Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization‚ÄìTime of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity
Authors: Anthony Levasseur
Yusuke Ominami
Anthony Fontanini
Akiko Hisada
Aurelia Caputo
Jean-Christophe Lagier
Didier Raoult
Jacques Bou Khalil
Souad Belkacemi
Saber Khelaifia
Journal: Journal of Clinical Microbiology (2019)
Discipline: MICROBIOLOGY
Keywords: Treponema; culture; microscopy; oral cavity; passive filtration.
Affiliations: Microbes Evolution Phylogeny, and Infections, Aix-Marseille Universit√©, Institut de Recherche pour le D√©veloppement, Assistance Publique-Hopitaux de Marseille, Marseille, France., Institut Hospitalo-Universitaire M√©diterran√©e Infection, Marseille, France., Science and Medical Systems Business Group, Hitachi High-Technologies Corp., Tokyo, Japan., Research and Development Group, Hitachi, Ltd., Saitama, Japan., H√¥pital de la Timone, Assistance Publique-Hopitaux de Marseille, Marseille, France., UMR VITROME, Aix Marseille Universit√©, Institut de Recherche pour le D√©veloppement, SSA, Assistance Publique-Hopitaux de Marseille, IHU-M√©diterran√©e Infection, Marseille, France., Microbes Evolution Phylogeny, and Infections, Aix-Marseille Universit√©, Institut de Recherche pour le D√©veloppement, Assistance Publique-Hopitaux de Marseille, Marseille, France .

Abstract:  We present here 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 from the human oral cavity. Of the 44 oral samples cultivated, 15 (34%) were found to be positive for Treponema using electron microscopy and were also culture positive. All were subcultured on agar plates; based on genome sequencing and analyses, 10 were strains of Treponema pectinovorum and 5 were strains of Treponema denticola The 29 samples that were negative for Treponema remained culture negative. In addition, 14 Treponema species ordered from the DSMZ collection were cultured in the T-Raoult culture medium optimized here. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used and 30 novel spectra were added to the MALDI-TOF MS database. We have successfully developed a new and effective method for treponemal detection, culture, and identification. 

Retracted: No

PubPeer comments:
 This paper mentioned an approval under the IHU ethics committee no. 2016-010. Several others papers, published in different type of journals, mention the same approval number, for different type of test, indications or patients. It would be in the public interest if the authors can explain how the approval number for one protocol can cover so many different types of situation. Otherwise, in this paper, the authors mentioned that a study has been performed in healthy participants. In France, prospective study on healthy participants MUST get an approval from certified IRB (Comitu00e9 de Protection des Personnes) before to start. The local IHU Ethics Committee is not on the list of the certified IRB ha. It would be in the public interest to understand if the authors have requested and obtained an approval from a Comitu00e9 de Protection des Personnes before to start the recruitment of participants. It would be in the public interest if the authors can explain why the study did not mention any approval from a Comitu00e9 de Protection des Personnes before to start the recruitment. According Editors guidances and Helsinki Declaration, a study should be in compliance with the national law and get the proper authorization before the study start date (recruitment of participants). I would be in the public interest if the editor can clarify these points. [Repertoire of the gut microbiota from stomach to colon using culturomics and next-generation sequencing] , 
 This paper is part of a set of 34 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 34 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 34 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,#21 and #32), the recruitment of participants have been done in 2015, before the ethical approval date in or after 2016. One 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 34 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 31 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 33 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 34 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 34 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] 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 - u2018Duodenibacillus massiliensisu2019 gen. nov., sp. nov., a new species identified in human duodenum] 18.t[New microbes and new infections - u2018Colibacter massiliensisu2019 gen. nov., sp. nov., a new bacterial species isolated from human left colon] 19.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] 20.t[New microbes and new infections - Genome sequence and description of Bacteroides bouchesdurhonensis sp. nov., a new anaerobic bacterium isolated from the human gut] 21.t[New microbes and new infections - Olsenella timonensis sp. nov., a new bacteria species isolated from the human gut microbiota] 22.t[New microbes and new infections - Massilicoli timonensis sp. nov., a new bacterium isolated from the human microbiota] 23.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] 24.t[New microbes and new infections - Corynebacterium urinapleomorphum sp. nov., a new bacterial species isolated from human urine sample] 25.t[New microbes and new infections - u2018Caecibacter massiliensisu2019 gen. nov., sp. nov., isolated from human right colon] 26.t[New microbes and new infections - u2018Colidextribacter massiliensisu2019 gen. nov., sp. nov., isolated from human right colon] 27.t[New microbes and new infections - u2018Merdibacter massiliensisu2019 gen. nov., sp. nov., isolated from human ileum] 28.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] 29.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] 30.t[New microbes and new infections - Massilistercora timonensis gen. nov., sp. nov., a new bacterium isolated from the human microbiota] 31.t[New microbes and new infections - Anaerococcus marasmi sp. nov., a new bacterium isolated from human gut microbiota] 32.t[New microbes and new infections - Massilimicrobiota timonensis gen. nov., sp. nov., a new bacterium isolated from the human gut microbiota] 33.t[Scientific reports - Draft genome and description of Merdibacter massiliensis gen. nov., sp. nov., a new bacterium genus isolated from the human ileum] 34.t[Scientific reports - Colibacter massiliensis gen. nov. sp. nov., a novel Gram-stain-positive anaerobic diplococcal bacterium, isolated from the human left colon] , 
 Expression of Concern, 7 September 2022: https: journals.asm.org doi 10.1128 jcm.01078-22 It reads:  *The American Society for Microbiology was notified that the above-mentioned paper is being reviewed as part of a u201cscientific misconduct investigationu201d by the University of Aix Marseille. This Expression of Concern is issued pending the outcome of the investigation and will be updated accordingly thereafter.* ,