Repertoire of the gut microbiota from stomach to colon using culturomics and next-generation sequencing
Authors: Morgane Mailhe
Anthony Levasseur
Pierre-Edouard Fournier
Emmanouil Angelakis
Jérémy Delerce
Catherine Robert
Jean-Christophe Lagier
Véronique Vitton
Davide Ricaboni
Didier Raoult
Dipankar Bachar
Jean-Michel Gonzalez
Frédéric Cadoret
Grégory Dubourg
Journal: BMC Microbiology (2018)
Discipline: MICROBIOLOGY
Keywords: Culturomics; Gut microbiota; Metagenomics; New species.
Affiliations: Aix Marseille Univ, IRD, MEPHI, IHU - Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005, Marseille, France., Department of Clinical and Biomedical Sciences Luigi Sacco, III Division of Infectious Diseases, University of Milano, Via GB Grassi, 74, 20157, Milan, Italy., Service de Gastroentérologie, Hôpital Nord, Assistance Publique-Hôpitaux de Marseille, 13915, Marseille, France., Aix Marseille Univ, IRD, VITROME, IHU - Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005, Marseillle, France., Aix Marseille Univ, IRD, MEPHI, IHU - Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005, Marseille, France. .

Abstract:  Background: Most studies on the human microbiota have analyzed stool samples, although a large proportion of the absorption of nutrients takes place in upper gut tract. We collected samples from different locations along the entire gastrointestinal tract from six patients who had simultaneously undergone upper endoscopy and colonoscopy, to perform a comprehensive analysis using culturomics with matrix assisted laser desorption ionisation - time of flight (MALDI-TOF) identification and by metagenomics targeting the 16S ribosomal ribonucleic acid (rRNA) gene. Results: Using culturomics, we isolated 368 different bacterial species, including 37 new species. Fewer species were isolated in the upper gut: 110 in the stomach and 106 in the duodenum, while 235 were isolated from the left colon (p &lt; 0.02). We isolated fewer aero-intolerant species in the upper gut: 37 from the stomach and 150 from the left colon (p &lt; 0.004). Using metagenomics, 1,021 species were identified. The upper gut microbiota was revealed to be less rich than the lower gut microbiota, with 37,622 reads from the stomach, 28,390 from the duodenum, and 79,047 from the left colon (p &lt; 0.009). There were fewer reads for aero-intolerant species in the upper gut (8,656 in the stomach, 5,188 in the duodenum and 72,262 in the left colon, p &lt; 0.02). Patients taking proton pump inhibitors (PPI) were then revealed to have a higher stomach pH and a greater diversity of species in the upper digestive tract than patients not receiving treatment (p &lt; 0.001). Conclusion: Significant modifications in bacterial composition and diversity exist throughout the gastrointestinal tract. We suggest that the upper gut may be key to understanding the relationship between the gut microbiota and health. 

Retracted: No

PubPeer comments:
 This paper is part of a set of 35 papers relating to the protocol approved by the by the ethics committee of the Institut Hospitalo-Universitaire Mu00e9diterranu00e9e Infection (IHU) under the **number 2016-010**. We found some of the same authors in the 35 papers. **One protocol approved under the number nu00b02016-010:** Authors mentioned this is the approval number of the IHU ethics committee: *u201cApproval (agreement no. 2016-010) was obtained from the local ethics committee of the IHU-Mediterranu00e9e Infection (Marseille, France)u201d u2013 source: Article #5.* In accordance with other articles and documents from the same institute, the number 2016u2013010 cover only one protocol using this number. The 35 papers should be considered as part of a unique protocol nu00b02016-010. **Ethics approval obtained in or after 2016.** In accordance with the approval number (2016-010), readers could assume with a good confidence that the approval number has been obtained in or after 2016. **When did the participants recruitment start? Is the ethics approval retrospective?** The recruitment period is different depending on the paper. In 4 papers (articles #2, #15,#22 and #33), the recruitment of participants have been done in 2015, before the ethical approval date in or after 2016. 1 of this paper (article #2) describe a study in 1 healthy participant. Study on healthy participant is prospective and an intervention (stool collection) was done. Retrospective ethics committee approval for a prospective interventional protocol is a serious breach of the Helsinki Declaration. In accordance with Editors guidances and Helsinki Declaration, if a prospective study has not been granted ethics committee approval prior to commencing, retrospective ethics approval cannot be obtained and it may not be possible to consider the manuscript for peer review. Accepting retrospective ethics approval for interventional prospective study would promote these practices and this risk should be considered very seriously by Editors. **Children in the study** In 2 papers, authors mentioned that children have been recruited (1 healthy child in 1 paper, and a 2-month-old child with rotavirus gastroenteritis in another paper). As a reminder, children are considered as a vulnerable population. **In France, only certified IRB (Comitu00e9 de Protection des Personnes) are qualified to approve prospective study in humans.** In accordance with French law, only a certified IRB can approve research in human. Approval from the certified IRB must be obtained before to recruit participants Furthermore, children are considered as a vulnerable population with additional protection. Among the 35 papers, no paper mentions any approval from a Comitu00e9 de Protection des Personnes. The IHU ethics committee is not on the list of the certified IRB in France and donu2019t have the ability to validate a prospective study in human, especially in healthy volunteers or children. In accordance with Helsinki Declaration and Editors guidances, a clinical research must be approved according to the relevant laws of the countries of the participants. The Editors should clarify this point and ensure that an approval from the Comitu00e9 de Protection des Personnes has been obtained for this protocol, especially considering the recruitment of health volunteers (adult and children). The date of the approval, the scope of the protocol and the recruitment dates of participants should be clarified for every paper. **Different type of participants:** -t1 paper describes a study recruiting 6 patients with an upper endoscopy (article #1) -t1 paper describes a study recruiting 2 patients with an ischemic heart disease and a digestive endoscopy (article #4) -t1 paper describes study recruiting 44 heathy participants (article #5) -tThe 32 other papers describe studies recruiting 1 single participant only. This single participant is not the same one in the 32 papers. Depending on the paper, the single participant can be a patient or a healthy volunteer, a man or a woman, an adult or a child. Participants are not recruited at the same time or during a common period of time. We can note that recruitment occurred in 2015, 2016 and 2017. **Different types of objectives and samples** -tIn one paper (article 5), the objective of the study is to build a new passive-filtration-based culture device combined with rapid identification with a new electron microscope (Hitachi TM4000) for the detection and culture of Treponema species. This study is done on 44 healthy participants and oral samples are collected -tFor the other 34 studies, the objectives are to describe new bacterial species, to present main characteristic of a bacteria. The studies are mostly done on 1 participant only. Urine sample, gastrointestinal tract sample or stools samples are collected depending on the paper. **Participants located in different countries?** The readers could presume that the samples have been collected in France for almost all the papers. However, for 2 papers, the authors have noted the nationality of the participant (Senegal in article #6 and Niger in article #32). There is no mention about the site where the sample have been collected and no mention about approval of the relevant Ethics Committees in Senegal and Niger. Could the authors clarify where the samples have been collected for these 2 papers and if a local and appropriate Ethics approval have been obtained. **How the same protocol can cover so many different interventions, different study designs and different types of participants?** Could the authors disclose the protocol of the study 2016-010 ? Or, at least, explain how a unique protocol can cover the diversity of situations described by the 35 papers ? The Editors should also clarify how the same protocol can cover so many different type of study design, inclusion criteria and exclusion criteria. Editors should also note that using the same Ethics Committee approval number for different types of design, interventions and participants are something already noted in pubpeer relating some scientific publications of some of the authors. [Pubpeer - Example 1] [Pubpeer - Example 2] **Salami publication** According to the article  [Salami publication: definitions and examples]  : *u201cSalami publication can be roughly defined as a publication of two or more articles derived from a single study. Articles of such type report on data collected from a single study split into several segments just large enough to gain reasonable results and conclusions, also known as u201cminimal publishable unitu201d.* (...) * From the publication ethics point of view, it gives undeserved credit to authors, misleads the scientific community and directly influences clinical practice by distorting medical evidence. * **LIST OF THE 35 PAPERS COVERED BY THE SAME ETHICS APPROVAL NUMBER 2016-010** 1.t[BMC microbiology - Repertoire of the gut microbiota from stomach to colon using culturomics and next-generation sequencing] 2.t[Current Microbiology - Fenollaria timonensis sp. nov., A New Bacterium Isolated from Healthy Human Fresh Stool] 3.t[Human Microbiome Journal - Peptoniphilus duodeni sp. nov., a new bacterial species identified in human duodenum] 4.t[Human Microbiome Journal - Olsenella provencensis sp. nov., Olsenella phocaeensis sp. nov., and Olsenella mediterranea sp. nov. isolated from the human colon] 5.t[Journal of Clinical Microbiology - Passive filtration, rapid scanning electron microscopy and MALDI-TOF MS for Treponema culture and identification from the oral cavity] 6.t[New microbes and new infections - Pseudoruminococcus massiliensis gen. nov., sp. nov., a new bacterium isolated from the human gut microbiota] 7.t[New microbes and new infections - Dialister massiliensis sp. nov., a new bacterium isolated from the human gut] 8.t[New microbes and new infections - Parabacteroides massiliensis sp. nov., a new bacterium isolated from a fresh human stool specimen] 9.t[New microbes and new infections - Genome sequence and description of Traorella massiliensis gen. nov., sp. nov., a new bacterial genus isolated from human left colon] 10.t[New Microbes and New Infections - u201cTidjanibacter massiliensisu201d gen. nov., sp. nov., a new bacterial species isolated from the 1 human colon 2] 11.t[New Microbes and New Infections - u201cTraorella massiliensisu201d gen. nov., sp. nov., a new bacterial species isolated from the left 1 colon of a 76-year-old woman 2] 12.t[New Microbes and New Infections - Non-contiguous finished genome sequence and description of Fusobacterium massiliense 1 sp. nov. isolated from the human duodenum 2] 13.t[New microbes and new infections - Massilioclostridium coli gen. nov., sp. nov., a new member of the Clostridiaceae family isolated from the left colon of a 27-year-old woman] 14.t[New microbes and new infections - Varibaculum timonense sp. nov., a new bacterial species isolated from human stool sample] 15.t[New microbes and new infections - Corynebacterium bouchesdurhonense sp. nov., and Corynebacterium provencense sp. nov., two new species isolated from obese patients] 16.t[New microbes and new infections - u2018Angelakisella massiliensisu2019 gen. nov., sp. nov., a new bacterial species isolated from human ileum] 17.t[New microbes and new infections - Noncontiguous finished genome sequence and description of Fusobacterium massiliense sp. nov. isolated from human duodenum] 18.t[New microbes and new infections - u2018Duodenibacillus massiliensisu2019 gen. nov., sp. nov., a new species identified in human duodenum] 19.t[New microbes and new infections - u2018Colibacter massiliensisu2019 gen. nov., sp. nov., a new bacterial species isolated from human left colon] 20.t[New microbes and new infections - u2018Ileibacterium massilienseu2019gen. nov., sp. nov., a new bacterial species isolated from human ileum of a patient with Crohn disease] 21.t[New microbes and new infections - Genome sequence and description of Bacteroides bouchesdurhonensis sp. nov., a new anaerobic bacterium isolated from the human gut] 22.t[New microbes and new infections - Olsenella timonensis sp. nov., a new bacteria species isolated from the human gut microbiota] 23.t[New microbes and new infections - Massilicoli timonensis sp. nov., a new bacterium isolated from the human microbiota] 24.t[New microbes and new infections - Noncontiguous finished genome sequence and description of Mediterranea massiliensis gen. nov., sp. nov., a new member of the Bacteroidaceae family isolated from human colon] 25.t[New microbes and new infections - Corynebacterium urinapleomorphum sp. nov., a new bacterial species isolated from human urine sample] 26.t[New microbes and new infections - u2018Caecibacter massiliensisu2019 gen. nov., sp. nov., isolated from human right colon] 27.t[New microbes and new infections - u2018Colidextribacter massiliensisu2019 gen. nov., sp. nov., isolated from human right colon] 28.t[New microbes and new infections - u2018Merdibacter massiliensisu2019 gen. nov., sp. nov., isolated from human ileum] 29.t[New Microbes and New Infections - Genome sequence and description of Urinicoccus timonensis gen. nov., sp. nov., a new bacterium isolated from a human stool sample] 30.t[New microbes and new infections - Description of Clostridium cagae sp. nov., Clostridium rectalis sp. nov. and Hathewaya massiliensis sp. nov., new anaerobic bacteria isolated from human stool samples] 31.t[New microbes and new infections - Massilistercora timonensis gen. nov., sp. nov., a new bacterium isolated from the human microbiota] 32.t[New microbes and new infections - Anaerococcus marasmi sp. nov., a new bacterium isolated from human gut microbiota] 33.t[New microbes and new infections - Massilimicrobiota timonensis gen. nov., sp. nov., a new bacterium isolated from the human gut microbiota] 34.t[Scientific reports - Draft genome and description of Merdibacter massiliensis gen. nov., sp. nov., a new bacterium genus isolated from the human ileum] 35.t[Scientific reports - Colibacter massiliensis gen. nov. sp. nov., a novel Gram-stain-positive anaerobic diplococcal bacterium, isolated from the human left colon] ,