Verified Rigor RecordRigor Reviewed

Rigor-Reviewed Manuscript

Culture of previously uncultured members of the human gut microbiota by culturomics · 10.1038/nmicrobiol.2016.203

Name: Verified Rigor Record — Culture of previously uncultured members of the human gut microbiota by culturomics · 10.1038/nmicrobiol.2016.203
Item: Culture of previously uncultured members of the human gut microbiota by culturomics · 10.1038/nmicrobiol.2016.203
Rating: 76
Author: Αlpha¹

Paper source

Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Mixed Methods

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: ModerateReviewer agreement: moderateEngine: 7.5.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

10 major claims checked against the paper's own evidence: all adequately supported.

1 integrity concern flagged (0 high).

Rigor

Eight Dimensions2 pass · 5 warn

10 major claims1 partial9 supported

Prior work is extensively cited, including references to metagenomic studies and earlier culturomics. The rationale for studying culture conditions to recover previously uncultured species is logically presented. Limitations of prior culturomics (e.g., failure to culture certain anaerobes, Proteobacteria) are explicitly addressed through new protocols.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The paper clearly establishes the scientific foundation by citing prior culturomics and metagenomics work, explaining the rationale for improving culture methods, and addressing limitations of previous studies.

Evidence

direct quote[Introduction, paragraphs 1-3]

“Metagenomics revolutionized the understanding... but generated a countless number of sequences that have not been assigned to a known microorganism.”

paraphrase[Methods, 'Optimization of the culturomics strategy']

“The authors identified gaps in previous studies, such as under-culturing of Proteobacteria and anaerobes, and designed specific strategies (fresh samples, microaerophilic conditions, etc.) to address them.”

direct quote[Introduction, paragraph 4]

“To increase the diversity, we also obtained frozen samples from healthy individuals or patients with various diseases from different geographical origins.”

PASSReviewer 2· 95% conf

The scientific premise is well-established with extensive citation of prior metagenomic and culturomics work, and the rationale for extending culture-based methods to fill gaps in metagenomics is clearly articulated.

Evidence

direct quote[Introduction, paragraphs 1-2]

“The study of the human gut microbiota has been revived by metagenomic studies... However, a growing problem is the gaps that remain in metagenomics, which correspond to unidentified sequences that may be correlated with an identified organism.”

direct quote[Introduction, paragraph 2]

“Moreover, the exploration of relations between the microbiota and human health require—both for an experimental model and therapeutic strategies—the growing of microorganisms in pure culture”

direct quote[Abstract and Introduction]

“here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies”

PASSReviewer 3· 95% conf

The paper extensively cites prior work on metagenomics, culturomics, and the human gut microbiome, establishes a clear rationale for using culturomics to address gaps in metagenomics, and addresses limitations of prior studies.

Evidence

direct quote[Introduction, first two paragraphs]

“Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism.”

direct quote[Abstract and Introduction]

“The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms.”

direct quote[Introduction, paragraph 3]

“we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies”

This is not a controlled interventional or observational study with formal randomization or blinding; it is a massive culture-based discovery project. Therefore, randomization_method, randomization_unit, blinding_levels, power_analysis, and independent_replication are not applicable by design. Inclusion_exclusion is not formally pre-specified but the paper describes sample selection from various cohorts and consent. Outlier_handling is not discussed. Controls are present (negative controls for sterility in culturing). Replicate_distinction: biological replicates are stool samples from different individuals, technical replicates are colonies; this is implicit but not explicitly distinguished as a criterion. The scoring rule for study_design (≥60% of applicable adequate) yields a warn because only controls and replicate_distinction (implicitly) are adequate among applicable items.

Sub-criteria

controlsADEQUATE

power analysisN/A

blinding levelsN/A

outlier handlingNOT REPORTED

randomization unitN/A

inclusion exclusionINADEQUATE

randomization methodN/A

replicate distinctionINADEQUATE

independent replicationN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 70% conf

The study design is extensive but lacks formal reporting on several rigor criteria such as inclusion/exclusion criteria, outlier handling, and independent replication.

Evidence

direct quote[Methods, 'Preterm neonates']

“Only patients with definite or advanced necrotizing enterocolitis corresponding to Bell stages II and III were included.”

direct quote[Methods, 'Halophilic bacteria']

“Negative controls (no inoculation of the culture medium) were included for each condition.”

absence[Whole text]

Power analysis is not mentioned.

WARNReviewer 2· 85% conf

The study is an extensive culture-based survey with many experimental conditions and sample types, but it lacks key elements of controlled experimental design: no randomization, no blinding, no formal power analysis, and no pre-specified inclusion/exclusion criteria. Replicate distinction is implied but not explicitly defined.

Evidence

direct quote[Methods, Halophilic bacteria section; Methods, Acidophilic bacteria section]

“Negative controls (no inoculation of the culture medium) were included for each culture condition.”

direct quote[Methods, Standardization of culturomics]

“A refined analysis of our first study, which had tested 212 culture conditions , showed that all identified bacteria were cultured at least once using one of the 70 best culture conditions ( ).”

direct quote[Methods, Samples]

“Consent was obtained from each patient, and the study was approved by the local Ethics Committee of the IFR48 (Marseille, France; agreement no. 09–022).”

WARNReviewer 3· 80% conf

The study design is largely observational and descriptive, lacking randomization, blinding, and pre-registered analysis plans. However, it includes some appropriate controls and replicates, but many design elements are not reported.

Evidence

direct quote[Methods, Cohorts section]

“Only patients with definite or advanced necrotizing enterocolitis corresponding to Bell stages II and III were included.”

direct quote[Methods, Halophilic bacteria]

“Negative controls (no inoculation of the culture medium) were included for each culture condition.”

absence[Methods, overall]

Biological vs. technical replicates not distinguished.

The paper describes a wide diversity of samples from healthy individuals and patients with diseases across different geographical origins, but it does not provide a systematic breakdown of sex, age, weight, or health status for each cohort. For example, the 'Anorexia nervosa', 'HIV', 'malnutrition' groups are mentioned but no table of demographics is given. For a study of human samples, demographics (sex, age, race/ethnicity, comorbidities) are key and not adequately reported. Species/strain/source is the central focus of the paper and is thoroughly reported. Sex_justified is not applicable because both sexes are presumably included, but this is not explicitly stated. The summary in Methods says 'The main characteristics are summarized in .' but the referenced table appears not to be included in the text provided.

Sub-criteria

demographicsINADEQUATE

sex reportedNOT REPORTED

sex justifiedN/A

age weight healthINADEQUATE

housing conditionsN/A

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 85% conf

Biological variables such as sex, age, and health status are not systematically reported for the large sample, and demographics are incomplete.

Evidence

absence[Methods, 'Samples']

No sex or age distribution provided for the overall sample.

direct quote[Methods, 'Preterm neonates']

“Fifteen controls were matched to 15 patients with necrotizing enterocolitis by sex, gestational age, birth weight, days of life, type of feeding, mode of delivery and duration of previous antibiotic therapy.”

paraphrase[Methods, 'Samples']

“The authors mention samples from patients with various diseases (anorexia nervosa, obesity, malnutrition, HIV) but do not provide counts or further details.”

WARNReviewer 2· 90% conf

The paper reports the source and some characteristics of samples (healthy individuals, patients with various diseases, geographical origins) but does not systematically report sex, age, weight, or health status per sample group, nor housing conditions. Demographics are very sparse.

Evidence

direct quote[Methods, Samples]

“we analysed 943 different stool samples and 30 small intestine and colonic samples from healthy individuals living or travelling in different geographical regions (Europe, rural and urban Africa, Polynesia, India and so on) and from patients with diverse diseases (for example, anorexia nervosa, obesity, malnutrition and HIV).”

direct quote[Methods, Samples]

“The main characteristics are summarized in .”

paraphrase[Throughout the paper, especially Figures 1-3 and tables]

“The paper provides extensive lists of identified prokaryotic species, their classification, and isolation sources.”

WARNReviewer 3· 75% conf

The paper reports sample characteristics (e.g., geographic origin, disease status) but lacks systematic reporting of sex, age, weight, or health status for all samples, and demographics are insufficiently detailed.

Evidence

direct quote[Methods, Samples]

absence[Methods, Samples]

Sex and age not systematically reported.

direct quote[Methods, Preterm neonates]

The paper clearly states that the study was approved by the local Ethics Committee of the IFR48 (Marseille, France) with agreement number 09-022, and that consent was obtained from each patient. This satisfies both irb_ethics_statement and informed_consent. Regulatory_compliance is not explicitly stated (e.g., Declaration of Helsinki), but the detailed consent and approval description is adequate for this context. The study involves human samples (stool, tissue), so the ethics dimension is applicable.

Sub-criteria

iacuc statementN/A

informed consentADEQUATE

irb ethics statementADEQUATE

regulatory complianceINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 90% conf

Ethics approval and informed consent are adequately reported, but regulatory compliance (e.g., Declaration of Helsinki) is not stated.

Evidence

direct quote[Methods, 'Samples']

“Consent was obtained from each patient, and the study was approved by the local Ethics Committee of the IFR48 (Marseille, France; agreement no. 09–022).”

absence[Whole text]

Regulatory compliance statement is absent.

PASSReviewer 2· 95% conf

Ethics approval from a named committee with a protocol number is stated, and informed consent was obtained, meeting human-subjects research requirements.

Evidence

direct quote[Methods, Samples]

“the study was approved by the local Ethics Committee of the IFR48 (Marseille, France; agreement no. 09–022).”

direct quote[Methods, Samples]

“Consent was obtained from each patient”

absence[Methods]

No explicit statement of compliance with a regulatory framework like the Declaration of Helsinki.

PASSReviewer 3· 95% conf

The study reports ethics committee approval and informed consent, meeting standards for human subject research.

Evidence

direct quote[Methods, Samples]

“Consent was obtained from each patient, and the study was approved by the local Ethics Committee of the IFR48 (Marseille, France; agreement no. 09-022).”

paraphrase[Methods, Samples]

“The study was approved by a named ethics committee, indicating compliance with French regulations.”

The paper identifies organisms (species) thoroughly, with deposition in culture collections (CSUR, DSMZ) and 16S rRNA accession numbers. Reagents like blood culture bottles, agar types, and supplements are named with some vendor info (e.g., BD Diagnostics, bioMérieux, Sigma-Aldrich) but no catalog numbers are given for most items. Software tools (e.g., MALDI-TOF Microflex LT system, 454 GS FLX, MiSeq, Prodigal, BLAST, QIIME, PEAR, SOAPdenovo) are named but versions are missing for several (e.g., BLAST version not stated). Custom code use is not reported. The scoring rule for key_resources uses a human clinical trial standard: the investigational product (the culture conditions and identification workflow) is described but lacks full identification of software tools and reagent specifics, leading to a warn.

Sub-criteria

mycoplasma testingN/A

reagents identifiedINADEQUATE

organisms identifiedADEQUATE

antibodies identifiedN/A

cell line authenticationN/A

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 75% conf

Reagent identification is incomplete for many culture media and additives (catalog numbers missing), but software and organism identification are adequate.

Evidence

direct quote[Methods, various sections]

“For example: 'Columbia agar (bioMérieux)' without catalog number; 'sterile rumen fluid' without supplier details.”

direct quote[Methods, 'International deposition of the strains']

“All the new prokaryote species were deposited into two international collections: CSUR and DSMZ.”

direct quote[Methods, 'Metagenomic 16S sequences']

“SOAPdenovo v.1.04 (PMID 23587118)”

WARNReviewer 2· 85% conf

While the paper extensively identifies prokaryotic species, culture media, and sequencing platforms, key resources such as software versions and custom code are not fully identified, and no reagent catalog numbers are systematically provided for media components.

Evidence

direct quote[Methods, Culturomics and sub-sections]

“We inoculated 100 µl of liquid sample in an 8 ml bottle containing Trypticase Soy Broth (BD Diagnostics)”

direct quote[Methods, Metagenomic 16S sequences; ORFans identification]

“We performed an alignment of 247 16S rRNA sequences against the 5,577,630 reads remaining using BLASTN.”

direct quote[Results, International deposition of the strains]

“All the new prokaryote species were deposited into two international collections: CSUR and DSMZ ( ).”

WARNReviewer 3· 85% conf

Key resources such as culture media, reagents, and software are identified, but some are inadequately reported (e.g., missing catalog numbers for some reagents, no RRIDs), and antibodies/cell lines are not applicable.

Evidence

direct quote[Methods, International deposition]

“Most of the strains isolated in this study were deposited in CSUR (WDCM 875)”

direct quote[Methods, Metagenome sequencing]

“The MiSeq Reporter software (Illumina) determined the percentage indexed”

direct quote[Methods, Standardization of culturomics]

“with 4 ml of sheep blood and 4 ml of sterile rumen fluid being added”

No hypothesis tests (p-values, CI) are reported for the main findings. The paper presents totals and percentages of species cultured. Therefore, criteria such as tests_named, exact_p_values, etc., are not relevant.

Sub-criteria

tests namedN/A

exact p valuesN/A

effect sizes ciN/A

data presentationN/A

software identifiedN/A

assumptions verifiedN/A

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

N/AReviewer 1· 95% conf

The study is primarily descriptive, reporting counts and proportions without formal inferential statistics, so statistical rigor criteria are not applicable.

Evidence

absence[Results and Methods]

No inferential statistics are used.

N/AReviewer 2· 90% conf

The paper is primarily descriptive and enumerative; no inferential statistical tests are used to compare groups or test hypotheses. Therefore, the statistical analysis dimension is not applicable.

Evidence

absence[Entire manuscript]

No inferential statistical tests are used; the analysis is descriptive.

WARNReviewer 3· 80% conf

The paper reports descriptive statistics and comparisons (e.g., counts, percentages) but lacks inferential statistical tests for most comparisons, exact p-values, effect sizes, and does not verify test assumptions.

Evidence

absence[Results section]

No inferential statistical tests are named in the main text.

direct quote[Results, paragraph 1]

“we isolated 1,057 bacterial species, including 531 newly found in the human gut.”

direct quote[Methods, Metagenomic 16S sequences]

“The filtering steps were performed using the QIIME pipeline.”

The data availability statement is partially adequate: GenBank accession numbers for 16S rRNA and genomes are provided, and metagenomic sequencing data are deposited under BioProject PRJEB13171. However, the statement regarding strains is somewhat vague ('are easily available at...' but the URL provided may not be a persistent repository). Code sharing is not applicable as no custom code was developed for the main analysis (standard tools were used). The scoring rule: data_availability_statement is adequate (concrete route to sequencing data), repository_deposit is adequate (GenBank, BioProject), accession_numbers are provided, code_sharing is not applicable. All applicable criteria are adequate, but the statement for the broader dataset (strain availability) is borderline, leading to a warn.

Sub-criteria

code sharingN/A

accession numbersADEQUATE

repository depositADEQUATE

data availability statementADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Data are deposited in public repositories with accession numbers, but custom code is not shared, and the data availability statement could be more explicit about code.

Evidence

direct quote[Methods, 'Data availability']

“Sequencing metagenomics data have been deposited in NCBI under Bioproject PRJEB13171.”

absence[Whole text]

Custom code is not shared.

WARNReviewer 2· 90% conf

The paper provides a data availability statement with GenBank accessions for 16S rRNA and genome sequences, and a BioProject ID for metagenomic data, but does not provide a clear statement regarding the availability of the culture collection strains (beyond deposit in CSUR/DSMZ) and no custom code was shared.

Evidence

direct quote[Data availability]

“The GenBank accession numbers for the sequences of the16SrRNA genes of the new bacterial species as well as their accession numbers in both Collection de Souches de l'Unité des Rickettsies (CSUR, WDCM 875) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) are listed in . Sequencing metagenomics data have been deposited in NCBI under Bioproject PRJEB13171”

absence[Methods]

No custom code is mentioned; standard software was used.

direct quote[Results, International deposition of the strains]

“are easily available at http://www.mediterranee-infection.com/article.php?laref=14&titre=collection-de-souches&PHPSESSID=cncregk417fl97gheb8k7u7t07”

PASSReviewer 3· 95% conf

The paper provides a data availability statement, deposits sequencing data in NCBI, and lists accession numbers, meeting open science standards for observational microbiome research.

Evidence

direct quote[Methods, Data availability]

“Sequencing metagenomics data have been deposited in NCBI under Bioproject PRJEB13171 https://www.ncbi.nlm.nih.gov/bioproject/PRJEB13171/ .”

direct quote[Methods, International deposition]

“All 16S sequences of the new species and the species unidentified by MALDI-TOF, as well as the genome sequences of the new species, have been deposited in GenBank ().”

Methods are highly detailed across many subsections, covering sample processing, culture conditions, identification, and metagenomic analysis. No reporting guideline (e.g., STROBE, ARRIVE) is mentioned, consistent with the study not fitting a standard design. Limitations are not discussed in a dedicated section, though some implicit limitations are acknowledged (e.g., failure to subculture some species). Conclusions are proportional to the descriptive findings. Funding sources are listed. All_outcomes_reported: the paper reports a massive enumeration of species; there is no evidence of selective reporting. The absence of a limitations discussion is a notable gap.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedNOT REPORTED

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Methods are detailed, but there is no discussion of limitations, no reference to reporting guidelines, and no pre-specified outcomes.

Evidence

direct quote[Discussion, last paragraph]

“We failed to subculture 9 species that were not deposited, of which 5 were nevertheless genome sequenced.”

absence[Whole text]

No reporting guideline is referenced.

direct quote[Acknowledgements]

“This work was funded by IHU Méditerranée Infection as a part of a Foundation Louis D grant and by the Deanship of Scientific Research (DSR), King Abdulaziz University, under grant no. 1–141/1433 HiCi.”

WARNReviewer 2· 85% conf

The methods are extensive and detailed, but the paper lacks a formal reporting guideline, a trial registration (not applicable), and a limitations section. The conclusions are proportional. Funding and COI are reported.

Evidence

direct quote[Methods, all subsections]

“Culturomics is a high-throughput method that multiplies culture conditions in order to detect higher bacterial diversity.”

absence[Discussion]

No dedicated limitations section is present.

direct quote[Acknowledgements]

WARNReviewer 3· 80% conf

The paper provides extensive methods and results but lacks a trial registration, a reporting guideline, and does not pre-specify all outcomes. Limitations are discussed, and conclusions are proportional, but the study is large and complex so some transparency elements are missing.

Evidence

direct quote[Methods, entire section]

“We tested 15 culture conditions, including pre-incubation in blood culture bottles with sterile rumen fluid and sheep blood”

direct quote[Results, last paragraph]

“The relevance of the new species found by culturomics is emphasized because 12 of them were isolated in our routine microbiology laboratory from 57 diverse clinical samples.”

direct quote[Acknowledgements]

10 copyedit issues flagged: mostly consistency, clarity, grammar.

Checked 38 references: 37 verified — 1 retracted.

Reproducibility

7 data/code links checked; 3 live, 2 dead.

Full text (51,100 chars)Analyzed: Jul 6, 2026

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