Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

Alaa Muayad Altaie; Basema Saddik; Mohammed Amjed Alsaegh; Sameh S M Soliman; Rifat Hamoudi; Lakshman P Samaranayake

doi:10.1371/journal.pone.0255485

. 2021 Aug 5;16(8):e0255485. doi: 10.1371/journal.pone.0255485

Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

Alaa Muayad Altaie ^1,², Basema Saddik ^1,³, Mohammed Amjed Alsaegh ^1,², Sameh S M Soliman ^1,⁴, Rifat Hamoudi ^1,^5,^6,^‡,^*, Lakshman P Samaranayake ^1,^2,^‡,^*

Editor: Andrej M Kielbassa⁷

PMCID: PMC8341601 PMID: 34351963

Abstract

Objective

To assess the prevalence of unculturable bacteria in periapical abscess, radicular cyst, and periapical granuloma.

Methods

PubMed, Scopus, Science Direct, and Ovid databases were systematically searched from January 1990 to May 2020. All the included studies were cross-sectional design. The risk of bias was assessed using Joanna Briggs Institute check-list. Heterogeneity was described using meta-regression and mixed-effects model for lesion, country, and sequence technique moderators. Funnel plot and unweighted Egger’s regression test were used to estimate the publication bias. Microbiome data on diversity, abundance, and frequency of unculturable bacteria in the periapical lesions were reviewed, analysed, and the principal component analysis (PCA) was performed.

Results

A total of 13 studies out of 14,780, were selected for the final analysis. These studies focused on the prevalence of unculturable bacteria in periapical abscesses and related lesions. Approximately 13% (95% CI: 7–23%) of the cumulative number of bacteria derived from periapical abscesses was unculturable. Country moderator significantly (P = 0.05) affects the diversity summary proportion. While the pooled frequency of unculturable bacteria was 8%; 95% CI: 5, 14%, the estimate of the pooled abundance of unculturable bacteria was 5%; 95% CI: 2, 12% with a significant (P = 0.05) country moderator that affects the abundance summary proportion. Of the 62 unculturable bacteria, 35 were subjected to PCA and Peptostreptococcus sp. oral clone CK035 was the most abundant species in periapical abscesses. Hybridization techniques were found to be the most reliable molecular methods in detecting the abundance and frequency of unculturable bacteria.

Conclusion

The significant prevalence of unculturable bacteria in the periapical abscess, suggests that they are likely to play, a yet unknown, critical role in the pathogenesis and progression of the disease. Further research remains to be done to confirm their specific contributions in the virulence and disease progression.

Introduction

Periapical abscesses are, by far, the most frequent infectious lesions of the alveolar bones [1, 2]. Periapical abscess occurs in and around the apex of a root, the periodontal membrane of a tooth, and the adjacent alveolar bone [2–4]. The spread of infection via the apical foramen and inflammation sets in a cascade of reactions attracting inflammatory chemical mediators to initiate the periapical pathology that eventually results in a periapical abscess, a radicular cyst, or a periapical granuloma [2, 5].

There are several classifications of periapical lesions [6, 7]. World Health Organization (WHO) [6] provides a classification of the periapical lesions including periapical abscess, radicular cyst, and periapical granuloma, according to the clinical signs, other than symptoms or the histopathological differences of the periapical lesions. Another classification was proposed by Nair [7] and depending on the histopathological findings of the periapical abscess, radicular cyst, and periapical granuloma. However, it was the best to combine the clinical findings of signs and symptoms in addition to the histopathological examination when classifying the periapical lesions. This was achieved by using the updated version of classification of the American Association of Endodontics (AAE) for periapical lesions [8]. Periapical abscesses were classified into acute and chronic periapical abscess according to onset type, pain intensity, swelling, and discharge of pus or sinus formation [8]. The histological identification of periapical granuloma differentiates it from radicular cyst in which the former characterized by chronic inflammatory cells such as macrophages, plasma cells and lymphocytes and sometimes a cluster of multinucleated giant cells, capillaries, fibroblasts, and collagen fibres are also present [7, 8].

In clinico-pathological terms, periapical abscess is defined as a localized collection of pus within the alveolar bone, at the root apex of a tooth [9]. As mentioned, the lesion is usually initiated as a sequel of extension of infection into the periapex of an affected tooth. Once the intact pulp chamber is breached, colonization of the root canal ensues with a diverse mix of bacteria [10]. After entering the periapical tissues, these bacteria induce an acute inflammatory reaction and pus formation leading to a periapical abscess [11]. A number of researchers, over the last few decades, have investigated the microbiome of the periapical abscess using conventional bacteriological culture and noted that Bacteroides sp., Streptococcus sp., anaerobic cocci, and Fusobacterium sp. are the major constituents of periapical abscess [12]. However, recent next generation sequencing (NGS) studies indicate that a profusion of unculturable bacteria are present in periapical abscess and are the likely co-contributors to the disease [13].

Using polymerase chain reaction (PCR) and cloning strategies that target 16S rRNA, it is possible to determine the bacterial composition and diversity of any given infection [14]. The 16S rRNA approach defines a species (or more precisely a phylotype) as strains or clones with > 98.5% identity [15]. Studies based on 16S rRNA gene clone libraries have shown that 40–60% of the oral microbiome is composed of as-yet-unculturable bacteria [16–18]. For instance, the Human Oral Microbiome Database (HOMD) lists approximately 220 oral taxa that have not been cultivated [19]. Thus, only 29–50% of the oral species-level taxa belonging to the phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria have been successfully cultivated. The number of cultivable members of the Spirochaetes and Synergistetes phyla is relatively low [16, 20]. Of 48 oral Spirochaetes listed in HOMD, only ten have been cultivated and named, while only three of oral Synergistetes, have been cultivated and named: Jonquetella anthropi [21], Fretibacterium fastidiosum [22], and Pyramidobacter piscolens [23]. Furthermore, it has been estimated that approximately 50% of the human oral flora is unculturable [24, 25], and the failure of curing of some infectious diseases speculated that at least some of the unculturable microbiome are involved in disease progression and may account for unknown antimicrobial resistance [26]. On the other hand, the importance of some unculturable bacteria represented by their production of secondary metabolites that may have a promising future as effective antimicrobial agents [27]. These findings indicate that a significant proportion of bacterial species inhabiting the oral ecosystem and causing dental diseases is yet to be described, and their contribution to diseases such periapical abscess is yet to be defined.

There are no reviews in the English Language literature, to our knowledge, on the unculturable bacteria in periapical abscess and other sequelae of pulp necrosis such as radicular cysts, and periapical granulomas. Hence, the primary aim of this study was to perform a systematic literature review and meta-analysis of the unculturable bacteria in periapical lesions. A secondary aim was to review the role of individual unculturable bacteria in terms of abundance and frequency in the foregoing periapical lesions, and to review the molecular techniques evaluating the latter. Therefore, the purpose of this study was to conduct a systematic review and meta-analysis that investigates the prevalence of unculturable bacteria in the periapical abscess, radicular cyst, and periapical granuloma using culture-independent methods. The followed question format was (CoCoPop) [28, 29] as condition, context, and population. The condition represents the unculturable bacterial profile, context represents all studies conducted on periapical abscesses, radicular cysts, and periapical granulomas using culture-independent methods, and population represents human clinical samples derived from periapical lesions (periapical abscess, radicular cyst, and periapical granuloma).

Methods

Protocol and registration

We performed the systematic review and meta-analysis in accordance with Cochrane Collaboration and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [30]. The study was registered in PROSPERO database https://www.crd.york.ac.uk/PROSPERO/ with the registration ID (CRD42020160557).

Search strategy and study selection

First, we systematically searched PubMed, Scopus, Science Direct, and Ovid databases using the MeSH (Medical Subject Headings) terms “dentoalveloar abscess”, “periapical lesion”, “periapical abscess”, “periapical granuloma”, “radicular cyst”, “unculturable bacteria”, and “sequencing” (S1 Table). Manual searching, and cross-reference checks were also conducted. The search was limited to human studies published over a decade, from 1990/01/01 to 2020/05/31. The inclusion criteria for the study were, English language, and original studies on microbiological investigations using culture-independent methods. The exclusion criteria were represented by any paper that used culture-dependent methods for bacterial identification, did not identify the unculturable bacteria, and histologically did not specify radicular cyst from periapical granuloma. Two independent reviewers (A.A.M, A.M.A) identified relevant studies based on the inclusion criteria and MeSH terms. Cohen’s kappa was used to assess the inter-rater reliability between the two reviewers (~ 20 for title and abstract screening, and ~10 for full text screening) during the selection of studies for inclusion. Cohen’s kappa less than 0.20 referred as slight agreement, between 0.21–0.40 as fair agreement, between 0.41–0.60 as moderate agreement, between 0.61–0.80 as substantial agreement, and between 0.81–1.00 as perfect agreement. Discrepancies between the reviewers were resolved after an iterative consensus process.

Data extraction

Full-text articles were reviewed in detail after a thorough screening of the titles and abstracts. For each selected article, the general characteristics which included the first author’s name and year, study design, country, sample size, gender, age, dentition type, numbers and lesion type, sample collection procedure and origin, and the molecular biology method for bacterial detection, were extracted to an Excel spreadsheet. When studies included more than one type of lesion, the corresponding information was recorded. Additional information including diversity, abundance, and frequency as well as the abundance and frequency of the individual organism were also reported.

Risk of bias assessment

Risk of bias assessment was performed using a customised checklist based on Critical Appraisal of Joanna Briggs Institute for Analytical Cross Sectional Studies [31] as per the selected study criteria. This was used to assess the risk of bias, or the quality of studies included. The standard Checklist for Analytical Cross Sectional Studies was used, and each article independently assessed by the two authors (A.A.M, and A.M.A) (S2 Table). Cohen’s kappa was used to check the inter-rater reliability for scoring the risk of bias assessment of the included studies. The final scores of the risk of bias assessment for the included studies were determined after consensus process between the reviewers.

Resources used to identify the unculturability of bacteria

The following web resources were utilized to ascertain whether the identified bacteria are culturable or not. Expanded Human Oral Microbiome Database (eHOMD) [32] http://www.homd.org/, NCBI Taxonomy Browser-NIH [33] https://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/, and the Culture Collection of the University of Gothenburg [34] https://ccug.se/ were used to check the culturability of the bacteria included in the study.

Statistical analysis

Meta-analysis was performed as primary outcomes for the diversity, abundance, and frequency of unculturable bacteria in the periapical abscess, using RStudio software version 1.2.5019 available at https://www.r-project.org/. Forest plot was conducted to graphically represent the consistency and reliability of the results. The analysis was done for three separate groups on the proportion (percentage) of unculturable bacteria [35]. Moderators were identified and included the lesion type, country of the study, and the molecular biological technique used for bacterial identification. The proportion in each group was set to less than 0.2 but as the study number was relatively low, data transformation was performed using logit transformation to obtain the approximated, binomial distribution [36, 37].

Transformed proportions were combined to get the pooled effect sizes. Random-effect model [38] with DerSimonian and Laird method were used for this purpose [39]. The level of heterogeneity was assessed by Cochran’s Q test and quantified using Higgins I² test [40] Heterogeneity was classified as low if the I² was (0–40%), moderate (30–60%), substantial (50–90%), or high (75–100%) [41, 42]. P-values were obtained by comparing the Chi-squared test with a statistic Q and k-1 degrees of freedom, using the confidence interval 95% as the cut-off for statistically significant of heterogeneity. The proportion of true heterogeneity across all studies was described using meta-regression of moderators and mixed-effects model with DerSimonian and Laird method to find the effect of these moderators on summary effect size.

Outlying studies were tested using studentized residuals that showed z-value of more than 2 or 3 in an absolute value. The decision of removing outlying studies was dependent on testing its influence on summary effect size [43]. Studentized residuals test has been used to estimate the change after removing the outliers [43] and to detect the normal distribution of studies [44]. To confirm the impact of the outlying studies, the following tests were employed to show the change in their corresponding estimate after removing the outliers. DFFITS and Cook’s distances values showed the change in the standard deviations [44], co-variance ratios (cov.r) showed the effects on the precision of estimates, and the amount of heterogeneity (tau².del) displayed tau² of each study [44]. The test statistics for heterogeneity (QE.del) showed the χ² test for homogeneity [44]. Hat values indicated that large values reveal large influence, and the weights test indicated the weight of a study in the overall meta-analytic average effect size. Lastly, funnel plot asymmetry and unweighted Egger’s regression test were used to estimate the publication bias [35].

Secondary outcomes were performed for individual unculturable bacterial abundance and frequency using principal component analysis (PCA) in RStudio software. Factoextra, magrittr, and dplyr libraries were used to determine the individual unculturable bacteria in relation to their abundance, frequency, and biological sequencing techniques [45].

Results

Study selection

A literature search of PubMed, Scopus, Science Direct, Ovid, as well as a manual search retrieved a total of 14,780 studies on periapical abscess, radicular cyst, and periapical granuloma. Deduplicating reveals the removal of 39 studies. Screening the titles and the abstracts of these studies leads to the exclusion of 11,220, and 3343, respectively. The remaining 217 full-text articles were perused and 201 were excluded (Fig 1). The excluded studies were unspecified lesions (absence of histological analysis for discrimination between periapical granuloma and cyst) (60 articles), detection of culturable bacteria through molecular methods (45 articles), case-reports (22 articles), samples unrelated to granuloma, cyst, and abscess (16 articles), culture methods for bacterial detection (10 articles), histobacteriological detection of bacteria (10 articles), narrative reviews (eight articles), detection of bacteria through culture media then molecular methods, mass spectrometry, or electron microscope (six articles), unculturable bacteria had not been recorded by molecular methods (five articles), case-series (three articles), full-text articles are unavailable (three articles), detection of virulence genes (three articles), no bacteriological detection (three articles), nondescript unculturable bacteria (two articles), immunological detection of culturable bacteria (two articles), full-text article in language other than English (one article), book chapter (one article), and duplicate publication (one article). Of the final records, 16 were included in the qualitative analysis of the systematic review [44, 46–60], from them, only 13 were included in the final quantitative meta-analysis [44, 46–52, 54–56, 59, 60] (Fig 1).

As none of the reviewed studies on periapical granuloma and radicular cyst reported the presence of unculturable bacteria, this systematic review and meta-analysis was confined only to the periapical abscess.

Study characteristics

A total of 16 studies out of 14,780 studies (0.11%) were selected for the systematic review, and only 13 (0.09%) were used for the final analysis. All the included studies were cross sectional study design and conducted between 2001 and 2020. Seven of the included studies were from Brazil [44, 50–54, 60], two from China [55, 56], two from USA [46, 47], and one each from South Korea [57], Republic of Estonia [59], UK [58], Brazil/USA [49], and Scotland [48]. The reviewed periapical lesions included 12 studies on acute periapical abscess [44, 46–56], two studies related to chronic periapical abscess [57, 58], while for the rest two studies [59, 60], the lesion type was recorded in our study as “periapical abscess” only without acute or chronic specification because one study mentioned the lesions as “abscess” [60], and the another represented an indeterminate analysis on acute/ chronic abscess [59] (Table 1).

Table 1. General characteristics of the included studies.

Author /year	Study design	Country	Sample size	Gender	Age	Dentition type	Number and lesion type	Sample collection procedure and origin	Molecular biology technique
Flynn et al. (2012) [46]	Cross sectional	USA	9	NM	NM	NM	9 Acute periapical abscesses	Aspiration/Purulent	ABI
George et al. (2016) [47]	Cross sectional	USA	18	NM	16–60 years	Permanent	18 Acute periapical abscesses	Aspiration/Purulent	Microbial microarrays
Jacinto et al. (2007) [60]	Cross sectional	Brazil	5	NM	NM*	NM	5 Periapical abscesses	Paper point/Root canal	Denaturing high-performance liquid chromatography
Riggio et al. (2007) [48]	Cross sectional	Scotland	4	1 Woman, 3 men	18-33years	Permanent	4 Acute periapical abscesses	Surgical drainage/Aspiration/Purulent	Terminal restriction fragment length polymorphism
Rôças and Siqueira (2009) [50]	Cross sectional	Brazil	52	NM	18–74 years	Permanent	21 Acute periapical abscesses	Aspiration/Purulent	Nested-PCR
Rôças and Siqueira (2018) [51]	Cross sectional	Brazil	133	48 Women, 85 men	16–75 years	Permanent	55 Acute periapical abscesses	Aspiration/Purulent	Semi-quantitative reverse-capture checkerboard assay
Rôças et al. (2006) [49]	Cross sectional	Brazil, USA	77	NM	NM	Permanent	77 Acute periapical abscesses	Aspiration/Purulent	ABI
Rolph et al. (2001) [58]	Cross sectional	UK	41	17 Women, 8 men	NM	NM	1 Chronic periapical abscess	Paper points/Root canal	Thermo Sequenase sequencing
Sakamoto et al. (2006) [52]	Cross sectional	Brazil	16	NM	18–44 years	Permanent	7 Acute periapical abscesses	Aspiration/Purulent	Terminal restriction fragment length polymorphism
Sakamoto et al. (2009) [44]	Cross sectional	Brazil	90	NM	NM	Permanent	6 Acute periapical abscesses	Aspiration/Purulent	ABI
Siqueira and Rôças (2007) [53]	Cross sectional	Brazil	50	NM	>18 years	Permanent	29 Acute periapical abscesses	Aspiration/Purulent	ABI
Siqueira and Rôças (2009) [54]	Cross sectional	Brazil	42	NM	NM*	Permanent	42 Acute periapical abscesses	Aspiration/Purulent	Checkerboard DNA-DNA hybridization
Vengerfeldt et al. (2014) [59]	Cross sectional	Republic of Estonia	12	7 Men, 5 women	27–66 years	Permanent	3 Acute and 1 chronic periapical abscess (periapical abscesses)	Paper points/Root canal	Illumina Sequencing
Yang et al. (2010) [55]	Cross sectional	China	11	5 Boys, 6 girls	5.4–7.6 years	Deciduous	11 Acute periapical abscesses	Puncturing the mucosa and paper points inserted into the mucosa	ABI
Yun et al. (2017) [57]	Cross sectional	South Korea	10	NM	2–7 years	Deciduous	2 Chronic periapical abscesses	Paper points/Root canal	Pyrosequencing
Zhang et al. (2020) [56]	Cross sectional	China	9	6 Boys, 3 girls	3–11 years	Deciduous	9 Acute periapical abscesses	Paper point/Root canal	ABI

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NM: not mentioned, NM*: not mentioned but checked by the authors.

Different molecular techniques were reported in regard to the detection of unculturable bacteria. Six (37.5%) out of 16 studies used the ABI SOLiD sequencing technology [44, 46, 49, 53, 55, 56], and two (12.5%) used terminal restriction fragment length polymorphism (T-RFLP) [48, 52]. Further, Illumine sequencer [59], pyrosequencing [57], denaturing high-performance liquid chromatography (dHPLC) [60], checker-board DNA-DNA hybridization [54], thermo sequenase sequencing [58], nested-polymerase chain reaction N-PCR [50], microbial array [47], and Semi-quantitative reverse-capture checkerboard assay [51] were used in only one study each (6.3%) (Table 1).

Risk of bias assessment

The Critical Appraisal of Joanna Briggs Institute for Analytical Cross Sectional Studies [31] was used to evaluate the risk of bias. Cohen’s kappa revealed that the observed agreement of inter-rater reliability for the risk of bias assessment was 94% and Cohen’s kappa was 0.88 (95% CI: 0.64–1.00). The final scores of the included studies were determined after consensus process between the reviewers. All studies with a minimum score of 6/8 were included in the review. Of the 16 included studies, eight reached a score of 8/8 [51–53, 55–57, 59, 60], while the other eight got (6/8) [44, 46–50, 54, 58] of the bias assessment (S3 Table).

Unculturable bacteria in periapical abscess

Acute periapical abscess

Of the 16 reviewed studies, 12 reported the presence of unculturable bacteria in acute periapical abscess [44, 46–56] (Table 2 and S4 Table). Briefly, in the acute periapical abscesses, 58 unculturable bacteria were identified from a total of 397 identified organisms (14.6%). Five studies reported both the abundance and frequency of isolated bacteria [44, 46, 52, 55, 56], and another five reported only the frequency of bacterial isolation [47, 49–51, 54], while only a single study provided the data on bacterial abundance [48].

Table 2. Extracted results from each study of periapical abscess.

Study	Number of unculturable bacteria	Total number of bacteria	Diversity %	Clones of unculturable bacterial	Total number of bacterial clones	Abundance %	Frequency of unculturable bacteria	Total frequency of bacteria	Frequency %
Flynn et al. (2012) [46]	5	25	20.00	6	391	1.53	6	67	8.96
George et al. (2016) [47]	4	41	9.76	_	_	_	54	550	9.82
Riggio et al. (2007) [48]	3	29	10.34	12	203	5.91	_	_	_
Rocas et al. (2006) [49]	1	10	10.00	_	_	_	2	267	0.75
Rocas and Siqueira (2009) [50]	1	3	33.33	_	_	_	3	9	33.33
Rocas and Siqueira (2018) [51]	2	39	5.13	_	_	_	11	455	2.42
Sakamoto et al. (2006) [52]	9	29	31.03	24	93	25.81	18	85	21.18
Sakamoto et al. (2009) [44]	15	24	62.50	18	287	6.27	16	37	43.24
Siqueira and Rocas (2009) [54]	11	55	20.00	_	_	_	39	354	11.02
Yang et al. (2010) [55]	1	17	5.88	8	424	1.89	1	62	1.61
Zhang et al. (2020) [56]	6	125	4.80	_	_	_	_	_	_
Jacinto et al. (2007) [60]	3	33	9.09	30	480	6.25	4	48	8.33
Vengerfeldt et al. (2014) [59]	1	54	1.85	_	_	_	1	136	0.74
Total	62	484		98	1878		155	2070

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There is a conflict in the literatures on the cultivability of the isolates from periapical abscess, as some reported the identical phylotypes are culturable, and others as unculturable. For instance, Siqueira and Rocas [53] reported Synergistes phylotype from periapical abscess as unculturable, but HOMD database indicates that all Synergistes sp., except for E3-33 E1, are culturable. Further, this study looked for only one phylotype of Synergistes and they did not mention neither the frequency nor the abundance of this unculturable bacterium and ultimately was excluded from the meta-analysis [53] (Table 1).

Acute/chronic periapical abscess

In general, only two studies [59, 60] revealed the prevalence of culturable and unculturable bacteria in the periapical abscesses without mentioning whether they are acute or chronic. For this reason, these studies were included in this systematic review and meta-analysis as “periapical abscess”.

The total number of periapical abscess samples, unassigned as either acute or chronic were nine, and four unculturable isolates were reported out of a total 87 identified bacteria (4.6%) form these samples. Thus, Vengerfeldt et al., recorded the frequency of uncultured TG5 group as 25% [59]. Jacinto et al., reported the frequency of uncultured Staphylococcus sp. clone pGA 2 as 40%; while 20% for the uncultured rape rhizosphere bacterium wr0200 and bacterium clone aab38e07 was noted among the periapical abscess samples [60]. Jacinto et al., also recorded the abundance of the uncultured bacterial clones among all other bacterial clones. The uncultured rape rhizosphere bacterium wr0200 was recorded as the most abundant 2.71%, while the Staphylococcus sp. clone pGA 2 and uncultured bacterium clones aab38e07 were 2.08% and 1.46%, respectively [60] (S4 Table).

Chronic periapical abscess

The unculturable bacteria from chronic periapical abscess were reported in two studies [57, 58]. Collectively, three samples of chronic periapical abscesses were reported and two unculturable bacteria were identified from a total of 23 culturable bacteria (8.7%). The chronic abscesses were samples from primary [57] and permanent teeth [58]. Yun et al., reported that the abundance of uncultured bacterium AF287795_g (Selenomonas sp. oral clone CS002/ Mitsuokella sp. Human Oral Taxon (HOT) 131) is 16% [57]. On the other hand, Rolph et al., reported the unidentified Eubacterium clone 3.3 [U43698] in one case of chronic periapical abscess, although its isolation frequency and the abundance were not reported [58]. These two studies [57, 58] were excluded from the meta-analysis due to low sample size as one of them included two cases [57], and the other study included only one case of chronic periapical abscesses [58].

Outcome measures: Primary outcome measures: Diversity, abundance, and frequency of unculturable bacteria in periapical abscess

In this meta-analysis, the diversity, abundance, and frequency of unculturable bacteria were studied. The total number of unculturable bacteria reported in all abscess samples was 62 out of a total 484 identified bacteria. The diversity of unculturable bacteria in periapical abscesses [59, 60] and in acute periapical abscess [44, 46–52, 54–56] were determined. The diversity of unculturable bacteria in all abscess samples was 13%, 95% CI: 7, 23% (Fig 2A).

Fig 2 — (A) Diversity of unculturable bacteria in periapical abscess was estimated to be 13% (95%, CI: 7, 23%) in 13 studies. (B) Abundance of unculturable bacteria was estimated to be 5% (95%, CI: 2, 12%) in six studies. (C) Frequency of unculturable bacteria in periapical abscess was estimated to be 8% (95%, CI: 5, 14%) in 11 studies.

The abundance of unculturable bacteria in periapical abscess [60] and in acute periapical abscess [44, 46–48, 52, 55] were calculated. The estimate of the pooled abundance of unculturable bacteria in all abscess samples was 5%; 95% CI: 2, 12% (Fig 2B).

The frequency of unculturable bacteria in periapical abscess [59, 60] and in acute periapical abscess [44, 46, 47, 49–52, 54, 55] were determined. The pooled frequency of unculturable bacteria in all abscess samples was 8%; 95% CI: 5, 14% (Fig 2C).

Heterogeneity of studies

The heterogeneity between studies were evaluated by meta-regression analysis. For this purpose, we estimated which specific moderator had a significant impact on the outcomes using three arbitrary categories, depending on the availability of data. Namely, i) the type of lesion, ii) the country of study origin, and iii) the sequencing technique employed. Lesion moderation was deemed dichotomous, as acute abscess, and abscess. Similarly, in the country of origin moderation, two divisions were evaluated, Brazil and other countries, as most data originated from Brazil, while for “sequence” moderation, the quality of DNA sequencing and hybridization were used as two separate categories. The term “sequence” represented a nonspecific 16S rRNA targeted gene sequencing of bacterial species using techniques such as ABI, pyrosequencing, Illumina sequencing, and thermo sequencing, while the “hybridization” refers to a specific 16S rRNA targeted gene identification using techniques such as T-RFLP, N-PCR, microbial microarrays, dHPLC, checkerboard DNA-DNA hybridization, and semi-quantitative reverse-capture checkerboard assay.

For diversity estimate, the heterogeneity (I²) was high (78%, 95% CI: 63, 87%), with tau² = 1.09/0.32; 3.5, and P < 0.01. As for lesion moderation, there were two studies denoting the lesions as “abscess” [59, 60], and 11 studies defined as “acute abscess” [44, 46–52, 54–56]. The heterogeneity (R²) computed for the latter was 3.86%, the test of moderator coefficient was [QM (df = 1) = 1.74, P = 0.19], and the significant slope coefficient was 1.30; Z (15) = 1.32, and P = 0.19. All the meta-regression analyses revealed that the lesion moderation did not significantly impact the summary proportions (S1A Fig). With the country moderation, the heterogeneity was 33.83%, the test of moderator coefficient was [QM (df = 1) = 3.86, P = 0.05], and the significant slope coefficient was 1.17; Z (15) = 1.96, and P = 0.05, representing a significant effect on summary proportion (S1B Fig). Regarding the sequence technique moderator, the amount of heterogeneity (R²) was 0.00%, the test of moderator coefficient was [QM (df = 1) = 0.04, P = 0.85], and the significant slope coefficient was -0.14; Z (15) = -0.19, and P = 0.85, depicting no significant effect of the sequence techniques on the summary proportion (S1C Fig).

For abundance estimate, the amount of heterogeneity (I²) was high (93%, 95% CI: 87.6, 96.1%), tau² = (0.96/0.36; 6.99), and P< 0.01. Meta-regression analysis was conducted to estimate which moderator had a considerable impact on the abundance summary proportion. With the lesion moderator, the abscess lesion was recorded in one study [60], and acute abscess in five studies [44, 46, 48, 52, 55]. The amount of heterogeneity (R²) was 0.00%, the test of moderator coefficient was [QM (df = 1) = 0.02, P = 0.9], and the significant slope coefficient was -0.19; Z (15) = -0.14, and P = 0.9 (S2A Fig). Regarding the country moderator, the amount of heterogeneity (R²) was 25.14%, the test of moderator coefficient was [QM (df = 1) = 3.86, P = 0.05], and the significant slope coefficient was 1.44; Z (15) = 1.96, and P = 0.05 (S2B Fig). For the sequence methods moderator, the amount of heterogeneity (R²) was 15.46%, the test of moderator coefficient was [QM (df = 1) = 3.26, P = 0.07], and the significant slope coefficient was -1.4; Z (15) = -1.8, and P = 0.07 (S2C Fig).

For frequency, the amount of heterogeneity (I²) was high (89.9%, 95% CI: 83.9, 93.6%), tau² = (0.87 /0.58; 5.49), and (P < 0.01). In meta-regression analysis, the lesion moderator for abscess studies [59, 60] and acute abscess studies [44, 46, 47, 49–52, 54, 55] revealed that the amount of heterogeneity (R²) was 0.00%, the test of moderator coefficient was [QM (df = 1) = 1.41, P = 0.24], and the significant slope coefficient was 1.09; Z(15) = 1.18, and P = 0.24. These results indicated that the lesion moderator did not significantly affect the summary proportion (S3A Fig). For the country moderator, the amount of heterogeneity (R²) was 0.00%, the test of moderator coefficient was [QM (df = 1) = 1.44, P = 0.23], and the significant slope coefficient was 0.96; Z (15) = 1.21, and P = 0.23 (S3B Fig). In the case of sequence methods moderator, the amount of heterogeneity (R²) was 0.00%, the test of moderator coefficient was [QM (df = 1) = 1.3, P = 0.25], and the significant slope coefficient was -0.78; Z (15) = -1.14, and P = 0.25 (S3C Fig).

Outlying studies

Several analyses were performed for detection the outlying studies. For the diversity of unculturable bacteria in periapical abscess, studentized residuals tests showed that study number 8 [44] has an outlying z-value 3.05. Leaving out this study [44] revealed some changes on the summary proportion from 13%; 95% CI: 7, 23% to 11%; 95% CI: 7, 18% (S4A Fig). Externally studentized residuals (rstudent) test, revealed that all studies are normally distributed except for the 8^th study which is located outside the range limit [44]. DFFITS values showed the standard deviations of the 8^th study [44] was the highest (0.82). Also, Cook’s distances (cook.d) was 0.34 for the 8^th study [44] and co-variance ratios (cov.r) was 0.62. The amount of heterogeneity (tau².del) displayed tau² after the omitting of the 8^th study [44] was 0.46. The test statistics for heterogeneity (QE.del) was 26.61 if the 8^th study [44] is omitted, hat values was 0.09, and lastly the weights was 9.33. All these tests confirmed that study [44] is an outlying study but not influential, so it was not removed (S4B Fig).

For abundance estimate, the outlying studies, studentized residuals tests and leaving out each study revealed that the study number 3 [52] is an outlier with a z-value of 3.23, and summary proportion has changed from 5%; 95% CI: 2, 12% to 4%; 95% CI: 2, 7% after removing the 3^rd study [52] (S5A Fig). The rstudent test was 3.23, DFFITS was 1.33, cook.d test was 0.63, cov. r test was 0.45, tau2.del test was 0.3, QE.del test was 19.52, the hat and weight tests were 0.17 and 17.18 after removing the 3^rd study [52], respectively. Although these tests reveal that the 3^rd study [52] is an outlier and influential study (S5B Fig), we did not exclude it because its effect was minor on abundance summary proportion to be only from 5% to 4% when leaving out this study (Fig 1B and S5A Fig).

For frequency estimate, studentized residuals tests showed that the study number 7 [44] has an outlying z-value 2.62. Leaving out this study revealed some changes on the summary proportion from 8%; 95% CI: 5, 14% to 7%; 95% CI: 4, 11% (S6A Fig). The rstudent test was 2.62, DFFITS was 0.77, cook.d test was 0.44, cov. r test was 0.85, tau2.del test was 0.61, QE.del test was 64.98, the hat and weight tests were 0.11 and 10.52 after removing the 7^th study [44]. The results confirmed that study [44] is an outlying study but did not have an influential effect (S6B Fig).

Publication bias of studies

The publication bias was determined visually through the funnel plot, and statistically, by the unweighted Egger’s regression test. S7A Fig showed the funnel plot of diversity estimate of unculturable bacteria and Egger’s regression test was z = -1.2905, and P = 0.1969, which revealed non-significant publication bias. For the abundance, Egger’s regression test was z = -1.92, and P = 0.06, which showed no significant publication bias (S7B Fig). On the other hand, funnel plot and Egger’s regression test showed a significant publication bias for the frequency estimate of unculturable bacteria (z = -2.17, and P = 0.03) (S7C Fig).

Outcome measures: Secondary outcome measures: Abundance and frequency of individual unculturable bacteria in periapical abscess

In periapical abscess, the abundance and frequency of unculturable bacteria were determined (S4 Table). The secondary outcomes were measured for 35 unculturable bacteria using three variables, the abundance and frequency as parametric variables and the sequence techniques as non-parametric variable (Table 3). PCA was performed, where the abundance and frequency were represented as active variables, while the sequence techniques as a supplementary-categorical variable. PCA for individuals (unculturable bacteria) and for variables have been calculated and visualized using biplot (Fig 3). The variance percentages were 60.04 and 39.96 for PC1 and PC2, respectively (Fig 3). Regarding the PCA for the individual unculturable bacteria, the acute angle between the abundance and frequency variables and the Pearson’s correlation between the abundance and frequency revealed a positive but non-significant correlation (0.2, P = 0.3) (Fig 3 and S8 Fig).

Table 3. Secondary outcome measures of individual unculturable bacteria with active abundance and frequency, and supplementary sequence technique variables.

Symbol number of bacteria	Studies of acute periapical abscess	Unculturable bacteria	Abundance%	Frequency %	Sequence techniques
1	Flynn et al. (2012) [46] (Abundance/Frequency)	Leptotrichia sp. AM420283	0.26	11.11	Sequence
2		Leptotrichia [G-1] sp. Oral Taxon 220	0.26	11.11	Sequence
3		Peptostreptococcaceae [G-4] sp. Oral Taxon 103	0.51	22.22	Sequence
4		Synergistes[G-3] sp. Oral Taxon 360	0.26	11.11	Sequence
5		Prevotella sp. Oral Taxon 315	0.26	11.11	Sequence
6	Sakamoto et al. (2006) [52] (Abundance/Frequency)	Bacteroidales oral clone MCE7_164/MCE3_262/MB4_G15	1.08	14	Hybridization
7		Prevotella sp. E9_42/Preqotella sp. oral clone PUS9.180	2.15	43	Hybridization
8		Uncultured Eubacterium E1-K13	1.08	14	Hybridization
9		Lachnospiraceae oral clone 55A-34	1.08	57	Hybridization
10		Lachnospiraceae oral clone MCE7-60	1.08	43	Hybridization
11		Peptostreptococcus sp. oral clone CK035	11.8	29	Hybridization
12		Selenomonas sp. oral clone 55A-7	1.08	29	Hybridization
13		Bacterium MDA2477/Bacterium MDA2477-like oral clone 51A-9	5.38	14	Hybridization
14		Pseudomonas sp. LCY11	1.08	14	Hybridization
15	Sakamoto et al. (2009) [44] (Abundance/Frequency)	Treponema sp. oral taxon IV:18:C9	0.70	33.3	Sequence
16		Treponema clone 142–10	0.35	16.7	Sequence
17		Treponema clone 142–21	0.70	16.7	Sequence
18		Treponema clone 142–82	0.35	16.7	Sequence
19		Treponema clone 18f-1	0.70	16.7	Sequence
20		Treponema clone 18f-6	0.35	16.7	Sequence
21		Treponema clone 18f-7	0.35	16.7	Sequence
22		Treponema clone 18f-22	0.35	16.7	Sequence
23		Treponema clone 18f-33	0.35	16.7	Sequence
24		Treponema clone 18f-35	0.35	16.7	Sequence
25		Treponema clone 18f-48	0.35	16.7	Sequence
26		Treponema clone 94A-72	0.35	16.7	Sequence
27		Treponema clone 94A-89	0.35	16.7	Sequence
28		Treponema clone 94A-92	0.35	16.7	Sequence
29		Treponema clone 94A-94	0.35	16.7	Sequence
30	Yang et al. (2010) [55] (Abundance/Frequency)	Bacteroidales genomosp. P4 oral clone MB2_G17	1.9	9.1	Sequence
31	Zhang et al. (2020) [56] (Abundance/Frequency)	Acinetobacter sp. Oral taxon 408	0.75	33	Sequence
32		Peptostreptococcaceae [XI][G-7] sp. oral taxon 081	0.56	11	Sequence
33	Jacinto et al. (2007) [60] (Abundance/Frequency)	Uncultured Staphylococcus sp. clone pGA 2	2.08	40	Hybridization
34		Uncultured rape rhizosphere bacterium wr0200	2.71	20	Hybridization
35		Uncultured bacterium clone aab38e07	1.46	20	Hybridization

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For better interpretation, bacteria were represented as numbers (Table 3). Biplot showed that bacterium 11 has the highest abundance and frequency followed by 9, 7, and 33. The other bacteria including 13, 10, 34, 15, 12, and 31 represented the second highest abundances in a descending order, while bacteria 10, 15, 13, 12, 31, and 34 considered as the second highest frequency. Conversely, 1, 2, and 5 showed the lowest abundance and with similar values. Bacteria 4, and 32 showed the second lowest abundance, while bacteria 16, 18, 21, 22, 23, 24, 25, 26, 27, 28, and 29 were the third lowest abundance. On the other hand, the frequency of bacteria 1, 2, and 5 were the lowest, while bacteria 4, and 32 were the second lowest frequency and bacterium 30 was the third. Bacteria 16, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29, 6, 8, and 14 showed the fourth lowest frequency. The other bacteria including 3, 17, 19, 20, and 35 were nearly the same average for both abundance and frequency (Fig 3).

The supplementary variable, sequence techniques, was employed for grouping the bacteria into two major groups, “sequence” or “hybridization” using confidence ellipse (Fig 4). The confidence ellipse of hybridization group was 1, -0.01, while the confidence ellipse of sequence group was -0.5, 0.01, indicating that the hybridization technique is more prominent.

Discussion

To the best of our knowledge, this is the first systematic review and meta-analysis on unculturable bacteria in, periapical abscess, periapical granulomas or cysts. In our study, the outcomes of the included studies on the identification of unculturable bacteria generally provided information about the diversity, abundance, and frequency of the bacterial phylotypes. Molecular identification have shown that the bacterial diversity in most environments is strongly underestimated in culture-based techniques [17, 61] and 34% of the identified bacterial taxa are uncultivated [62], indicating that these unrecognized bacterial species present in the periapical lesions may participate in persistent apical periodontitis [62], periapical abscess [63] or the pathogenesis of other oral diseases [17, 18, 64]. We noted that the diversity score of unculturable bacteria in the acute periapical abscess was 13%. This figure is different from previous studies where the diversity percentage of unculturable bacteria in periapical abscess was 24% [46] while in another study it was 55% [13]. On the other hand, a low diversity score (2%) was noted in one study where the lesion was simply defined as a ‘dental abscess’ without referring to the acute/chronic nature of the lesion [59]. This discrepancy in the diversity score could depend on the country where the experiments were conducted, as for instance, we noted a significant higher diversity score in Brazil when compared to other countries (P = 0.05) (S1B Fig). In addition, it has been reported previously that geographical locations have an impact on the bacterial community [65, 66] and hence bacterial diversity. Furthermore, the molecular methods used in the studies may influence the sensitivity of unculturable phylotypes. For example, a study reported the diversity score of 55% when using T-RFLP (hybridization technique) [13], while another one reported this figure as 2% on using the proprietary Illumina sequencing method [59]. The others have noted that the increasingly advanced techniques used to detect the unculturable bacterial phylotypes may affect the diversity score due to newly included phylotypes of unculturable bacteria [67–70].

Bacterial abundance and their interactions are significantly correlated to the severity of acute periapical abscesses [51, 65]. However, in term of abundance, different teeth with acute apical periodontitis represent heterogeneous bacterial abundance [71, 72]. In the current meta-analysis, the abundance of unculturable bacterial phylotypes was computed to be 5%, concurring with a study noted a similar abundance of 6% [48]. On the other hand, a higher abundance of 26% was found [13], and a lower percentages of 1.5% [46] and 2% [55] were also reported. We also noted a significant difference the abundance of phylotypes depending on the country of study origin. For instance, studies performed in Brazil [44, 52, 60] showed a higher phylotype abundance when compared to those conducted in other countries [46, 48, 55] (S2B Fig). One possible explanation for this finding is the variation in analytical techniques used in different jurisdictions. Thus, more unculturable clones were detected in Brazilian studies using T-RFLP analysis [13, 48] in contrast to lower detection rates noted by others using ABI sequencing method [46, 55].

The frequency of bacterial species and its correlation to periapical lesions was studied for the first time in 2018 [51]. This study revealed a significant correlation between the frequency of specific bacterial taxa and the asymptomatic form of apical periodontitis [51]. The frequency of unculturable bacteria in periapical abscess was 8% in our study. This figure is intermediate with regards to previous data where both a low frequency of 1–2% [49, 51, 55, 59], and higher frequencies of 43% [44] and 33% [50] have been noted. Significant publication bias was detected during the identification of the frequency of unculturable bacteria in periapical abscess. Frequency is positively correlated to diversity [73] and the type of sequencing methods may have an impact on the frequency. According to our results, we found that the high frequency of unculturable bacteria in some studies were related to their high diversity in the same studies and vice versa (Table 2). Furthermore, the significant publication bias in frequency can be due to the different approach for sequencing. In our study, the lowest frequencies were detected in three studies [49, 55, 59] that used sequencing methods, while the highest frequencies were detected in another study [50] that used the hybridization technique. Although another highest frequency was detected by Sakamoto et al. [44], this study focused on a specific genus (Treponema), for that reason the frequency was high despite using a sequencing technique.

Regarding the identity of unculturable bacteria, their abundance could be positively correlated with the identification frequency. An exponential increase in the abundance and frequency of unculturable bacteria, Leptotrichia sp. AM420283 and Peptostreptococcaceae [G-4] sp. Oral Taxon 103, was reported [46]. Another study [52] also reported a high frequency of isolation of Lachnospiraceae oral clone 55A-34, Lachnospiraceae oral clone MCE7_60, and Selenomonas sp. oral clone 55A-7 to be 57%, 43%, and 29%, respectively. While the abundance was 1.8% for all the foregoing bacteria.

In the present study, the highest variable for abundance and frequency of unculturable bacteria was reported for Peptostreptococcus sp. oral clone CK035. Accordingly, a study found that its abundance was 11.8% in acute periapical abscess [52] compared to 15% in chronic periapical lesions [74]. The first detection of this bacterium was figured out in chronic apical periodontitis with 25% for its frequency [75].

Confidence ellipse of hybridization and sequence of individual unculturable bacteria clearly separated the bacteria into two groups according to their values of abundance and frequency. As shown in Fig 4, the hybridization techniques used, including T-RFLP, N-PCR, microbial microarrays, dHPLC, checkerboard DNA-DNA hybridization, or semi-quantitative reverse-capture checkerboard assay appear to be more powerful and sensitive tools for recording the total number of clones than the sequencing techniques including ABI, pyrosequencing, illumina sequencing, or thermos-sequenase sequencing. Comparing the T-RFLP with illumina sequencing method [76, 77] or with Ion Torrent PGM [78] revealed that T-RFLP is a powerful and highly reliable method for microbial screening. However, further studies are needed to confirm this result specifically if the aim of the study was targeted towards low or moderate abundant bacteria like unculturable phylotypes as shown in our study.

In this systematic review and meta-analysis, the unculturable bacteria in periapical lesions including periapical abscess, granuloma, and cyst were investigated. Only one article used a molecular method in detecting organisms in both the periapical granulomas and cysts, but only to the genus level of the isolates [79]. Some studies focused on a specific, culturable or unculturable phylotypes of a bacterial genus, while ignoring the in-depth screening of other bacterial genera or unculturable phylotypes [44]. The review indicates that there is a dearth of research on unculturable bacteria in periapical granuloma and radicular cysts. Therefore, more research is warranted on chronic periapical abscess, periapical granuloma, and radicular cyst with an emphasize on the diversity, abundance, and frequency of the unculturable bacteria. This will allow us to understand the role of these newly described organisms in the disease process and pave the way for more effective and efficacious therapeutic approaches not only for periapical lesions but also for other infections in general.

Conclusion

This review indicates that unculturable bacteria are moderately exist in the periapical abscess and such prevalence may significantly contribute to their pathogenesis. However, their role in the pathogenesis is yet to be determined since their prevalence and abundance may not necessarily reflect their activity. Some organisms may be dormant or inactive while others may be highly active. Nevertheless, we noted a positive correlation between the abundance and frequency of individual unculturable bacterium Peptostreptococcus sp. oral clone CK035 in periapical abscesses. In terms of methodology, hybridization techniques appear to be more reliable in detecting the abundance and frequency of individual unculturable bacteria in periapical lesions. Collectively, careful analysis of the available data regarding the uncultivable bacteria indicates their importance in the progression of periapical abscess and hence therapeutic strategy, while more experimental screening is still required.

Supporting information

S1 Checklist. PRISMA 2009 checklist.

(DOC)

Click here for additional data file.^{(63.5KB, doc)}

S1 Table. Literature search strategy and terms for the prevalence of unculturable bacteria in periapical lesions.

(PDF)

Click here for additional data file.^{(40.5KB, pdf)}

S2 Table. Checklist of critical appraisal of Joanna Briggs Institute for analytical cross sectional studies.

(PDF)

Click here for additional data file.^{(171.1KB, pdf)}

S3 Table. Bias assessment of included studies using Joanna Briggs Institute for analytical cross sectional studies.

(PDF)

Click here for additional data file.^{(70.3KB, pdf)}

S4 Table. Extracted results from each study of individual bacteria in periapical abscess.

(PDF)

Click here for additional data file.^{(101.7KB, pdf)}

S1 Fig. Scatter plot depicting the moderators’ effect on summary proportion of diversity of unculturable bacteria in periapical abscess.

(A) Lesion moderator. (B) Country moderator. (C) Sequence technique moderator.

(TIF)

Click here for additional data file.^{(284.5KB, tif)}

S2 Fig. Scatter plot depicting the moderators’ effect on summary proportion of abundance of unculturable bacteria in periapical abscess.

(A) Lesion moderator. (B) Country moderator. (C) Sequence technique moderator.

(TIF)

Click here for additional data file.^{(258.7KB, tif)}

S3 Fig. Scatter plot depicting the moderators’ effect on summary proportion of frequency of unculturable bacteria in periapical abscess.

(A) Lesion moderator. (B) Country moderator. (C) Sequence technique moderator.

(TIF)

Click here for additional data file.^{(230.5KB, tif)}

S4 Fig. Results for outlying studies of diversity of unculturable bacteria in periapical abscess.

(A) Forest plot depicting summary proportion after leaving out each study. (B) Influential analysis plot of diversity of unculturable bacteria in thirteen studies of periapical abscess.

(TIF)

Click here for additional data file.^{(1,008.6KB, tif)}

S5 Fig. Results for outlying studies of abundance of unculturable bacteria in periapical abscess.

(A) Forest plot depicting summary proportion after leaving out each study. (B) Influential analysis plot of abundance of unculturable bacteria in six studies of periapical abscess.

(TIF)

Click here for additional data file.^{(760.9KB, tif)}

S6 Fig. Results for outlying studies of frequency of unculturable bacteria in periapical abscess.

(A) Forest plot depicting summary proportion after leaving out each study. (B) Influential analysis plot of frequency of unculturable bacteria in eleven studies of periapical abscess.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S7 Fig. Funnel plot of publication bias of unculturable bacteria in periapical abscess.

(A) Diversity. (B) Abundance. (C) Frequency.

(TIF)

Click here for additional data file.^{(182.7KB, tif)}

S8 Fig. Scatter plot of abundance and frequency correlation for individual unculturable bacteria.

(TIF)

Click here for additional data file.^{(284.8KB, tif)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

References

1.Peters E, Lau M. Histopathologic examination to confirm diagnosis of periapical lesions: A review. J Can Dent Assoc. 2003;69(9):598–600. . [PubMed] [Google Scholar]
2.Cotti E, Campisi G. Advanced radiographic techniques for the detection of lesions in bone. Endod Topics. 2004;7(1):52–72. 10.1111/j.1601-1546.2004.00064.x. [DOI] [Google Scholar]
3.Ramanpreet B, Simarpreet S, Rajat B, Amandeep B, Shruti G. Histopathological insight into periapical lesions: An institutional study from Punjab. Int J Oral Maxillofac Pathol. 2012;3(3):2–7. [Google Scholar]
4.Safi L, Adl A, Azar MR, Akbary R. A twenty-year survey of pathologic reports of two common types of chronic periapical lesions in Shiraz Dental School. J Dent Res Dent Clin Dent Prospects. 2008;2(2):63–70. doi: 10.5681/joddd.2008.013 . [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Soares JA, Leonardo MR, da Silva LA, Tanomaru Filho M, Ito IY. Histomicrobiologic aspects of the root canal system and periapical lesions in dogs’ teeth after rotary instrumentation and intracanal dressing with Ca(OH)2 pastes. J Appl Oral Sci. 2006;14(5):355–64. doi: 10.1590/s1678-77572006000500011 . [DOI] [PMC free article] [PubMed] [Google Scholar]
6.World Health Organization. Application of the international classification of diseases to dentistry and stomatology: ICD-DA, 3rd ed, Geneva. 1995. Available from: https://apps.who.int/iris/handle/10665/40919. [Google Scholar]
7.Nair PN. Apical periodontitis: A dynamic encounter between root canal infection and host response. Periodontol 2000. 1997;13:121–48. doi: 10.1111/j.1600-0757.1997.tb00098.x . [DOI] [PubMed] [Google Scholar]
8.American Association of Endodontics. Glossary of endodontic terms. 10th ed. 2020. Available from: https://www.aae.org/specialty/download/glossary-of-endodontic-terms/. [Google Scholar]
9.Gutmann JL, Baumgartner JC, Gluskin AH, Hartwell GR, Walton RE. Identify and define all diagnostic terms for periapical/periradicular health and disease states. J Endod. 2009;35(12):1658–74. doi: 10.1016/j.joen.2009.09.028 . [DOI] [PubMed] [Google Scholar]
10.Shu M, Wong L, Miller JH, Sissons CH. Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol. 2000;45(1):27–40. doi: 10.1016/s0003-9969(99)00111-9 . [DOI] [PubMed] [Google Scholar]
11.Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med. 2004;15(6):348–81. doi: 10.1177/154411130401500604 . [DOI] [PubMed] [Google Scholar]
12.Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol. 1991;6(2):123–5. doi: 10.1111/j.1399-302x.1991.tb00464.x [DOI] [PubMed] [Google Scholar]
13.Sakamoto M, Rôças IN, Siqueira JF, Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol. 2006;21(2):112–22. doi: 10.1111/j.1399-302X.2006.00270.x . [DOI] [PubMed] [Google Scholar]
14.Hugenholtz P, Pace NR. Identifying microbial diversity in the natural environment: A molecular phylogenetic approach. Trends Biotechnol. 1996;14(6):190–7. doi: 10.1016/0167-7799(96)10025-1 . [DOI] [PubMed] [Google Scholar]
15.Paster BJ, Dewhirst FE. Molecular microbial diagnosis. Periodontol 2000. 2009;51:38–44. doi: 10.1111/j.1600-0757.2009.00316.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, et al. The human oral microbiome. J Bacteriol. 2010;192(19):5002–17. doi: 10.1128/JB.00542-10 . [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol. 2001;183(12):3770–83. doi: 10.1128/JB.183.12.3770-3783.2001 . [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005;43(11):5721–32. doi: 10.1128/JCM.43.11.5721-5732.2005 . [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Teles R, Teles F, Frias-Lopez J, Paster B, Haffajee A. Lessons learned and unlearned in periodontal microbiology. Periodontol 2000. 2013;62(1):95–162. doi: 10.1111/prd.12010 . [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Vartoukian SR, Palmer RM, Wade WG. The division "Synergistes". Anaerobe. 2007;13(3–4):99–106. doi: 10.1016/j.anaerobe.2007.05.004 . [DOI] [PubMed] [Google Scholar]
21.Jumas-Bilak E, Carlier JP, Jean-Pierre H, Citron D, Bernard K, Damay A, et al. Jonquetella anthropi gen. nov., sp. nov., the first member of the candidate phylum ’Synergistetes’ isolated from man. Int J Syst Evol Microbiol. 2007;57(12):2743–8. doi: 10.1099/ijs.0.65213-0 . [DOI] [PubMed] [Google Scholar]
22.Vartoukian SR, Downes J, Palmer RM, Wade WG. Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol. 2013;63(2):458–63. doi: 10.1099/ijs.0.041038-0 . [DOI] [PubMed] [Google Scholar]
23.Downes J, Vartoukian SR, Dewhirst FE, Izard J, Chen T, Yu WH, et al. Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum ’Synergistetes’ isolated from the human oral cavity. Int J Syst Evol Microbiol. 2009;59(5):972–80. doi: 10.1099/ijs.0.000364-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Arank A, Syed SA, Kenney EB, Freter R. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl Microbiol. 1969;17(4):568–76. doi: 10.1128/am.17.4.568-576.1969 . [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Socransky SS, Gibbons RJ, Dale AC, Bortnick L, Rosenthal E, Macdonald JB. The microbiota of the gingival crevice area of man. I. Total microscopic and viable counts and counts of specific organisms. Arch Oral Biol. 1963;8:275–80. doi: 10.1016/0003-9969(63)90019-0 . [DOI] [PubMed] [Google Scholar]
26.Dymock D, Weightman AJ, Scully C, Wade WG. Molecular analysis of microflora associated with dentoalveolar abscesses. J Clin Microbiol. 1996;34(3):537–42. doi: 10.1128/jcm.34.3.537-542.1996 . [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Lewis K, Epstein S, D’Onofrio A, Ling LL. Uncultured microorganisms as a source of secondary metabolites. J Antibiot. 2010;63(8):468–76. doi: 10.1038/ja.2010.87 . [DOI] [PubMed] [Google Scholar]
28.Munn Z, Stern C, Aromataris E, Lockwood C, Jordan Z. What kind of systematic review should I conduct? A proposed typology and guidance for systematic reviewers in the medical and health sciences. BMC Med Res Methodol. 2018;18(1):5. doi: 10.1186/s12874-017-0468-4 . [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Munn Z, Moola S, Lisy K, Riitano D, Murphy F. Claustrophobia in magnetic resonance imaging: A systematic review and meta-analysis. Radiography. 2015;21(2):e59–e63. 10.1016/j.radi.2014.12.004. [DOI] [Google Scholar]
30.Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4(1):1. doi: 10.1186/2046-4053-4-1 . [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Moola S, Munn Z, Tufanaru C, Aromataris E, Sears K, Sfetcu R, et al. Chapter 7: Systematic reviews of etiology and risk. In: Aromataris E, Munn Z (editors). Joanna Briggs Institute Reviewer’s Manual. The Joanna Briggs Institute, 2017. Available from: https://wiki.jbi.global/display/MANUAL/Chapter+7%3A+Systematic+reviews+of+etiology+and+risk. [Google Scholar]
32.Expanded Human Oral Microbiome Database. eHOMD. Available from: http://www.homd.org/.
33.The NCBI Taxonomy Homepage. Available from: https://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/.
34.Culture Collection University of Gothenburg. CCUG. Available from: https://ccug.se/.
35.Wang N. How to conduct a meta-analysis of proportions in R: A comprehensive tutorial: Preprint; 2018. Available from: https://www.researchgate.net/publication/325486099_How_to_Conduct_a_Meta-Analysis_of_Proportions_in_R_A_Comprehensive_Tutorial. [Google Scholar]
36.Barendregt JJ, Doi SA, Lee YY, Norman RE, Vos T. Meta-analysis of prevalence. J Epidemiol Community Health. 2013;67(11):974–8. doi: 10.1136/jech-2013-203104 . [DOI] [PubMed] [Google Scholar]
37.Wang D, Liu Q. Learning to draw samples: With application to amortized MLE for generative adversarial learning. A conference paper at ICLR. 2016. Available from: https://openreview.net/pdf/d1cab3a9a4271a9003c492bb98269ec3e66a8fb4.pdf. [Google Scholar]
38.Card NA. Applied meta-analysis for social science research. New York, USA: Guilford Press. 2012. Available from: https://books.google.ae/books?id=hSYVGMrJgpkC&printsec=frontcover#v=onepage&q&f=false. [Google Scholar]
39.DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials. 1986;7(3):177–88. doi: 10.1016/0197-2456(86)90046-2 . [DOI] [PubMed] [Google Scholar]
40.Higgins JPT, Green S. Cochrane handbook for systematic reviews of interventions. London, United Kingdom: Cochrane Collaboration. 2011. Available from: https://handbook-5-1.cochrane.org/. [Google Scholar]
41.Deeks JJ, Higgins JPT, Altman DG. Chapter 9: Analysing data and undertaking meta-anlysis. In: Higgins JPT, Green S (editors). Cochrane handbook for systematic reviews of interventions. 2011. Available from: https://handbook-5-1.cochrane.org/index.htm#chapter_9/9_analysing_data_and_undertaking_meta_analyses.htm. [Google Scholar]
42.Huedo-Medina TB, Sánchez-Meca J, Marín-Martínez F, Botella J. Assessing heterogeneity in meta-analysis: Q statistic or I2 index? Psychol Methods. 2006;11(2):193–206. doi: 10.1037/1082-989X.11.2.193 . [DOI] [PubMed] [Google Scholar]
43.Viechtbauer W, Cheung MW-L. Outlier and influence diagnostics for meta-analysis. Res Synth Methods. 2010;1(2):112–25. doi: 10.1002/jrsm.11 . [DOI] [PubMed] [Google Scholar]
44.Sakamoto M, Siqueira JF Jr., Rocas IN, Benno Y. Diversity of spirochetes in endodontic infections. J Clin Microbiol. 2009;47(5):1352–7. doi: 10.1128/JCM.02016-08 . [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Statistical tools for high-throughput data analysis. (STHDA). Quick principal component analysis data visualization—R software and data mining. Available from: http://www.sthda.com/english/.
46.Flynn TR, Paster BJ, Stokes LN, Susarla SM, Shanti RM. Molecular methods for diagnosis of odontogenic infections. J Oral Maxillofac Surg. 2012;70(8):1854–9. doi: 10.1016/j.joms.2011.09.009 . [DOI] [PubMed] [Google Scholar]
47.George N, Flamiatos E, Kawasaki K, Kim N, Carriere C, Phan B, et al. Oral microbiota species in acute apical endodontic abscesses. J Oral Microbiol. 2016;8:30989. doi: 10.3402/jom.v8.30989 . [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Riggio MP, Aga H, Murray CA, Jackson MS, Lennon A, Hammersley N, et al. Identification of bacteria associated with spreading odontogenic infections by 16S rRNA gene sequencing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;103(5):610–7. doi: 10.1016/j.tripleo.2006.08.009 . [DOI] [PubMed] [Google Scholar]
49.Rôças IN, Baumgartner JC, Xia T, Siqueira JF. Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations. J Endod. 2006;32(12):1135–8. doi: 10.1016/j.joen.2006.05.001 . [DOI] [PubMed] [Google Scholar]
50.Rôças IN, Siqueira JF. Prevalence of new candidate pathogens Prevotella baroniae, Prevotella multisaccharivorax and as-yet-uncultivated Bacteroidetes clone X083 in primary endodontic infections. J Endod. 2009;35(10):1359–62. doi: 10.1016/j.joen.2009.05.033 . [DOI] [PubMed] [Google Scholar]
51.Rôças IN, Siqueira JF. Frequency and levels of candidate endodontic pathogens in acute apical abscesses as compared to asymptomatic apical periodontitis. PLoS One. 2018;13(1). doi: 10.1371/journal.pone.0190469 . 10.1371/journal.pone.0190469. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Sakamoto M, Rocas IN, Siqueira JF Jr., Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol. 2006;21(2):112–22. doi: 10.1111/j.1399-302X.2006.00270.x . [DOI] [PubMed] [Google Scholar]
53.Siqueira JF, Rôças IN. Molecular detection and identification of Synergistes phylotypes in primary endodontic infections. Oral Dis. 2007;13(4):398–401. doi: 10.1111/j.1601-0825.2006.01301.x . [DOI] [PubMed] [Google Scholar]
54.Siqueira JF Jr., Rocas IN. The microbiota of acute apical abscesses. J Dent Res. 2009;88(1):61–5. doi: 10.1177/0022034508328124 . [DOI] [PubMed] [Google Scholar]
55.Yang Q-B, Fan L-N, Shi Q. Polymerase chain reaction-denaturing gradient gel electrophoresis, cloning, and sequence analysis of bacteria associated with acute periapical abscesses in children. J Endod. 2010;36(2):218–23. doi: 10.1016/j.joen.2009.11.001 . [DOI] [PubMed] [Google Scholar]
56.Zhang W, Chen Y, Shi Q, Hou B, Yang Q. Identification of bacteria associated with periapical abscesses of primary teeth by sequence analysis of 16S rDNA clone libraries. Microb Pathog. 2020;141:103954. doi: 10.1016/j.micpath.2019.103954 . [DOI] [PubMed] [Google Scholar]
57.Yun KH, Lee H-S, Nam OH, Moon CY, Lee J-H, Choi SC. Analysis of bacterial community profiles of endodontically infected primary teeth using pyrosequencing. Int J Paediatr Dent. 2017;27(1):56–65. doi: 10.1111/ipd.12226 . [DOI] [PubMed] [Google Scholar]
58.Rolph HJ, Lennon A, Riggio MP, Saunders WP, MacKenzie D, Coldero L, et al. Molecular identification of microorganisms from endodontic infections. J Clin Microbiol. 2001;39(9):3282–9. doi: 10.1128/JCM.39.9.3282-3289.2001 . [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Vengerfeldt V, Špilka K, Saag M, Preem J-K, Oopkaup K, Truu J, et al. Highly diverse microbiota in dental root canals in cases of apical periodontitis (data of illumina sequencing). J Endod. 2014;40(11):1778–83. doi: 10.1016/j.joen.2014.06.017 . [DOI] [PubMed] [Google Scholar]
60.Jacinto RC, Gomes BP, Desai M, Rajendram D, Shah HN. Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography. Oral Microbiol Immunol. 2007;22(6):403–10. doi: 10.1111/j.1399-302X.2007.00378.x . [DOI] [PubMed] [Google Scholar]
61.Hugenholtz P, Pitulle C, Hershberger KL, Pace NR. Novel division level bacterial diversity in a Yellowstone hot spring. J Bacteriol. 1998;180(2):366–76. doi: 10.1128/JB.180.2.366-376.1998 . [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Handal T, Caugant DA, Olsen I, Sunde PT. Bacterial diversity in persistent periapical lesions on root-filled teeth. J Oral Microbiol. 2009;1. doi: 10.3402/jom.v1i0.1946 . [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Siqueira JF Jr., Rôças IN. As-yet-uncultivated oral bacteria: Breadth and association with oral and extra-oral diseases. J Oral Microbiol. 2013;5. doi: 10.3402/jom.v5i0.21077 . [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Pace N.R. SDA, Lane D.J., Olsen G.J. The analysis of natural microbial populations by ribosomal RNA sequences. In: Marshall KC (editors). Advances in microbial ecology. Springer, Boston. 1986. 10.1007/978-1-4757-0611-6_1. [DOI] [Google Scholar]
65.Siqueira JF Jr., Rôças IN. Microbiology and treatment of acute apical abscesses. Clin Microbiol Rev. 2013;26(2):255–73. doi: 10.1128/CMR.00082-12 . [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Gupta VK, Paul S, Dutta C. Geography, ethnicity or subsistence-specific variations in human microbiome composition and diversity. Front Microbiol. 2017;8(1162). doi: 10.3389/fmicb.2017.01162 . [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Bodor A, Bounedjoum N, Vincze GE, Erdeiné Kis Á, Laczi K, Bende G, et al. Challenges of unculturable bacteria: Environmental perspectives. Rev Environ Sci Biotechnol. 2020;19(1):1–22. 10.1007/s11157-020-09522-4. [DOI] [Google Scholar]
68.Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature. 2016;533(7604):543–6. doi: 10.1038/nature17645 . [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Puspita ID, Kamagata Y, Tanaka M, Asano K, Nakatsu CH. Are uncultivated bacteria really uncultivable? Microbes Environ. 2012;27(4):356–66. doi: 10.1264/jsme2.me12092 . [DOI] [PMC free article] [PubMed] [Google Scholar]
70.Harper-Owen R, Dymock D, Booth V, Weightman AJ, Wade WG. Detection of unculturable bacteria in periodontal health and disease by PCR. J Clin Microbiol. 1999;37(5):1469–73. doi: 10.1128/JCM.37.5.1469-1473.1999 . [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Siqueira JF Jr., Rôças IN, Rosado AS. Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by denaturing gradient gel electrophoresis fingerprinting approach. Oral Microbiol Immunol. 2004;19(6):363–70. doi: 10.1111/j.1399-302x.2004.00170.x [DOI] [PubMed] [Google Scholar]
72.Machado de Oliveira JC, Siqueira JF Jr., Rôças IN, Baumgartner JC, Xia T, Peixoto RS, et al. Bacterial community profiles of endodontic abscesses from Brazilian and USA subjects as compared by denaturing gradient gel electrophoresis analysis. Oral Microbiol Immunol. 2007;22(1):14–8. doi: 10.1111/j.1399-302X.2007.00311.x [DOI] [PubMed] [Google Scholar]
73.Borşan S-D, Ionică AM, Galon C, Toma-Naic A, Peştean C, Sándor AD, et al. High diversity, prevalence, and co-infection rates of tick-borne pathogens in ticks and wildlife hosts in an urban area in Romania. Front Microbiol. 2021;12(351). 10.3389/fmicb.2021.645002. . [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Saito D, de Toledo Leonardo R, Rodrigues JLM, Tsai SM, Höfling JF, Gonçalves RB. Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries. J Med Microbiol. 2006;55(1):101–7. doi: 10.1099/jmm.0.46212-0 [DOI] [PubMed] [Google Scholar]
75.Rôças IN, Siqueira JF Jr., Root canal microbiota of teeth with chronic apical periodontitis. J Clin Microbiol. 2008;46(11):3599–606. doi: 10.1128/JCM.00431-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
76.De Vrieze J, Ijaz UZ, Saunders AM, Theuerl S. Terminal restriction fragment length polymorphism is an "old school" reliable technique for swift microbial community screening in anaerobic digestion. Sci Rep. 2018;8(1):16818. doi: 10.1038/s41598-018-34921-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Lindström S, Rowe O, Timonen S, Sundström L, Johansson H. Trends in bacterial and fungal communities in ant nests observed with terminal-restriction fragment length polymorphism (T-RFLP) and next generation sequencing (NGS) techniques-validity and compatibility in ecological studies. PeerJ. 2018;6:e5289. 10.7717/peerj.5289. . [DOI] [PMC free article] [PubMed] [Google Scholar]
78.de la Fuente G, Belanche A, Girwood SE, Pinloche E, Wilkinson T, Newbold CJ. Pros and cons of ion-torrent next generation sequencing versus terminal restriction fragment length polymorphism T-RFLP for studying the rumen bacterial community. PLoS One. 2014;9(7):e101435. 10.1371/journal.pone.0101435 . [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Mussano F, Ferrocino I, Gavrilova N, Genova T, Dell’Acqua A, Cocolin L, et al. Apical periodontitis: Preliminary assessment of microbiota by 16S rRNA high throughput amplicon target sequencing. BMC oral health. 2018;18(1):55. 10.1186/s12903-018-0520-8. . [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0255485.r001

Decision Letter 0

Andrej M Kielbassa

21 May 2021

PONE-D-20-39104

Prevalence of unculturable bacteria in the dentoalveolar abscess and related periapical lesions: A systematic review and meta-analysis

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #1: The study brings new information and its objective can be relevant. Although a lot of work has been done, this submission has concerns that are listed below:

- Abstract:

Line 22: “To assess the prevalence of unculturable bacteria in dentoalveolar abscess (DAA) and related periapical lesions.” What lesions?

Line 26: Which month of 2020 was the search conducted?

Methods: Which were the eligibility criteria? What type of studies was included? Were the risk of bias, meta-regression and publication bias assessed? How was the effect measured? What type of statistical analysis was performed?

I suggest rewriting the results as follows: Approximately 13% (%95 CI: 7-23%) of the cumulative...

On the results the author affirms “Country moderator significantly ( P -value = 0.05) affects the diversity summary proportion.”, but it was not mentioned on the methods.

The conclusion is not in accordance with the objective of the study.

I suggest rewriting the conclusion as follows: “The moderate, yet significant prevalence of unculturable bacteria in such lesions, SUGGESTS that they are likely to play,...”

- Introduction

Authors should provide more information about the importance of the knowledge on the prevalence of unculturable bacteria in the dentoalveolar abscess and related periapical lesions to improve the background and also enrich the discussion of the results.

Line 51: “initiate the periapical pathology that eventually results in a DAA and its sequelae” What sequelae?

I think the authors should discuss DAA and the related periapical lesions.

Why did the author choose the Nair’s classification?

Line 60: “Periapical granuloma is a misnomer as it does not contain granulomatous tissue”. I suggest that the authors include new and current references to confirm this statement.

Line 62: Authors could include referenced to this statement.

Authors could use a single terminology to DAA, apical abscess and periapical abscess throughout the study.

I think it is necessary to clarify why it would be important to compile data on this topic.

According to PRISMA checklist “Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS).” Therefore, authors could include the terms “DAA and related periapical lesions” on the objective of the study (line 90). If possible, specify which “related periapical lesions”.

- Materials and Methods

The study could be rewritten and qualified according to the PRISMA Preferred reporting items for systematic reviews and meta‐analyses, available at https://onlinelibrary.wiley.com/doi/full/10.1111/iej.13118

Lines 96 and 98 – Fig 1 should be moved to the results section.

Line 105 – Authors could review the search period. In the abstract, the authors affirm that “databases were systematically searched from 1990 to 2020”. In the methodology they affirm that “the search was limited to human studies published 105 over a decade, from 1990/01/01 to 2019/12/31”. The reference number 40 corresponds to a study published in April 2020.

Have reviewers been previously calibrated to ensure inter‐rater reliability for the search and for the risk of bias assessment?

The exclusion criteria must be selected previously to conducting the study, and not according to the results. This would characterize a selection bias. The criteria presentation leaves doubt whether the criteria were previously established or not. In addition, the number of articles excluded according to each criterion must be presented in the results, not in the methods.

Line 123, 129, 131 and 132: Fig 1, Table 1, 2, 3 and S2 should be moved to the results section.

S2 Table and Table 3 are redundant.

Line 163: in this section, please include the customized checklist used to Risk of bias assessment (S3 Table).

Line 169: “Finally, all studies with a minimum score of 6/8 for 170 were included in the review.” Was it a criteria of inclusion or the result of the Risk of bias assessment?

Line 195: Please specify what is PCA in the first time you use this term on the text.

Line 201: Authors do not mention the duplicates removal. In fact, according to the flowchart, 14,819 studies were identified. 39 were duplicates and, therefore, removed.

Authors identified more than 14,000 potentially relevant articles during the electronic search, but at the end of the process only 16 articles were included.

A more refined search strategy should have been used for a more accurate result.

Why did the authors include only 13 studies in the meta-analysis? Why were 3 studies excluded?

Line 201 and 209: “dentoalveolar abscess (DAA) and related periapical lesions.” What lesions?

Line 232: S3 table should be moved to the methods section.

Lines 240 to 245: wouldn't this paragraph be a DISCUSSION of the results?

Why did the authors include acute abscess AND abscess? Why were the studies that evaluated Chronic DAA. not included in the meta-analysis? Couldn't it be a bias?

Line 274, 277, 280: The author refers to figure 2A, 2B and 2C, but this figure does not specify which is ABC.

Meta regression: Is there any limitation by using this analysis? Because there are few studies that evaluated unculturable bacteria in “periapical abscess” and “acute DAA” (type of lesion). Data would not show the real effect of the lesion type on prevalence of unculturable bacteria. Besides that, could the sample collection procedure be a specific moderator?

Line 338 to 347: Wouldn't this paragraph be a METHODOLOGY?

Line 348 to 373: For the diversity of unculturable bacteria in periapical abscess, the study number 8 “is an outlying study but not influential, so it was not removed.” But, for abundance estimation, the “tests reveal that the 3rd study [33] is an influential study on the effect of summary proportion”. Why this study was not removed?

Discussion:

Since the number of bacteria is normally greater and more virulent in symptomatic and purulent infections than in chronic and asymptomatic infections, could the inclusion of studies that do not specify what type of ADA in the meta-analysis characterize a bias in the results?

Line 417: This was the diversity found for all abscesses.

Lines 418 and 419: Authors could rewrite this sentence for better understanding.

Line 424 AND 439: Are authors suggesting any explanation for the country's interference? Is the collection method important?

More information regarding the importance of the knowledge on the prevalence of unculturable bacteria in dentoalveolar abscess and related periapical lesions should be included in this section. The authors could improve the discussion. What is the importance of knowing the frequency, abundance and diversity of these bacteria? What are the suggestions for new studies? Do the authors suggest an explanation for the significant publication bias for the frequency estimate of unculturable bacteria and for the outlier? Regarding chronic ADA, can different results be found?

What is the contribution of this study? There could be a last paragraph summarizing the findings.

Conclusions:

Authors state that the prevalence of unculturable bacteria is ABUNDANT and at the conclusion of the abstract they state that it is MODERATE. Please, review these statements.

The conclusion could be more objective and in accordance with the aim of the study.

Reviewer #2: This manuscript discusses the role unculturable bacteria in the dentoalveolar abscess and how it effects on the periapical lesions in depth through systematic review and Meta-analysis as study design. The meta analysis for this study is very good. The number of included studies in this study is expected due to new idea and the number of studies related to the topic is very small and future studies are required in this field. I think this paper eligible to publish in peer review journal like this journal.

Reviewer #3: The authors should revise the nomenclature of periapical lesions and acute apical abscess and change them in entire manuscript according to the guidelines of the AAE (diagnosis).

Dentoalveolar abscess may involve lesions of not only endodontic origin

Two studies (Jacinto et al. 2007 and Vengerfeldt et al. 2014) included in the review did not have their data clarified regarding the diagnosis. Have the authors contacted the corresponding authors to clarify the diagnosis gap? It seems that in the Jacinto et al (2007) the paper point samples were collected from root canals of symptomatic teeth (i.e. with acute apical abscess).

Great number of the studies included in this review were from Brazil. The authors highlighted a high discrepancy in the results found in these studies. Is there any hypothesis for this diversity of uncultivable bacteria found in studies carried out in the Brazilian population?

Please clarify the origin of the sample collected. For instance, Jacinto el al collected samples from the root canals of symptomatic teeth. On the other hand, Siqueira and Rocas (2007) and many other authors collected samples from the purulent collection.

Another point that needs to clarify is the presence of abscess in primary or in adult dentition. For example, Zhang et al studied the presence of abscess in primary teeth (children).

It would be interesting to explain the differences between uncultivable, unculturable and uncultivated bacteria.

Reviewer #4: Review Comments to the Author:

Could you please see my attached review, I have added all my comments to the Author in an attachment.

Reviewer #5: Abstract

- Revise for uniformity, and stick to the Journal's Guidelines. "( P -value = 0.05)" must read "(p = 0.050)". Revise thoroughly.

- With your conclusions, please stick exclusively to your aims. See: "To assess the prevalence of uncultivable bacteria in dentoalveolar abscess (DAA) and related periapical lesions." Now, you conclude that "unculturable bacteria in such lesions (...) are likely to play, a yet unknown, critical role in the pathogenesis and progression of the disease". This is something different, and you surely will agree.

- "More search remains to be done on the communal behaviour, virulence, and pathogenicity in this ecosystem." This is a common phrase only, but not an answer to your research question.

- Please stick to the word maximum allowed here, to provide complete information.

Intro

- With respect to the research question, this section is considered much too long. Please shorten significantly, and elaborate both aims and objectives more clearly.

- What about your null hypothesis?

Meths

- Why did you restrict your inclusion criteria to English?

Results

- This has been meticulously elaborated.

Disc

- Please do not use authors' names with your text (these will be acknowledged with your reference list). Instead, do focus on your main thoughts.

- This section is considered easily intelligible.

Concl

- Please see comments given above. With your Conclusions, please stick exclusively to your revised aims. Do not simply repeat your results here. Instead, provide a reasonable and generalizable extension of your outcome.

- See, for example, "Despite the remarkable diversity of uncultivable bacteria in these lesions, their role in the pathogenesis is yet to be determined. A positive correlation was shown between the abundance and frequency of individual uncultivable bacteria. Peptostreptococcus sp. oral clone CK035 showed the highest abundance and frequency. Hybridization techniques appear to be more reliable in detecting the abundance and frequency of individual unculturable bacteria in periapical lesions." All these aspects would seem right, and, thus, these thoughts might be copied & pasted to your Disc section. However, these are not considered conclusions deducible from your study.

Refs

- Full of minor and major shortcomings. Please revise for uniform formatting.

- Again, stick to the Journal's guidelines, and consult some recently published Plos One papers.

- doi numbers are missing.

**********

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Reviewer #1: No

Reviewer #2: Yes: Faisal Turki Alghamdi

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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PLoS One. 2021 Aug 5;16(8):e0255485. doi: 10.1371/journal.pone.0255485.r002

Author response to Decision Letter 0

1 Jul 2021

30th June 2021

Editor-in-Chief

PLOS One

Dear Editor,

Re: Manuscript entitled Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

Thank you for the five reviewer reports on the above manuscript. We have now addressed virtually all the points raised by the reviewers as described in the point-to-point rebuttal in the attached file. Some of the reasons why we preferred the described methodology as well as the narrative edits are explained therein.

We acknowledge that the comments of the reviewers have significantly improved the manuscript and hope that it will now be acceptable for publication in PLOS One.

Thank you again.

Best regards,

Rifat Hamoudi

On behalf of all authors

Point-To-Point Response

We thank the reviewers for the valuable and insightful comments. Responses to each of the comments raised are outlined below in addition to other minor changes that have been done throughout the manuscript.

Reviewer #1

- Abstract

Comment #1: Line 22: “To assess the prevalence of unculturable bacteria in dentoalveolar abscess (DAA) and related periapical lesions.” What lesions?

Response: Thank you for this comment. Taking into consideration your comment #13 in the introduction, the text was modified on lines 22 to 23.

Comment #2: Line 26: Which month of 2020 was the search conducted?

Response: The text was modified on lines 25 to 26.

More clarification for the reviewer: At first, we conducted a Boolean search for published research from 1990/01/01 to 2019/12/31. Subsequent publications, if any, up to the date of the manuscript submission was also alerted by an activated research engine, and an additional manual search which was conducted for publications between 2020/01/01 to 2020/05/31 (the period of statistical analysis and drafting the manuscript).

Comment #3: Methods: Which were the eligibility criteria? What type of studies was included? Were the risk of bias, meta-regression and publication bias assessed? How was the effect measured? What type of statistical analysis was performed?

Response: Thank you for this comment. All these points were modified and clarified further in the abstract, except for eligibility criteria. Given the limited word count of 300 words and based on previous systematic review and meta-analysis article formats used in PLOS One journal, we did not include this information in the abstract, but it is outlined extensively in the methods section. In the abstract, the text on lines 26 to 32 was modified.

Comment #4: I suggest rewriting the results as follows: Approximately 13% (%95 CI: 7-23%) of the cumulative…

Response: The text was modified on line 36.

Comment #5: On the results the author affirms “Country moderator significantly ( P -value = 0.05) affects the diversity summary proportion.”, but it was not mentioned on the methods.

Response: We thank the reviewer for this comment. According to your previous comment #3 in the abstract section, we mentioned the meta-regression and moderators on lines 27 to 29.

Comment #6: The conclusion is not in accordance with the objective of the study. I suggest rewriting the conclusion as follows: “The moderate, yet significant prevalence of unculturable bacteria in such lesions, SUGGESTS that they are likely to play,…”

Response: We thank the reviewer for this comment. According to this comment and reviewer #5/ comment #2, we have modified the conclusion on lines 46 to 49.

- Introduction

Comment #7: Authors should provide more information about the importance of the knowledge on the prevalence of unculturable bacteria in the dentoalveolar abscess and related periapical lesions to improve the background and also enrich the discussion of the results.

Response: Thank you for this comment. We have now added a paragraph to the introduction on lines 96 to 101.

Comment #8: Line 51: “initiate the periapical pathology that eventually results in a DAA and its sequelae” What sequelae?

Response: We thank the reviewer for this important point. Taking into consideration reviewer #3/comment #1. The text on lines 53 to 56 was modified.

Comment #9: I think the authors should discuss DAA and the related periapical lesions.

Response: We thank the reviewer for this comment, and we have also considered your comment #13 and reviewer #3/comment #1 about DAA term. On lines 52 to 56, the text was modified, and it briefly described the periapical abscess, radicular cyst, and periapical granuloma formation.

On lines 72 to 83, we have described the formation of periapical abscess.

We did not describe the formation of radicular cyst and periapical granuloma in any detail as they have not been included in the meta-analysis.

Comment #10: Why did the author choose the Nair’s classification?

Response: Thanks for this important question. According to this comment and reviewer #4/comment #2 we have modified the text on lines 57 to 71 and it is clearly now answering this comment.

Comment #11: Line 60: “Periapical granuloma is a misnomer as it does not contain granulomatous tissue”. I suggest that the authors include new and current references to confirm this statement.

Response: We have included another new reference Shah et al. (2017) [1] as they discussed the types of the diseases that contain granulomatous inflammation, and periapical granuloma was not included in this classification. However, according to reviewer #4/comment #2, we have deleted this sentence because we revised the classification paragraph on lines 57 to 71.

Comment #12: Line 62: Authors could include referenced to this statement.

Response: Thank you for this suggestion. We have now included an additional reference Gutmann et al (2009) [2] on line 73.

Comment #13: Authors could use a single terminology to DAA, apical abscess and periapical abscess throughout the study.

Response: We agree with the reviewer on this suggestion. In accordance with the reviewer #3/comment #1 and as most of the included studies used the term “periapical abscess” we used “periapical abscess” throughout the manuscript.

Comment #14: I think it is necessary to clarify why it would be important to compile data on this topic.

Response: Thank you for your comment. According to your comment #7, we have responded by editing the paragraph which explains the importance of unculturable bacteria in generally and specifically in the oral cavity on lines 87 to 104.

Comment #15: According to PRISMA checklist “Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS).” Therefore, authors could include the terms “DAA and related periapical lesions” on the objective of the study (line 90). If possible, specify which “related periapical lesions”.

Response: We thank the reviewer for this comment. As our research question did not include an intervention, we did not strictly use the PICOS research question framework and as Plos One journal’s guidelines accept other research question like CoCoPop [3]. However, we mentioned our research question on lines 111 to 118.

- Materials and Methods

Comment #16: The study could be rewritten and qualified according to the PRISMA Preferred reporting items for systematic reviews and meta‐analyses, available at https://onlinelibrary.wiley.com/doi/full/10.1111/iej.13118

Response: We thank the reviewer for this comment and accordingly modification have been performed to further follow the PRISMA guideline throughout the manuscript.

Comment #17: Lines 96 and 98 – Fig 1 should be moved to the results section.

Response: Fig 1 was moved to the results section line 210 as indicated in the manuscript, and Fig 1 legend was moved to the results section lines 228 to 229.

Comment #18: Line 105 – Authors could review the search period. In the abstract, the authors affirm that “databases were systematically searched from 1990 to 2020”. In the methodology they affirm that “the search was limited to human studies published 105 over a decade, from 1990/01/01 to 2019/12/31”. The reference number 40 corresponds to a study published in April 2020. Have reviewers been previously calibrated to ensure inter‐rater reliability for the search and for the risk of bias assessment?

Response: Thank you for this important comment. We have added a clarification in our response to your comment #2. The text on lines 129 to 131 was modified.

The time periods that have been mentioned in S1 Table were also modified to be “1990/01/01 to 2020/05/31”.

Inter‐rater reliability was done using Cohen's kappa coefficient and it was included in the method section on lines 136 to 142 and on lines 157 to 160. Then in the result section we added the results of Cohen's kappa on lines 260 to 263.

Comment #19: The exclusion criteria must be selected previously to conducting the study, and not according to the results. This would characterize a selection bias. The criteria presentation leaves doubt whether the criteria were previously established or not. In addition, the number of articles excluded according to each criterion must be presented in the results, not in the methods.

Response: Thank you for this comment and we agree with the reviewer. We did in fact decide on our exclusion criteria before conducting the research, but we found that mentioning them again caused some repetition in the text when justifying the reasons for exclusion for some studies. However, a paragraph about the exclusion criteria was added to the method section on lines 132 to 135.

The paragraph that explains the reasons and the number of the excluded articles was moved from the methods section to the results section lines 210 to 222.

Comment #20: Line 123, 129, 131 and 132: Fig 1, Table 1, 2, 3 and S2 should be moved to the results section.

Response: Thank you for this comment. Figure and tables were moved as suggested to the results section to be first mentioned as follows: (Fig 1) was moved to the result section, line 210, (Table 1) was moved to the result section line 240, (Table 2) was moved to results section line 268, (Table 3) was moved to the result section line 418, and (S2 Table) which after changing the order became (S4 Table ) and was moved to the result section line 268.

Comment #21: S2 Table and Table 3 are redundant.

Response: We thank the reviewer for this comment. Table 3 and S2 Table (which is now S4 Table after changing the order of supplementary tables) are slightly redundant, but it is essential to be kept separated and we have included one of them as supplementary data. Table 3 included information and data used for secondary outcomes, which have been analysed using PCA, and this table is a part of S4 Table, but it is essential to be kept alone as the unculturable bacteria were given symbols (from 1 to 35). These have been used to indicate the correspondent’s bacterial name, which have not been mentioned in the text and indicated the distribution of the bacteria relative to abundance, frequency, and sequence technique variables in Fig 3 and Fig 4. S4 Table included the forest plot figures for diversity, abundance, and frequency and explained how some studies were included in one forest plot but not in another forest plot due to the missing data in abundance or frequency for example.

Comment #22: Line 163: in this section, please include the customized checklist used to Risk of bias assessment (S3 Table).

Response: We thank the reviewer for this important suggestion. S3 Table becomes now S2 Table after changing the order of supplementary tables and was added to the method section/ Risk of bias assessment/ Line 157.

Comment #23: Line 169: “Finally, all studies with a minimum score of 6/8 for were included in the review.” Was it a criteria of inclusion or the result of the Risk of bias assessment?

Response: We thank the reviewer for this important comment. The mentioned sentence was moved from methods section to the results section because this was not our selection criterion; it was a subsequent criterion for quality check assessment with some modification on lines 263 to 264.

Comment #24: Line 195: Please specify what is PCA in the first time you use this term on the text.

Response: Thank you for this comment. The text on lines 200 to 201 was modified.

Comment #25: Line 201: Authors do not mention the duplicates removal. In fact, according to the flowchart, 14,819 studies were identified. 39 were duplicates and, therefore, removed.

Response: Thank you for this important comment. Information about deduplication was added to lines 206 to 209.

Comment #26: Authors identified more than 14,000 potentially relevant articles during the electronic search, but at the end of the process only 16 articles were included. A more refined search strategy should have been used for a more accurate result.

Response: This is an important point that was carefully considered, but in the current context we are providing the results that were elicited by diligent analyses by the authors. Indeed, at the beginning, we decided to include all the possibilities that allowed us to source phylotypes of unculturable bacteria. As the reviewer is aware, molecular microbiology sequencing and the study of unculturable bacteria are relatively new subject, and hence most of the older articles devoid of this term had to be rejected (i.e., 45 articles rejected as they did not state the ‘unculturable phylotypes’ despite that they used culture-independent methods for bacterial identification). In addition, as we have explained in the text the perusal of full-texts revealed approximately 60 articles that used molecular analytical methods of periapical lesions, but the vast proportion of these did not specify the histological lesion type, i.e., whether it is a radicular cyst or periapical granuloma. This led to an elimination of a majority of the filtered articles as well leading to the final result of 16 articles that were evaluated.

Comment #27: Why did the authors include only 13 studies in the meta-analysis? Why were 3 studies excluded?

Response: Thank you for this comment. For the first excluded study Siqueira and Rocas [4], we edited the paragraph to be clearer, on lines 273 to 279. For the other two studies Yun et al. [5] and Rolph et al. [6], the paragraph was edited on lines 302 to 304.

Comment #28: Line 201 and 209: “dentoalveolar abscess (DAA) and related periapical lesions.” What lesions?

Response: Thank you for this comment. We have clarified this further on lines 206 to 208.

Comment #29: Line 232: S3 table should be moved to the methods section.

Response: Thank you for this comment. S3 Table which is now S2 Table after changing the order was moved to the method section line 157.

Comment #30: Lines 240 to 245: wouldn't this paragraph be a DISCUSSION of the results?

Response: Thank you for this suggestion. The mentioned paragraph which is now on lines 273 to 279 explains why some studies were excluded from the meta-analysis, we are not discussing the results of these studies in comparison to other studies. Furthermore, on lines 302 to 304, you can find that we explained the reasons for the exclusion of other studies Yun et al [5] and Rolph et al. [6] from meta-analysis. We believe this paragraph cannot be included in the discussion.

Comment #31: Why did the authors include acute abscess AND abscess? Why were the studies that evaluated Chronic DAA. not included in the meta-analysis? Couldn't it be a bias?

Response: Thank you for this comment. In this systematic review and meta-analysis, we included both acute and chronic forms of periapical abscess. Two studies Jacinto et al. [7] and Vengerfeldt et al. [8] did not mention in their paper whether the abscess samples were acute or chronic and therefore we termed them throughout our study simply as “abscess” and on lines 280 to 283.

Our answer for the second part of this comment (Why were the studies that evaluated Chronic DAA. not included in the meta-analysis? Couldn't it be a bias?), is that: According to our answer to your comment #27: Why did the authors include only 13 studies in the meta-analysis? Why were 3 studies excluded?), we modified the text on lines 294 to 304 to clarify why these two studies on chronic abscess were excluded from the meta-analysis.

Comment #32: Line 274, 277, 280: The author refers to figure 2A, 2B and 2C, but this figure does not specify which is ABC.

Response: We thank the reviewer for this very important comment. We have corrected this in Figure 2 as uploaded during the resubmission.

Comment #33: Meta regression: Is there any limitation by using this analysis? Because there are few studies that evaluated unculturable bacteria in “periapical abscess” and “acute DAA” (type of lesion). Data would not show the real effect of the lesion type on prevalence of unculturable bacteria. Besides that, could the sample collection procedure be a specific moderator?

Response: We thank the reviewer for this comment. We chose meta-regression for the following reasons: We looked for variables or moderators that may significantly affect our summary proportion such as lesion type, country of study origin, and the type of sequencing techniques. In addition, when determining heterogeneity, we preferred to use moderators over subgrouping, because using subgrouping for lesion types will yield a forest plot with only two studies, Vengerfeldt et al and Jacinto et al [59, 60] which is statistically inaccurate.

On the other hand, we did not investigate sampling procedure as a moderator because sequencing methods are sensitive techniques and need only a very small microbial sample to be investigated if we compared between paper point and aspiration procedure, taking into consideration an aseptic condition in both procedures had to be guaranteed. Guentsch et al. found a similar microbial load when using different sampling procedures of the gingival crevicular fluid [9]

Comment #34: Line 338 to 347: Wouldn't this paragraph be a METHODOLOGY?

Response: Thanks for this important comment. This paragraph was moved to the methods section lines 189 to 199.

Comment #35: Line 348 to 373: For the diversity of unculturable bacteria in periapical abscess, the study number 8 “is an outlying study but not influential, so it was not removed.” But, for abundance estimation, the “tests reveal that the 3rd study [33] is an influential study on the effect of summary proportion”. Why this study was not removed?

Response: We thank the reviewer for this important comment. The reviewer is correct, the 3rd study [10] is an influential outlying study, but we decided to keep it because the summary proportion when leaving out this study revealed minor change on abundance summary proportion from 5% to 4%, while leaving out other studies like Flynn et al. [11] and Yang et al. [12] had a more prominent effect on abundance summary proportion from 5% to 7% in both studies (Flynn et al. [11] and Yang et al. [12]) although they are not influential outlying studies. For this reason, we decided to keep this study. Accordingly, it is important to investigate all these parameters to reach the better decision of exclusion. We have clarified this further on lines 395 to 398.

-Discussion:

Comment #36: Since the number of bacteria is normally greater and more virulent in symptomatic and purulent infections than in chronic and asymptomatic infections, could the inclusion of studies that do not specify what type of ADA in the meta-analysis characterize a bias in the results?

Response: We thank the reviewer for this important comment. We included the studies that did not mentioned the type of abscess whether acute or chronic in this review because in our research question we looked for periapical abscess in general. Furthermore, we have tested the heterogeneity using lesion type moderator to resolve this bias and whether it has a significant effect on diversity, abundance, and frequency of unculturable bacteria in periapical abscess.

Comment #37: Line 417: This was the diversity found for all abscesses.

Response: Yes. We measured the diversity of unculturable bacteria in periapical abscess in 13 studies as illustrated in Table 2, line 306.

Comment #38: Lines 418 and 419: Authors could rewrite this sentence for better understanding.

Response: Thank you. The text was modified on lines 472 to 474.

Comment #39: Line 424 AND 439: Are authors suggesting any explanation for the country's interference? Is the collection method important?

Response: Thank you for this important comment. We added references to explain the significance in country moderator for diversity on lines 476 to 481.

Regarding our explanation whether the collection methods is important? is that; It could be important, but when we performed the analysis, we expected that sampling procedure does not have a significant effect on our results according to a previous study [9] which found that the type of sampling procedure did not affect the bacterial load. Taking into consideration that the methods used in the included studies have used molecular methods, which are very sensitive methods for bacterial detection including unculturable bacteria.

Comment #40: More information regarding the importance of the knowledge on the prevalence of unculturable bacteria in dentoalveolar abscess and related periapical lesions should be included in this section. The authors could improve the discussion. What is the importance of knowing the frequency, abundance and diversity of these bacteria? What are the suggestions for new studies? Do the authors suggest an explanation for the significant publication bias for the frequency estimate of unculturable bacteria and for the outlier? Regarding chronic ADA, can different results be found?

Response: We thank the reviewer for this comment, the requested information was added and divided into four sections as follows:

1- More information regarding the importance of the knowledge on the prevalence of unculturable bacteria in dentoalveolar abscess and related periapical lesions should be included in this section. The authors could improve the discussion. What is the importance of knowing the frequency, abundance and diversity of these bacteria?

Response: A paragraph on the importance of knowledge on bacterial diversity in oral cavity and periapical lesions was added to the discussion section on lines 464 to 471.

A paragraph on the importance of knowledge on bacterial abundance in oral cavity and periapical lesions was added to the discussion on lines 487 to 489.

A paragraph on the importance of knowledge on bacterial frequency in oral cavity and periapical lesions was added to the discussion in lines 500 to 502.

2- What are the suggestions for new studies?

Response: We have added a paragraph as a suggestion for new studies on lines 545 to 551.

3- Do the authors suggest an explanation for the significant publication bias for the frequency estimate of unculturable bacteria and for the outlier?

Response: We thank the reviewer for this important comment. We added our explanation in the discussion section on lines 505 to 516.

4- Regarding chronic ADA, can different results be found?

Response: Generally, yes, we are expecting a different unculturable microbial profile between acute and chronic periapical abscess, but whether this difference is significant or not, this needs more research to be done on the chronic periapical abscess with larger sample size because still the real identification of unculturable bacteria is less than the culturable one as indicated in our study. Santos et al. [13] found a significant higher diversity in microbial content in acute periapical abscess in comparison to the chronic periapical abscess with the possibility that more diverse microbial content is very likely to be related to the collective pathogenicity for many endogenous diseases [14, 15].

Comment #41: What is the contribution of this study? There could be a last paragraph summarizing the findings.

Response: We thank the reviewer for this suggestion. We have added a last paragraph in lines 540 to 551.

- Conclusions

Comment #42: Authors state that the prevalence of unculturable bacteria is ABUNDANT and at the conclusion of the abstract they state that it is MODERATE. Please, review these statements. The conclusion could be more objective and in accordance with the aim of the study.

Response: Thank you for all the valuable comments. The text on lines 553 to 563 was modified.

Reviewer #2

Comment: This manuscript discusses the role unculturable bacteria in the dentoalveolar abscess and how it effects on the periapical lesions in depth through systematic review and Meta-analysis as study design. The meta analysis for this study is very good. The number of included studies in this study is expected due to new idea and the number of studies related to the topic is very small and future studies are required in this field. I think this paper eligible to publish in peer review journal like this journal.

Response: We thank the reviewer for their valuable opinion and positive comments.

Reviewer #3

Comment #1: The authors should revise the nomenclature of periapical lesions and acute apical abscess and change them in entire manuscript according to the guidelines of the AAE (diagnosis). Dentoalveolar abscess may involve lesions of not only endodontic origin

Response: American Association of Endodontists (AAE) guidelines (https://www.aae.org/specialty/wp-content/uploads/sites/2/2017/07/endodonticdiagnosisfall2013.pdf) were revised and all dentoalveolar abscess (DAA) terms were replaced with periapical abscess as also recommended by the reviewer #1/comment #13.

Comment #2: Two studies (Jacinto et al. 2007 and Vengerfeldt et al. 2014) included in the review did not have their data clarified regarding the diagnosis. Have the authors contacted the corresponding authors to clarify the diagnosis gap? It seems that in the Jacinto et al (2007) the paper point samples were collected from root canals of symptomatic teeth (i.e. with acute apical abscess).

Response: We thank the reviewer for this comment. For these two studies, we did not contact the authors as the necessary information for the diagnosis was obtained from their presented data but after consensus understanding and decision between the two dentists (A.M.A, and M.A.A) included in this systematic review and meta-analysis as follows:

In the first study (Jacinto et al. 2007) [7], we are including what exactly they have written in their article and then, we explain what they mean as follows: “The selection of patients was accomplished by anamnesis, and by clinical and radiographic examination. The following features were noted for each patient: age, gender, tooth and pulpal status, nature of pain, previous pain, tenderness to percussion, pain on palpation, mobility, presence of a sinus and its origin, presence of swelling of the periodontal tissues, depth of periodontal pocket, previous antibiotic therapy and internal condition of the root canal (e.g. presence of clear, haemorrhagic or purulent exudates). Patients who had not received antibiotic therapy in the last 6 months and who had teeth with primary endodontic infections, periapical lesions and absence of periodontal diseases were selected for this study. All the cases selected showed clinical features of endodontic abscesses.”

Then they mentioned in Results and Discussion “ The study comprised samples from teeth with primary endodontic infections, no periodontal disease, presence of periapical radiolucency, and features of endodontic abscesses.” Our explanation for Jacinto et al. patient selection criteria is as follow:

1. They mentioned that they “noted” but they did not specify whether these features are available in all, some, or not available at all. In another words, the results of their examination were not clearly presented but they performed the required dental examination.

2. We have found that the word “noted” which means “examined” was applied to the following features {age, gender, tooth and pulpal status, nature of pain, previous pain, tenderness to percussion, pain on palpation, mobility, presence of a sinus and its origin, presence of swelling of the periodontal tissues, depth of periodontal pocket, previous antibiotic therapy and internal condition of the root canal (e.g. presence of clear, haemorrhagic or purulent exudates)}, which are all general features and are not specified characteristics for the sample or the clinical status). So, we cannot say that “we noted (age and tenderness to percussion)” and explain this sentence that there is tenderness to percussion (positive response) and apply this meaning to age and say “there is an age” this is not a clear meaning. Therefore, we decided that “noted” mean “examined or checked” but not “found, or available”.

Then they mentioned “The study comprised samples from teeth with primary endodontic infections, no periodontal disease, presence of periapical radiolucency, and features of endodontic abscesses”. In this paragraph we took each sentence alone, e.g., samples are primary endodontic infection/ there is no periodontal disease/ there is a periapical radiolucency/ there are features of endodontic abscess. Accordingly, we understood and decided that these samples belong to periapical abscess and not periodontal abscess, but we were not sure whether it is an acute or a chronic form.

In the second study (Vengerfeldt et al. 2014) [8], the authors were extensively and comprehensively performed the dental examination and mentioned the features of each group in Table 1 as follow: “The subject population was composed of 12 antibiotic-naive patients (ages ranging from 27–66 years) attending the Clinic of Stomatology at the University of Tartu, Estonia. Patients came to the clinic for root canal treatment or extraction between September 2010 and April 2011. Thorough anamnesis (systemic and local diseases, previous treatment, hygiene habits, allergy, and so on), intraoral status, and periapical x-rays were taken, which were all necessary for the upcoming treatment. To be included in the study, subjects had to have good systemic health. A thermal test was performed using cold and hot. A cold test was performed with Endo-Frost (-50⸰C) (Roeko, Langenau, Germany) and a cotton pellet (size 00) (Roeko); the frozen cotton pellet was held on the isolated and dried tooth on the restoration-free surface for about 2–5 seconds or until pain was felt. The hot test was performed using silicone polisher (HiLuster; Kerr Corp, Orange, CA) with a 1:1 contra-angle handpiece (W&H, B€urmoos, Austria) without air and water cooling by touching the restoration-free tooth surface with about 4000 rpm for about 5 seconds or until the patient felt pain. The electric pulp test or the vitality test was performed with the Elements Diagnostic Unit (Sybron Endo, Orange, CA) according to the manufacturer’s instructions or until pain was felt. The probe was touched to the restoration-free part of the isolated tooth until pain was felt. A percussion test was performed with a mirror handle using gentle and uniform tapping on the occlusal and horizontal side of each tooth, and sound teeth were registered as zero feeling; the same kind of tapping was performed on the accused tooth, and the feeling of the patient was compared and described. The palpation test was performed using uniform and solid pressure with the right index finger on the tip area of the root on both sides of the alveolar bone; the tooth was palpated by applying pressure on the tooth both vertically and horizontally. This was done bilaterally on both sides of the jaw to consider anatomic differences. Periapical radiographs were taken by an experienced radiologist using the Planmeca Prostile Intra X-Ray unit (Planmeca OY, Helsinki, Finland) with the RVG 6100 sensor (Carestream Dental LLC, Atlanta, GA) at a parallel angle with RINN yellow (posterior) or blue (anterior) sensor holder (Dentsply Rinn, Elgin, IL). PA radiographs were analyzed using the Trophy DICOM program (Kodak). Exclusion criteria were as follows: the presence of periodontal pockets greater than 5 mm, horizontal and vertical root fracture, deep carious lesions that made the tooth unrestorable, roots with previously apicectomized root tips with or without retrograde fillings, or any severe systemic condition like diabetes or immune suppression. In addition, subjects who received antibiotic or anti-inflammatory therapy in the previous 6 months were excluded. None of the sampled teeth presented posts, crowns, or bridges. Five of the investigated teeth were diagnosed with primary chronic apical periodontitis (pCAP) and 3 with secondary apical periodontitis (sCAP). Four teeth with a periapical abscess (evolved from apical periodontitis) were included as the controls. Clinical data are presented in Table 1.”

Accordingly, the necessary dental examination was mentioned in the text above in their study and the results of their diagnosis were presented in Table 1, so we believe that they did the data presentation of dental examination but without any discrimination of acute periapical abscess from chronic periapical abscess when they mentioned the list of bacteria derived from each sample. In their Table 2, as you can see that all periapical abscess acute or chronic are listed as periapical abscess. For that reason, we decided to include them as “periapical abscess” only during our systematic review and meta-analysis.

Comment #3: Great number of the studies included in this review were from Brazil. The authors highlighted a high discrepancy in the results found in these studies. Is there any hypothesis for this diversity of uncultivable bacteria found in studies carried out in the Brazilian population?

Response: We thank the reviewer for this comment. We have already included in the discussion our explanation for this heterogeneity in diversity scores between studies with some modification on lines 476 to 481.

Comment #4: Please clarify the origin of the sample collected. For instance, Jacinto el al collected samples from the root canals of symptomatic teeth. On the other hand, Siqueira and Rocas (2007) and many other authors collected samples from the purulent collection.

Response: We thank the reviewer for this comment. In Table 1, line 254, we mentioned the sample collection procedure including aspiration and paper point. Whenever there is an aspiration there is a purulent collection, while using paper point is related to sample collection from the root canal. However, for more clarification we added to column 9, sampling origin as indicated in the manuscript.

Comment #5: Another point that needs to clarify is the presence of abscess in primary or in adult dentition. For example, Zhang et al studied the presence of abscess in primary teeth (children).

Response: We thank the reviewer for this comment. Another column (column #7) was added to Table 1, line 254 including dentition type as indicated in the manuscript.

Comment #6: It would be interesting to explain the differences between uncultivable, unculturable and uncultivated bacteria.

Response: We thank the reviewer for this comment. This is an important point, and we agree it would be very interesting, however we believe it could be addressed in another narrative review. For this systematic review and meta-analysis, we think that this clarification will distract the reader from our goal and objectives of the study, and this should be discussed in another forum.

Reviewer #4

- Title:

Comment #1: This systematic review focused on apical abscesses. As no data on “related periapical lesions” was provided, I would suggest to remove this part of the title.

Response: We thank the reviewer for this suggestion. We have modified the title on lines 1 to 2.

- Introduction:

Comment #2: Lines 52-61: The authors used Nair (1997) as a reference for the classification of apical diseases. However, as the diagnosis in Endodontics is based on clinical signs/ symptoms and not on histopathological findings, an updated classification of apical diseases would be more appropriate (https://www.aae.org/specialty/clinical-resources/glossary-endodontic-terms/).

Response: We thank the reviewer for this comment, as this is a very important point for the updated classification of the periapical abscess. We have modified the text according to the reviewer’s recommendation to use the tenth edition of the AAE Glossary of Endodontic Terms which was updated in 2020 on lines 57 to 71.

Comment #3: Line 69: The “foregoing organisms” were not mentioned the text. Please revise this sentence.

Response: We thank the reviewer for this comment. The text was modified, and the “foregoing organisms” was replaced by the names of these organisms on lines 77 to 80.

Comment #4: Line 88: Granuloma and cysts are not sequelae of apical abscess. Please revise this sentence.

Response: We thank the reviewer for this comment. The text was modified on lines 105 to 107.

Comment #5: Lines 90-91: The secondary aim of this study was to review the possible role of non-cultivable microorganisms in the pathogenesis of apical diseases. However, the presence or abundance of bacterial DNA does not necessarily represent the bacterial activity in the disease process, as the DNA of dead bacteria can be detected by molecular methods. In addition, it has been found that bacteria with low abundance can be active in oral infections. Therefore, the secondary objective cannot be achieved due to the limitations of the studies included in this review.

Response: We thank the reviewer for the comment. The text on lines 109 to 111 was modified.

- Materials and Methods:

Comment #6: Lines 112-113: If the AAE classification of apical periodontitis (i.e., asymptomatic apical periodontitis) had been used instead of the classification based on histological findings (i.e., periapical granuloma and cyst), important information could have been extracted from the 60 articles that were excluded.

Response: In this systematic review and meta-analysis, we have focused our search strategy to include all the bacteria that have been detected in periapical abscess, radicular cyst, and periapical granuloma using molecular methods. There was a published systematic review that has been done on apical periodontitis and focused on the microbiota detected using sequencing strategy and entitled “A Systematic Review of the Root Canal Microbiota Associated with Apical Periodontitis: Lessons from Next-Generation Sequencing” [16].

Comment #7: Line 209: Please explain which periapical lesions are associated with DAA.

Response: Thank you for your comment. This paragraph was modified on lines 225 to 227.

- Outcome measures:

Comment #8: Lines 395-405: Due to the high diversity of the microbiome among individuals, most bacterial taxa were found only once. Therefore, the frequency and abundance analysis at the species level may not provide representative data.

Response: Thank you for this comment. Mentioning the taxa name of the unculturable bacteria instead of species name will be rather vague as a taxon may contain both culturable and unculturable species or phylotypes. For example, Vengerfeldt et al. (2014) [8] found in Synergistetes phylotype Pyramidobacter piscolens as culturable, while TG5 group which has a genus name only, is unculturable. Hence, we believe, it is more accurate to be specific in identifying the unculturable bacteria at their genus or species level rather than taxon level.

Comment #9: Lines 406-409: The statement that hybridization techniques are the most reliable molecular methods for detecting noncultivable bacteria should be analyzed with caution. First, it is important to note that only 5 studies were included in this analysis. Second, this finding is in contrast to the literature on microbiological methods, showing that sequencing is a reliable method for studying the oral microbiome. These issues should be further discussed.

Response: In response to the second point, our editing is on lines 334 to 339.

This mean that the “nonspecific 16S rRNA targeted gene” sequencing methods using 16S rRNA gene sequences as a general target for sequencing both the high and low abundant species wherein the high abundant species may mask the low abundant species that are usually represented by unculturable bacterial phylotypes or species [17]. On the other hand, nested PCR, restriction fragment length polymorphisms [18], and other hybridization methods [19] represented very sensitive methods for detecting even the low abundant microbial species because in these methods we are focusing on a specific and targeted area of the 16s rRNA genes that is not universal for all bacterial species but for a specific group or genus of bacteria.

Our response for the first point: We agree with the reviewer that six studies (not five studies) may not be enough for systematically concluding and affirming this result, but we think it is necessary to mention it for the first time in the context of detecting unculturable bacteria. We have modified the paragraph on lines 537 to 539.

- Conclusion:

Comment #10: Line 479: Bacterial abundance may not be correlated with their activity (please see the comments above: Lines 90-91). Please revise this sentence.

Response: We thank the reviewer for this comment. The conclusion has been modified based on your comment as well as reviewer #1/comment #42, and reviewer #5/comment #11. The conclusion now on lines 553 to 563.

Reviewer #5

- Abstract:

Comment #1: Revise for uniformity, and stick to the Journal's Guidelines. "( P -value = 0.05)" must read "(p = 0.050)". Revise thoroughly.

Response: We thank the reviewer for this important comment. We have revised the paper thoroughly and made the necessary changes to ensure uniformity.

Comment #2: With your conclusions, please stick exclusively to your aims. See: "To assess the prevalence of uncultivable bacteria in dentoalveolar abscess (DAA) and related periapical lesions." Now, you conclude that "unculturable bacteria in such lesions (...) are likely to play, a yet unknown, critical role in the pathogenesis and progression of the disease". This is something different, and you surely will agree.

Response: We thank the reviewer for this comment and yes do in fact agree with them. We have edited the conclusion according to this comment and reviewer #1/comment #6, on lines 46 to 49.

Comment #3: More search remains to be done on the communal behaviour, virulence, and pathogenicity in this ecosystem." This is a common phrase only, but not an answer to your research question.

Response: We thank the reviewer for this suggestion. The text on lines 48 to 49 was modified.

Comment #4: Please stick to the word maximum allowed here, to provide complete information.

Response: We thank the reviewer for the comment. We have revised the word maximum whilst ensuring complete information is maintained.

- Introduction:

Comment #5: With respect to the research question, this section is considered much too long. Please shorten significantly, and elaborate both aims and objectives more clearly.

Response: Thank you for this comment. Here we refer to another contrasting comment from a reviewer #1/comment #7 who stated, “Authors should provide more information (my emphasis) about the importance of the knowledge on the prevalence of unculturable bacteria in the dentoalveolar abscess and related periapical lesions to improve the background and also enrich the discussion of the results”. Introduction section has been revised and edited accordingly. We have edited each section according to the objective and the research question as follows:

Paragraph #1: Lines 51 to 56 explain a brief background about periapical lesions (abscess, cyst, and granuloma).

Paragraph #2: Lines 57 to 71 explain the best matched classification of periapical lesions for our study.

Paragraph #3: Lines 72 to 83 explain how the periapical abscess is formed, the bacterial role, how previously the culturable bacterial investigation has been extensively done, and the role of NGS sequencing methods in discovering the unculturable bacteria.

Paragraph #4: Lines 84 to 104 describe the universal gene sequences (16s rRNA) used in molecular methods for bacterial identification, its importance, and how it allows us to know the expected percentage of unculturable bacteria, what is the most database that have been used in this systematic review to discriminate between culturable and unculturable bacteria which is HOMD. In this paragraph, we added the importance of the unculturable bacteria and why we are studying their prevalence in periapical abscesses.

Paragraph #5: Lines 105 to 118 explain the objectives, aims, and research question of this study.

Comment #6: What about your null hypothesis?

Response: We thank the reviewer for this comment. According to PRISMA checklist and the reviewer #1/comment #15, we have included our research question in addition to some editing to the objectives on lines 105 to 118, therefore, we believe that adding the hypothesis will cause some repetition in the information.

- Methods

Comment #7: Why did you restrict your inclusion criteria to English?

Response: We thank the reviewer for this comment. Most available data were extracted from well-recognized publications which were mostly in English. In addition, all authors are more familiar with the English language, so to ensure the consistency of data extraction and to eliminate potential biases due to translation, selecting only papers in English was seen to be sufficient for the purposes of this systematic review.

- Results

Comment #8: This has been meticulously elaborated.

Response: We thank the reviewer and are very grateful for their positive comment.

- Discussion

Comment #9: Please do not use authors' names with your text (these will be acknowledged with your reference list). Instead, do focus on your main thoughts.

Response: We thank the reviewer for this comment. We have deleted authors names in the discussion as per the reviewer’s comment.

Comment #10: This section is considered easily intelligible.

Response: We thank the reviewer for their positive comment.

- Conclusion

Comment #11: Please see comments given above. With your Conclusions, please stick exclusively to your revised aims. Do not simply repeat your results here. Instead, provide a reasonable and generalizable extension of your outcome. See, for example, "Despite the remarkable diversity of uncultivable bacteria in these lesions, their role in the pathogenesis is yet to be determined. A positive correlation was shown between the abundance and frequency of individual uncultivable bacteria. Peptostreptococcus sp. oral clone CK035 showed the highest abundance and frequency. Hybridization techniques appear to be more reliable in detecting the abundance and frequency of individual unculturable bacteria in periapical lesions." All these aspects would seem right, and, thus, these thoughts might be copied & pasted to your Disc section. However, these are not considered conclusions deducible from your study.

Response: Thank you for this comment. According to this comment, reviewer #1/comment #42, and reviewer #4/comment #10, the conclusion was modified on lines 553 to 563.

- References

Comment #12:

- Full of minor and major shortcomings. Please revise for uniform formatting.

- Again, stick to the Journal's guidelines, and consult some recently published Plos One papers.

- doi numbers are missing.

Response: We thank the reviewer for the valuable comments. The reference list has been extensively checked and made sure to follow the journal style.

References:

1. Shah KK, Pritt BS, Alexander MP. Histopathologic review of granulomatous inflammation. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases. 2017;7:1-12.

2. Gutmann JL, Baumgartner JC, Gluskin AH, Hartwell GR, Walton RE. Identify and define all diagnostic terms for periapical/periradicular health and disease states. Journal of endodontics. 2009;35(12):1658-74.

3. Jörns-Presentati A, Napp A-K, Dessauvagie AS, Stein DJ, Jonker D, Breet E, et al. The prevalence of mental health problems in sub-Saharan adolescents: A systematic review. PLoS One. 2021;16(5):e0251689.

4. Siqueira JF, Rôças IN. Molecular detection and identification of Synergistes phylotypes in primary endodontic infections. Oral diseases. 2007;13(4):398-401.

5. Yun KH, Lee H-S, Nam OH, Moon CY, Lee J-H, Choi SC. Analysis of bacterial community profiles of endodontically infected primary teeth using pyrosequencing. International journal of paediatric dentistry. 2017;27(1):56-65.

6. Rolph HJ, Lennon A, Riggio MP, Saunders WP, MacKenzie D, Coldero L, et al. Molecular identification of microorganisms from endodontic infections. Journal of clinical microbiology. 2001;39(9):3282-9.

7. Jacinto RC, Gomes BP, Desai M, Rajendram D, Shah HN. Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography. Oral microbiology and immunology. 2007;22(6):403-10.

8. Vengerfeldt V, Špilka K, Saag M, Preem J-K, Oopkaup K, Truu J, et al. Highly diverse microbiota in dental root canals in cases of apical periodontitis (data of illumina sequencing). Journal of endodontics. 2014;40(11):1778-83.

9. Guentsch A, Kramesberger M, Sroka A, Pfister W, Potempa J, Eick S. Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis. Journal of periodontology. 2011;82(7):1051-60.

10. Sakamoto M, Rocas IN, Siqueira JF, Jr., Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral microbiology and immunology. 2006;21(2):112-22.

11. Flynn TR, Paster BJ, Stokes LN, Susarla SM, Shanti RM. Molecular methods for diagnosis of odontogenic infections. Journal of oral and maxillofacial surgery. 2012;70(8):1854-9.

12. Yang Q-B, Fan L-N, Shi Q. Polymerase chain reaction-denaturing gradient gel electrophoresis, cloning, and sequence analysis of bacteria associated with acute periapical abscesses in children. Journal of endodontics. 2010;36(2):218-23.

13. Santos AL, Siqueira JF, Jr., Rôças IN, Jesus EC, Rosado AS, Tiedje JM. Comparing the bacterial diversity of acute and chronic dental root canal infections. PLoS One. 2011;6(11):e28088.

14. Siqueira JF, Jr., Rôças IN. Community as the unit of pathogenicity: an emerging concept as to the microbial pathogenesis of apical periodontitis. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics. 2009;107(6):870-8.

15. Kuramitsu HK, He X, Lux R, Anderson MH, Shi W. Interspecies interactions within oral microbial communities. Microbiology and molecular biology reviews. 2007;71(4):653-70.

16. Manoil D, Al-Manei K, Belibasakis GN. A systematic review of the root canal microbiota associated with apical periodontitis: Lessons from next-generation sequencing. Proteomics clinical applications. 2020;14(3):1900060.

17. McLean JS, Lombardo M-J, Badger JH, Edlund A, Novotny M, Yee-Greenbaum J, et al. Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum. Proceedings of the national academy of sciences. 2013;110(26):E2390-E9.

18. Clusa L, Ardura A, Fernández S, Roca AA, García-Vázquez E. An extremely sensitive nested PCR-RFLP mitochondrial marker for detection and identification of salmonids in eDNA from water samples. PeerJ. 2017;5:e3045-e.

19. Podar M, Abulencia CB, Walcher M, Hutchison D, Zengler K, Garcia JA, et al. Targeted access to the genomes of low-abundance organisms in complex microbial communities. Applied and environmental microbiology. 2007;73(10):3205-14.

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PLoS One. doi: 10.1371/journal.pone.0255485.r003

Decision Letter 1

Andrej M Kielbassa

19 Jul 2021

Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

PONE-D-20-39104R1

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PLoS One. doi: 10.1371/journal.pone.0255485.r004

Acceptance letter

Andrej M Kielbassa

27 Jul 2021

PONE-D-20-39104R1

Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

Dear Dr. Hamoudi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Checklist. PRISMA 2009 checklist.

(DOC)

Click here for additional data file.^{(63.5KB, doc)}

S1 Table. Literature search strategy and terms for the prevalence of unculturable bacteria in periapical lesions.

(PDF)

Click here for additional data file.^{(40.5KB, pdf)}

S2 Table. Checklist of critical appraisal of Joanna Briggs Institute for analytical cross sectional studies.

(PDF)

Click here for additional data file.^{(171.1KB, pdf)}

S3 Table. Bias assessment of included studies using Joanna Briggs Institute for analytical cross sectional studies.

(PDF)

Click here for additional data file.^{(70.3KB, pdf)}

S4 Table. Extracted results from each study of individual bacteria in periapical abscess.

(PDF)

Click here for additional data file.^{(101.7KB, pdf)}

S1 Fig. Scatter plot depicting the moderators’ effect on summary proportion of diversity of unculturable bacteria in periapical abscess.

(A) Lesion moderator. (B) Country moderator. (C) Sequence technique moderator.

(TIF)

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S2 Fig. Scatter plot depicting the moderators’ effect on summary proportion of abundance of unculturable bacteria in periapical abscess.

(A) Lesion moderator. (B) Country moderator. (C) Sequence technique moderator.

(TIF)

Click here for additional data file.^{(258.7KB, tif)}

S3 Fig. Scatter plot depicting the moderators’ effect on summary proportion of frequency of unculturable bacteria in periapical abscess.

(A) Lesion moderator. (B) Country moderator. (C) Sequence technique moderator.

(TIF)

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S4 Fig. Results for outlying studies of diversity of unculturable bacteria in periapical abscess.

(A) Forest plot depicting summary proportion after leaving out each study. (B) Influential analysis plot of diversity of unculturable bacteria in thirteen studies of periapical abscess.

(TIF)

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S5 Fig. Results for outlying studies of abundance of unculturable bacteria in periapical abscess.

(A) Forest plot depicting summary proportion after leaving out each study. (B) Influential analysis plot of abundance of unculturable bacteria in six studies of periapical abscess.

(TIF)

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S6 Fig. Results for outlying studies of frequency of unculturable bacteria in periapical abscess.

(A) Forest plot depicting summary proportion after leaving out each study. (B) Influential analysis plot of frequency of unculturable bacteria in eleven studies of periapical abscess.

(TIF)

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S7 Fig. Funnel plot of publication bias of unculturable bacteria in periapical abscess.

(A) Diversity. (B) Abundance. (C) Frequency.

(TIF)

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S8 Fig. Scatter plot of abundance and frequency correlation for individual unculturable bacteria.

(TIF)

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0255485.ref001] 1.Peters E, Lau M. Histopathologic examination to confirm diagnosis of periapical lesions: A review. J Can Dent Assoc. 2003;69(9):598–600. . [PubMed] [Google Scholar]

[pone.0255485.ref002] 2.Cotti E, Campisi G. Advanced radiographic techniques for the detection of lesions in bone. Endod Topics. 2004;7(1):52–72. 10.1111/j.1601-1546.2004.00064.x. [DOI] [Google Scholar]

[pone.0255485.ref003] 3.Ramanpreet B, Simarpreet S, Rajat B, Amandeep B, Shruti G. Histopathological insight into periapical lesions: An institutional study from Punjab. Int J Oral Maxillofac Pathol. 2012;3(3):2–7. [Google Scholar]

[pone.0255485.ref004] 4.Safi L, Adl A, Azar MR, Akbary R. A twenty-year survey of pathologic reports of two common types of chronic periapical lesions in Shiraz Dental School. J Dent Res Dent Clin Dent Prospects. 2008;2(2):63–70. doi: 10.5681/joddd.2008.013 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref005] 5.Soares JA, Leonardo MR, da Silva LA, Tanomaru Filho M, Ito IY. Histomicrobiologic aspects of the root canal system and periapical lesions in dogs’ teeth after rotary instrumentation and intracanal dressing with Ca(OH)2 pastes. J Appl Oral Sci. 2006;14(5):355–64. doi: 10.1590/s1678-77572006000500011 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref006] 6.World Health Organization. Application of the international classification of diseases to dentistry and stomatology: ICD-DA, 3rd ed, Geneva. 1995. Available from: https://apps.who.int/iris/handle/10665/40919. [Google Scholar]

[pone.0255485.ref007] 7.Nair PN. Apical periodontitis: A dynamic encounter between root canal infection and host response. Periodontol 2000. 1997;13:121–48. doi: 10.1111/j.1600-0757.1997.tb00098.x . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref008] 8.American Association of Endodontics. Glossary of endodontic terms. 10th ed. 2020. Available from: https://www.aae.org/specialty/download/glossary-of-endodontic-terms/. [Google Scholar]

[pone.0255485.ref009] 9.Gutmann JL, Baumgartner JC, Gluskin AH, Hartwell GR, Walton RE. Identify and define all diagnostic terms for periapical/periradicular health and disease states. J Endod. 2009;35(12):1658–74. doi: 10.1016/j.joen.2009.09.028 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref010] 10.Shu M, Wong L, Miller JH, Sissons CH. Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol. 2000;45(1):27–40. doi: 10.1016/s0003-9969(99)00111-9 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref011] 11.Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med. 2004;15(6):348–81. doi: 10.1177/154411130401500604 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref012] 12.Brook I, Frazier EH, Gher ME. Aerobic and anaerobic microbiology of periapical abscess. Oral Microbiol Immunol. 1991;6(2):123–5. doi: 10.1111/j.1399-302x.1991.tb00464.x [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref013] 13.Sakamoto M, Rôças IN, Siqueira JF, Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol. 2006;21(2):112–22. doi: 10.1111/j.1399-302X.2006.00270.x . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref014] 14.Hugenholtz P, Pace NR. Identifying microbial diversity in the natural environment: A molecular phylogenetic approach. Trends Biotechnol. 1996;14(6):190–7. doi: 10.1016/0167-7799(96)10025-1 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref015] 15.Paster BJ, Dewhirst FE. Molecular microbial diagnosis. Periodontol 2000. 2009;51:38–44. doi: 10.1111/j.1600-0757.2009.00316.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref016] 16.Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, et al. The human oral microbiome. J Bacteriol. 2010;192(19):5002–17. doi: 10.1128/JB.00542-10 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref017] 17.Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol. 2001;183(12):3770–83. doi: 10.1128/JB.183.12.3770-3783.2001 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref018] 18.Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005;43(11):5721–32. doi: 10.1128/JCM.43.11.5721-5732.2005 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref019] 19.Teles R, Teles F, Frias-Lopez J, Paster B, Haffajee A. Lessons learned and unlearned in periodontal microbiology. Periodontol 2000. 2013;62(1):95–162. doi: 10.1111/prd.12010 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref020] 20.Vartoukian SR, Palmer RM, Wade WG. The division "Synergistes". Anaerobe. 2007;13(3–4):99–106. doi: 10.1016/j.anaerobe.2007.05.004 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref021] 21.Jumas-Bilak E, Carlier JP, Jean-Pierre H, Citron D, Bernard K, Damay A, et al. Jonquetella anthropi gen. nov., sp. nov., the first member of the candidate phylum ’Synergistetes’ isolated from man. Int J Syst Evol Microbiol. 2007;57(12):2743–8. doi: 10.1099/ijs.0.65213-0 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref022] 22.Vartoukian SR, Downes J, Palmer RM, Wade WG. Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol. 2013;63(2):458–63. doi: 10.1099/ijs.0.041038-0 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref023] 23.Downes J, Vartoukian SR, Dewhirst FE, Izard J, Chen T, Yu WH, et al. Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum ’Synergistetes’ isolated from the human oral cavity. Int J Syst Evol Microbiol. 2009;59(5):972–80. doi: 10.1099/ijs.0.000364-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref024] 24.Arank A, Syed SA, Kenney EB, Freter R. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl Microbiol. 1969;17(4):568–76. doi: 10.1128/am.17.4.568-576.1969 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref025] 25.Socransky SS, Gibbons RJ, Dale AC, Bortnick L, Rosenthal E, Macdonald JB. The microbiota of the gingival crevice area of man. I. Total microscopic and viable counts and counts of specific organisms. Arch Oral Biol. 1963;8:275–80. doi: 10.1016/0003-9969(63)90019-0 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref026] 26.Dymock D, Weightman AJ, Scully C, Wade WG. Molecular analysis of microflora associated with dentoalveolar abscesses. J Clin Microbiol. 1996;34(3):537–42. doi: 10.1128/jcm.34.3.537-542.1996 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref027] 27.Lewis K, Epstein S, D’Onofrio A, Ling LL. Uncultured microorganisms as a source of secondary metabolites. J Antibiot. 2010;63(8):468–76. doi: 10.1038/ja.2010.87 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref028] 28.Munn Z, Stern C, Aromataris E, Lockwood C, Jordan Z. What kind of systematic review should I conduct? A proposed typology and guidance for systematic reviewers in the medical and health sciences. BMC Med Res Methodol. 2018;18(1):5. doi: 10.1186/s12874-017-0468-4 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref029] 29.Munn Z, Moola S, Lisy K, Riitano D, Murphy F. Claustrophobia in magnetic resonance imaging: A systematic review and meta-analysis. Radiography. 2015;21(2):e59–e63. 10.1016/j.radi.2014.12.004. [DOI] [Google Scholar]

[pone.0255485.ref030] 30.Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4(1):1. doi: 10.1186/2046-4053-4-1 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref031] 31.Moola S, Munn Z, Tufanaru C, Aromataris E, Sears K, Sfetcu R, et al. Chapter 7: Systematic reviews of etiology and risk. In: Aromataris E, Munn Z (editors). Joanna Briggs Institute Reviewer’s Manual. The Joanna Briggs Institute, 2017. Available from: https://wiki.jbi.global/display/MANUAL/Chapter+7%3A+Systematic+reviews+of+etiology+and+risk. [Google Scholar]

[pone.0255485.ref032] 32.Expanded Human Oral Microbiome Database. eHOMD. Available from: http://www.homd.org/.

[pone.0255485.ref033] 33.The NCBI Taxonomy Homepage. Available from: https://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/.

[pone.0255485.ref034] 34.Culture Collection University of Gothenburg. CCUG. Available from: https://ccug.se/.

[pone.0255485.ref035] 35.Wang N. How to conduct a meta-analysis of proportions in R: A comprehensive tutorial: Preprint; 2018. Available from: https://www.researchgate.net/publication/325486099_How_to_Conduct_a_Meta-Analysis_of_Proportions_in_R_A_Comprehensive_Tutorial. [Google Scholar]

[pone.0255485.ref036] 36.Barendregt JJ, Doi SA, Lee YY, Norman RE, Vos T. Meta-analysis of prevalence. J Epidemiol Community Health. 2013;67(11):974–8. doi: 10.1136/jech-2013-203104 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref037] 37.Wang D, Liu Q. Learning to draw samples: With application to amortized MLE for generative adversarial learning. A conference paper at ICLR. 2016. Available from: https://openreview.net/pdf/d1cab3a9a4271a9003c492bb98269ec3e66a8fb4.pdf. [Google Scholar]

[pone.0255485.ref038] 38.Card NA. Applied meta-analysis for social science research. New York, USA: Guilford Press. 2012. Available from: https://books.google.ae/books?id=hSYVGMrJgpkC&printsec=frontcover#v=onepage&q&f=false. [Google Scholar]

[pone.0255485.ref039] 39.DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials. 1986;7(3):177–88. doi: 10.1016/0197-2456(86)90046-2 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref040] 40.Higgins JPT, Green S. Cochrane handbook for systematic reviews of interventions. London, United Kingdom: Cochrane Collaboration. 2011. Available from: https://handbook-5-1.cochrane.org/. [Google Scholar]

[pone.0255485.ref041] 41.Deeks JJ, Higgins JPT, Altman DG. Chapter 9: Analysing data and undertaking meta-anlysis. In: Higgins JPT, Green S (editors). Cochrane handbook for systematic reviews of interventions. 2011. Available from: https://handbook-5-1.cochrane.org/index.htm#chapter_9/9_analysing_data_and_undertaking_meta_analyses.htm. [Google Scholar]

[pone.0255485.ref042] 42.Huedo-Medina TB, Sánchez-Meca J, Marín-Martínez F, Botella J. Assessing heterogeneity in meta-analysis: Q statistic or I2 index? Psychol Methods. 2006;11(2):193–206. doi: 10.1037/1082-989X.11.2.193 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref043] 43.Viechtbauer W, Cheung MW-L. Outlier and influence diagnostics for meta-analysis. Res Synth Methods. 2010;1(2):112–25. doi: 10.1002/jrsm.11 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref044] 44.Sakamoto M, Siqueira JF Jr., Rocas IN, Benno Y. Diversity of spirochetes in endodontic infections. J Clin Microbiol. 2009;47(5):1352–7. doi: 10.1128/JCM.02016-08 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref045] 45.Statistical tools for high-throughput data analysis. (STHDA). Quick principal component analysis data visualization—R software and data mining. Available from: http://www.sthda.com/english/.

[pone.0255485.ref046] 46.Flynn TR, Paster BJ, Stokes LN, Susarla SM, Shanti RM. Molecular methods for diagnosis of odontogenic infections. J Oral Maxillofac Surg. 2012;70(8):1854–9. doi: 10.1016/j.joms.2011.09.009 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref047] 47.George N, Flamiatos E, Kawasaki K, Kim N, Carriere C, Phan B, et al. Oral microbiota species in acute apical endodontic abscesses. J Oral Microbiol. 2016;8:30989. doi: 10.3402/jom.v8.30989 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref048] 48.Riggio MP, Aga H, Murray CA, Jackson MS, Lennon A, Hammersley N, et al. Identification of bacteria associated with spreading odontogenic infections by 16S rRNA gene sequencing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;103(5):610–7. doi: 10.1016/j.tripleo.2006.08.009 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref049] 49.Rôças IN, Baumgartner JC, Xia T, Siqueira JF. Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations. J Endod. 2006;32(12):1135–8. doi: 10.1016/j.joen.2006.05.001 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref050] 50.Rôças IN, Siqueira JF. Prevalence of new candidate pathogens Prevotella baroniae, Prevotella multisaccharivorax and as-yet-uncultivated Bacteroidetes clone X083 in primary endodontic infections. J Endod. 2009;35(10):1359–62. doi: 10.1016/j.joen.2009.05.033 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref051] 51.Rôças IN, Siqueira JF. Frequency and levels of candidate endodontic pathogens in acute apical abscesses as compared to asymptomatic apical periodontitis. PLoS One. 2018;13(1). doi: 10.1371/journal.pone.0190469 . 10.1371/journal.pone.0190469. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref052] 52.Sakamoto M, Rocas IN, Siqueira JF Jr., Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol. 2006;21(2):112–22. doi: 10.1111/j.1399-302X.2006.00270.x . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref053] 53.Siqueira JF, Rôças IN. Molecular detection and identification of Synergistes phylotypes in primary endodontic infections. Oral Dis. 2007;13(4):398–401. doi: 10.1111/j.1601-0825.2006.01301.x . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref054] 54.Siqueira JF Jr., Rocas IN. The microbiota of acute apical abscesses. J Dent Res. 2009;88(1):61–5. doi: 10.1177/0022034508328124 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref055] 55.Yang Q-B, Fan L-N, Shi Q. Polymerase chain reaction-denaturing gradient gel electrophoresis, cloning, and sequence analysis of bacteria associated with acute periapical abscesses in children. J Endod. 2010;36(2):218–23. doi: 10.1016/j.joen.2009.11.001 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref056] 56.Zhang W, Chen Y, Shi Q, Hou B, Yang Q. Identification of bacteria associated with periapical abscesses of primary teeth by sequence analysis of 16S rDNA clone libraries. Microb Pathog. 2020;141:103954. doi: 10.1016/j.micpath.2019.103954 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref057] 57.Yun KH, Lee H-S, Nam OH, Moon CY, Lee J-H, Choi SC. Analysis of bacterial community profiles of endodontically infected primary teeth using pyrosequencing. Int J Paediatr Dent. 2017;27(1):56–65. doi: 10.1111/ipd.12226 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref058] 58.Rolph HJ, Lennon A, Riggio MP, Saunders WP, MacKenzie D, Coldero L, et al. Molecular identification of microorganisms from endodontic infections. J Clin Microbiol. 2001;39(9):3282–9. doi: 10.1128/JCM.39.9.3282-3289.2001 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref059] 59.Vengerfeldt V, Špilka K, Saag M, Preem J-K, Oopkaup K, Truu J, et al. Highly diverse microbiota in dental root canals in cases of apical periodontitis (data of illumina sequencing). J Endod. 2014;40(11):1778–83. doi: 10.1016/j.joen.2014.06.017 . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref060] 60.Jacinto RC, Gomes BP, Desai M, Rajendram D, Shah HN. Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography. Oral Microbiol Immunol. 2007;22(6):403–10. doi: 10.1111/j.1399-302X.2007.00378.x . [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref061] 61.Hugenholtz P, Pitulle C, Hershberger KL, Pace NR. Novel division level bacterial diversity in a Yellowstone hot spring. J Bacteriol. 1998;180(2):366–76. doi: 10.1128/JB.180.2.366-376.1998 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref062] 62.Handal T, Caugant DA, Olsen I, Sunde PT. Bacterial diversity in persistent periapical lesions on root-filled teeth. J Oral Microbiol. 2009;1. doi: 10.3402/jom.v1i0.1946 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref063] 63.Siqueira JF Jr., Rôças IN. As-yet-uncultivated oral bacteria: Breadth and association with oral and extra-oral diseases. J Oral Microbiol. 2013;5. doi: 10.3402/jom.v5i0.21077 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref064] 64.Pace N.R. SDA, Lane D.J., Olsen G.J. The analysis of natural microbial populations by ribosomal RNA sequences. In: Marshall KC (editors). Advances in microbial ecology. Springer, Boston. 1986. 10.1007/978-1-4757-0611-6_1. [DOI] [Google Scholar]

[pone.0255485.ref065] 65.Siqueira JF Jr., Rôças IN. Microbiology and treatment of acute apical abscesses. Clin Microbiol Rev. 2013;26(2):255–73. doi: 10.1128/CMR.00082-12 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref066] 66.Gupta VK, Paul S, Dutta C. Geography, ethnicity or subsistence-specific variations in human microbiome composition and diversity. Front Microbiol. 2017;8(1162). doi: 10.3389/fmicb.2017.01162 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref067] 67.Bodor A, Bounedjoum N, Vincze GE, Erdeiné Kis Á, Laczi K, Bende G, et al. Challenges of unculturable bacteria: Environmental perspectives. Rev Environ Sci Biotechnol. 2020;19(1):1–22. 10.1007/s11157-020-09522-4. [DOI] [Google Scholar]

[pone.0255485.ref068] 68.Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature. 2016;533(7604):543–6. doi: 10.1038/nature17645 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref069] 69.Puspita ID, Kamagata Y, Tanaka M, Asano K, Nakatsu CH. Are uncultivated bacteria really uncultivable? Microbes Environ. 2012;27(4):356–66. doi: 10.1264/jsme2.me12092 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref070] 70.Harper-Owen R, Dymock D, Booth V, Weightman AJ, Wade WG. Detection of unculturable bacteria in periodontal health and disease by PCR. J Clin Microbiol. 1999;37(5):1469–73. doi: 10.1128/JCM.37.5.1469-1473.1999 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref071] 71.Siqueira JF Jr., Rôças IN, Rosado AS. Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by denaturing gradient gel electrophoresis fingerprinting approach. Oral Microbiol Immunol. 2004;19(6):363–70. doi: 10.1111/j.1399-302x.2004.00170.x [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref072] 72.Machado de Oliveira JC, Siqueira JF Jr., Rôças IN, Baumgartner JC, Xia T, Peixoto RS, et al. Bacterial community profiles of endodontic abscesses from Brazilian and USA subjects as compared by denaturing gradient gel electrophoresis analysis. Oral Microbiol Immunol. 2007;22(1):14–8. doi: 10.1111/j.1399-302X.2007.00311.x [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref073] 73.Borşan S-D, Ionică AM, Galon C, Toma-Naic A, Peştean C, Sándor AD, et al. High diversity, prevalence, and co-infection rates of tick-borne pathogens in ticks and wildlife hosts in an urban area in Romania. Front Microbiol. 2021;12(351). 10.3389/fmicb.2021.645002. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref074] 74.Saito D, de Toledo Leonardo R, Rodrigues JLM, Tsai SM, Höfling JF, Gonçalves RB. Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries. J Med Microbiol. 2006;55(1):101–7. doi: 10.1099/jmm.0.46212-0 [DOI] [PubMed] [Google Scholar]

[pone.0255485.ref075] 75.Rôças IN, Siqueira JF Jr., Root canal microbiota of teeth with chronic apical periodontitis. J Clin Microbiol. 2008;46(11):3599–606. doi: 10.1128/JCM.00431-08 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref076] 76.De Vrieze J, Ijaz UZ, Saunders AM, Theuerl S. Terminal restriction fragment length polymorphism is an "old school" reliable technique for swift microbial community screening in anaerobic digestion. Sci Rep. 2018;8(1):16818. doi: 10.1038/s41598-018-34921-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref077] 77.Lindström S, Rowe O, Timonen S, Sundström L, Johansson H. Trends in bacterial and fungal communities in ant nests observed with terminal-restriction fragment length polymorphism (T-RFLP) and next generation sequencing (NGS) techniques-validity and compatibility in ecological studies. PeerJ. 2018;6:e5289. 10.7717/peerj.5289. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref078] 78.de la Fuente G, Belanche A, Girwood SE, Pinloche E, Wilkinson T, Newbold CJ. Pros and cons of ion-torrent next generation sequencing versus terminal restriction fragment length polymorphism T-RFLP for studying the rumen bacterial community. PLoS One. 2014;9(7):e101435. 10.1371/journal.pone.0101435 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255485.ref079] 79.Mussano F, Ferrocino I, Gavrilova N, Genova T, Dell’Acqua A, Cocolin L, et al. Apical periodontitis: Preliminary assessment of microbiota by 16S rRNA high throughput amplicon target sequencing. BMC oral health. 2018;18(1):55. 10.1186/s12903-018-0520-8. . [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

Alaa Muayad Altaie

Basema Saddik

Mohammed Amjed Alsaegh

Sameh S M Soliman

Rifat Hamoudi

Lakshman P Samaranayake

Roles

Abstract

Objective

Methods

Results

Conclusion

Introduction

Methods

Protocol and registration

Search strategy and study selection

Data extraction

Risk of bias assessment

Resources used to identify the unculturability of bacteria

Statistical analysis

Results

Study selection

Fig 1. PRISMA flowchart of the study selection process showing inclusion and exclusion of the identified studies.

Study characteristics

Table 1. General characteristics of the included studies.

Risk of bias assessment

Unculturable bacteria in periapical abscess

Acute periapical abscess

Table 2. Extracted results from each study of periapical abscess.

Acute/chronic periapical abscess

Chronic periapical abscess

Outcome measures: Primary outcome measures: Diversity, abundance, and frequency of unculturable bacteria in periapical abscess

Fig 2. Forest plot depicting the pooled estimate of unculturable bacteria in periapical abscess.

Heterogeneity of studies

Outlying studies

Publication bias of studies

Outcome measures: Secondary outcome measures: Abundance and frequency of individual unculturable bacteria in periapical abscess

Table 3. Secondary outcome measures of individual unculturable bacteria with active abundance and frequency, and supplementary sequence technique variables.

Fig 3. Biplot of abundance and frequency dimensions of individual unculturable bacteria in periapical abscesses.

Fig 4. PCA of individual unculturable bacteria in periapical abscesses categorized by sequence techniques supplementary variable.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Andrej M Kielbassa

Roles

Author response to Decision Letter 0

Decision Letter 1

Andrej M Kielbassa

Roles

Acceptance letter

Andrej M Kielbassa

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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