endodontic microbiology. Endodontic Topics, 30, 3–22. root canal flora. Oral Surgery, Oral Medicine, Oral Pathology,
Siqueira, J.F., Jr., Rôças, I.N., Alves, F.R. & Santos, K.R. (2004) Oral Radiology, and Endodontology, 78, 522–530.
Selected endodontic pathogens in the apical third of infected Takahama, A., Jr., Rôças, I.N., Faustino, I.S.P., Alves, F.R.F., Azevedo,
root canals: a molecular investigation. Journal of Endodontics, R.S., Gomes, C.C. et al. (2018) Association between bacteria
30, 638–643. occurring in the apical canal system and expression of bone-­
Siqueira, J.F., Jr., Rôças, I.N. & Rosado, A.S. (2004) Investigation resorbing mediators and matrix metalloproteinases in apical
of bacterial communities associated with asymptomatic and periodontitis. International Endodontic Journal, 51, 738–746.
symptomatic endodontic infections by denaturing gradient gel Tatikonda, A., Sudheep, N., Biswas, K.P., Gowtham, K., Pujari, S. &
electrophoresis fingerprinting approach. Oral Microbiology and Singh, P. (2017) Evaluation of bacteriological profile in the api-
Immunology, 19, 363–370. cal root segment of the patients with primary apical periodon-
Siqueira, J.F., Jr., Alves, F.R. & Rocas, I.N. (2011) Pyrosequencing titis. The Journal of Contemporary Dental Practice, 18, 44–48.
analysis of the apical root canal microbiota. Journal of Wang, J., Jiang, Y., Chen, W., Zhu, C. & Liang, J. (2012) Bacterial
Endodontics, 37, 1499–1503. flora and extraradicular biofilm associated with the apical seg-
Siqueira, J.F., Jr., Antunes, H.S., Perez, A.R., Alves, F.R.F., Mdala, I., ment of teeth with post-­treatment apical periodontitis. Journal
Silva, E.J.N.L. et al. (2020) The apical root canal system of teeth of Endodontics, 38, 954–959.
with posttreatment apical periodontitis: correlating microbio- Zandi, H., Kristoffersen, A.K., Ørstavik, D., Rôças, I.N., Siqueira, J.F.,
logic, tomographic, and histopathologic findings. Journal of Jr. & Enersen, M. (2018) Microbial analysis of endodontic in-
Endodontics, 46, 1195–1203. fections in root-­filled teeth with apical periodontitis before and
Siqueira, J.F., Jr., Antunes, H.S., Rocas, I.N., Rachid, C.T. & Alves, after irrigation using pyrosequencing. Journal of Endodontics,
F.R. (2016) Microbiome in the apical root canal system of 44, 372–378.
teeth with post-­treatment apical periodontitis. PLoS One, 11,
e0162887.
Siqueira, J.F., Jr. & Rôças, I.N. (2022a) Treatment of endodontic infec- SUPPORTING INFORMATION
tions, 2nd edition. London: Quintessence Publishing. Additional supporting information can be found online
Siqueira, J.F., Jr. & Rôças, I.N. (2022b) A critical analysis of research in the Supporting Information section at the end of this
methods and experimental models to study the root canal article.
microbiome. International Endodontic Journal, 55(Suppl 1),
46–71.
Siqueira, J.F., Jr. & Rôças, I.N. (2022c) Present status and future di-
rections: microbiology of endodontic infections. International How to cite this article: Siqueira, J.F. Jr, Silva,
Endodontic Journal, 55(Suppl 3), 512–530. W.O., Romeiro, K., Gominho, L.F., Alves, F.R.F. &
Siqueira, J.F., Jr., Rôças, I.N., Alves, F.R. & Silva, M.G. (2009) Bacteria Rôças, I.N. (2024) Apical root canal microbiome
in the apical root canal of teeth with primary apical periodonti- associated with primary and posttreatment apical
tis. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology,
periodontitis: A systematic review. International
and Endodontology, 107, 721–726.
Endodontic Journal, 00, 1–16. Available from:
Siqueira, J.F., Jr., Rôças, I.N., Debelian, G.J., Carmo, F.L., Paiva,
S.S.M., Alves, F.R.F. et al. (2008) Profiling of root canal bacterial https://doi.org/10.1111/iej.14071
communities associated with chronic apical periodontitis from