Alcohol pretreatment of stools effect on culturomics
Authors: Niokhor Dione
Issa Isaac Ngom
Sara Bellali
Edmond Kuete
Jean-Christophe Lagier
Thi-Phuong-Thao Pham
Didier Raoult
Marie Hocquart
Gr√©gory Dubourg
Camille Valles
Pamela Afouda
Journal: Scientific Reports (2020)
Discipline: Multidisciplinary
Keywords: ethanol stool disinfection; bacteriotherapy trials; Clostridium difficile infections; culturomic approach; cultivable organisms
Affiliations: Aix Marseille Universit√©, IRD, AP-HM, MEPHI, Marseille, France., IHU M√©diterran√©e Infection, Marseille, France., Aix Marseille Universit√©, IRD, AP-HM, MEPHI, Marseille, France. ., IHU M√©diterran√©e Infection, Marseille, France. .

Abstract:  Recent studies have used ethanol stool disinfection as a mean of promoting valuable species' cultivation in bacteriotherapy trials for Clostridium difficile infections (CDI) treatment with a particular focus on sporulating bacteria. Moreover, the culturomic approach has considerably enriched the repertoire of cultivable organisms in the human gut in recent years. This study aimed to apply this culturomic approach on fecal donor samples treated with ethanol disinfection to evidence potential beneficial microbes that could be used in bacteriotherapy trials for the treatment of CDI. Thereby, a total of 254 bacterial species were identified, 9 of which were novel. Of these, 242 have never been included in clinical trials for the treatment of CDIs, representing potential new candidates for bacteriotherapy trials. While non-sporulating species were nevertheless more affected by the ethanol pretreatment than sporulating species, the ethanol disinfection technique did not specifically select bacteria able to sporulate, as suggested by previous studies. Furthermore, some bacteria previously considered as potential candidates for bacteriotherapy have been lost after ethanol treatment. This study, while enriching the bacterial repertoire of the human intestine, would nevertheless require determining the exact contribution of each of species composing the bacterial consortia intended to be administered for CDI treatment. 

Retracted: No

PubPeer comments:
 This paper is part of the set of 38 papers relating to the protocol nu00b02016-011 approved by the ethics committee of the Institut Hospitalo-Universitaire Mu00e9diterranu00e9e Infection (IHU). In the 38 papers, we found the same authors and or authors from the same Institute. **SPECIFIC COMMENT ON THE PAPER** This study recruited 3 patients and 9 healthy volunteers in France. Fecal Microbiota Transplantation has been performed. It is an experimental procedure. This is a prospective study with intervention on healthy volunteers and patients. In France, prospective study, especially in healthy volunteers requires to obtain an approval from a certified IRB in France (Comitu00e9 de Protection des Personnes) before to start the recruitment of participants. The paper don t mention any approval from a Comitu00e9 de Protection des Personnes. The authors only mention the approval from the Ethics Committee of their institute which is not a certified IRB. Editors should clarify this point to ensure the relevant approval has been obtained in accordance with French laws. Furthermore, there is no mention about the dates of the recruitment of participants. Editors should verify these dates to ensure there is no retrospective ethics approval. **GENERAL COMMENT ON the 38 PAPERS WITH THE SAME PROTOCOL #** **The protocol nu00b02016-011:** In Plos One article, the authors mentioned the number 2016-011 is the number of the protocol. As quoted (see #29): *This study was carried out in accordance with the recommendations and approved by the clinical research ethics committee, IFR 48, Aix-Marseille University (protocol Nu00b0 2016u2013011)* In other articles, authors mentioned that it is the approval number of the IHU ethics committee: *The study was approved by the ethics committee of the Institut Hospitalo-Universitaire Mu00e9diterranu00e9e Infection (approval 2016-011)* In accordance with the IRB document provided by the IHU in PlosOne for another study in 2020, we can see that 2016u2013011 seems to be the approval # of the ethics committee. And this approval number cover only one protocol. The 38 papers should be considered as part of a unique protocol. Even if one study mentioned an approval date the 29 September 2019, according to the approval number, we can presume that the approval number has been obtained in 2016 (see #13). The 38 papers also mention different types of scope which are not linked to each others : bacteria characterisation, dental care, fecal infusion, tobacco effectu2026 **How the same protocol can cover different interventions and study design?** The 38 papers mentioned the following types of interventions and samples: - Fecal infusion (2 papers) - Oral fluid (4 papers) - Sputum (2 papers) - Stools (20 papers) - Stools, oral fluid and urine (1 paper) - Subgingival dental plaque and X-Ray (3 papers) - Urine (3 papers) - Vagine swab (2 papers) - Multiple samples (sputum, bronchial aspirates, and bronchoalveolar lavage samples u2013 1 paper) **How the same protocol can cover different type of participant recruitments?** The 38 papers covers different types of recruitment -t19 papers describe the recruitment of healthy participants only -t7 papers describe the recruitment of patients only -t12 papers describes the recruitment of patients and healthy participants. The number of patients recruited change according the paper. -tOnly 1 participant for study on bacteria -tUp to hundred for a study on periodontitis. The Editors should 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 which seems frequent in this Institute and certain of the authors. [Example: Pubpeer comment] **Participants located in different countries** It should be also noted that this protocol cover participants in different countries: - France only (25 papers) - Senegal only (8 papers) - Multiple countries (4 papers) It should be noted that 1 paper donu2019t provide any information regarding the location of the participants to the study (see #22). **Children in the study** Authors mentioned in 7 papers that healthy children or newborns have been recruited with this protocol. **All papers cover prospective studies** Several papers explicitly mention that the recruitment is prospective. The papers also mention the recruitment of healthy volunteers which qualified the recruitment as prospective. **Retrospective ethics approval** As explained, the ethic approval number indicates that it has been obtained in 2016. However, we can note that 4 papers mention that the recruitment of participants started in 2015. All these papers concern an interventional prospective study. Retrospective ethics committee approval for a prospective interventional study 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 (see #7; #15; #29; #33) As the 38 studies are covered by the same protocol, it may be considered that the 38 papers had a retrospective ethics approval. It should be clarified by the Editors. **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 38 papers, no one mention 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 has no ability to validate 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 and cover the 38 papers. The date of the approval, the scope of the protocol and the recruitment date of participants should be clarified for every papers. **Ethics committee approvals are not mentioned for certain papers** 9 out 38 papers donu2019t provide any information regarding the approval of an ethics approval in the country of the participants. The Editors should clarify this point and ensure that for these 9 papers, an ethic committee approval have been obtained in the country of each participant. **LIST OF THE 38 PAPERS COVERED BY THE ETHICS APPROVAL NUMBER 2016-011** 1.t[Anaerobe: Culture of salivary methanogens assisted by chemically produced hydrogen] 2.t[Antonie van Leeuwenhoek: Sediminibacillus massiliensis sp. nov., a moderately halophilic, Gram-positive bacterium isolated from a stool sample of a young Senegalese man] 3.t[Antonie van Leeuwenhoek: Oceanobacillus timonensis sp. nov. and Oceanobacillus senegalensis sp. nov., two new moderately halophilic, Gram-stain positive bacteria isolated from stools sample of healthy young Senegalese] 4.t[Antonie van Leeuwenhoek: Description and genomic characterization of Massiliimalia massiliensis gen. nov., sp. nov., and Massiliimalia timonensis gen. nov., sp. nov., two new members of the family Ruminococcaceae isolated from the human gut] 5.t[bioRxiv: Lactobacillus reuteri: direct passage from ingested yogurts to urine microbiota] 6.t[Clinical Microbiology and Infection: Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates] 7.t[Frontiers in microbiology: Deciphering the Urinary Microbiota Repertoire by Culturomics Reveals Mostly Anaerobic Bacteria From the Gut] 8.t[Frontiers in microbiology: Among live and dead bacteria, the optimization of sample collection and processing remains essential in recovering gut microbiota components] 9.t[Frontiers in public health: Tobacco smoking affects the salivary gram-positive bacterial population] 10.t[Gut Microbes: Running after ghosts: are dead bacteria the dark matter of the human gut microbiota?] 11.t[Journal of investigative and clinical dentistry: Peri-implantitis risk factors: A prospective evaluation] 12.t[Microbiological research: A new protectant medium preserving bacterial viability after freeze drying] 13.t[Microorganisms: Detection of Methanobrevobacter smithii and Methanobrevibacter oralis in Lower Respiratory Tract Microbiota] 14.t[New microbes and new infections: massiliensisu2019 sp. nov. andu2019Virgibacillus ndiopensisu2019 sp. nov., three halophilic species isolated from salty human stools by culturomics.] 15.t[New microbes and new infections: Urinicoccus massiliensis gen. nov., sp. nov., a new bacterium isolated from a human urine sample from a 7-year-old boy hospitalized for dental care] 16.t[New microbes and new infections: Selenomonas felix sp. nov., a new bacterium isolated from human sputum] 17.t[New microbes and new infections: Parabacteroides bouchesdurhonensis sp. nov., a new bacterium isolated from the stool of a healthy adult] 18.t[New microbes and new infections: Clostridium transplantifaecale sp. nov., a new bacterium isolated from patient with recurrent Clostridium difficile infection] 19.t[New microbes and new infections: Brachybacterium timonense sp. nov., a new bacterium isolated from human sputum] 20.t[New microbes and new infections: Prevotella marseillensis sp. nov., a new bacterium isolated from a patient with recurrent Clostridium difficile infection] 21.t[new microbes and new infections: Genomic and phenotypic description of Streptococcus resistens sp. nov., Streptococcus buccae sp. nov. and Streptococcus mediterraneus sp. nov., three new members of the Streptococcus genus isolated from the human oral cavity] 22.t[New microbes and new infections: Corynebacterium pacaense sp. nov., Alistipes megaguti sp. nov., Alistipes provencensis sp. nov., 3 new bacteria isolated from fresh human stool specimens] 23.t[New microbes and new infections: Brachybacterium massiliense sp. nov., a new bacterium isolated from stool from a healthy Senegalese child] 24.t[New microbes and new infections: Virgibacillus ndiopensis sp. nov., a new halophilic bacterium isolated from the stool of a healthy 11-year-old boy] 25.t[New microbes and new infections: Description of Gracilibacillus phocaeensis sp. nov., a new halophilic bacterium isolated from Senegalian human stool] 26.t[New microbes and new infections: Helcococcus massiliensis sp. nov., a new bacterial species isolated from the vaginal sample of a woman with bacterial vaginosis living in Dielmo, Senegal] 27.t[New microbes and new infections: Arcanobacterium ihumii sp. nov., Varibaculum vaginae sp. nov. and Tessaracoccus timonensis sp. nov., isolated from vaginal swabs from healthy Senegalese women] 28.t[Open forum infectious diseases: Successful fecal microbiota transplantation in a patient suffering from irritable bowel syndrome and recurrent urinary tract infections] 29.t[PloS one: Specific clones of Trichomonas tenax are associated with periodontitis] 30.t[PloS one: Extensive culturomics of 8 healthy samples enhances metagenomics efficiency] 31.t[PLoS One: Metabarcoding analysis of eukaryotic microbiota in the gut of HIV-infected patients] 32.t[Research Square: Prevalence of Treponema species in the Gut Microbiome is Linked to Bifidobacterium sp. and Bacteroides sp.] 33.t[Scientific reports: Peri-implantitis-associated methanogens: a preliminary report] 34.t[Scientific reports: Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota] 35.t[Scientific reports: Alcohol pretreatment of stools effect on culturomics] 36.t[Scientific reports: Metagenomic and culturomic analysis of gut microbiota dysbiosis during Clostridium difficile infection] 37.t[Scientific reports: Optimization and standardization of the culturomics technique for human microbiome exploration] 38.t[Scientific reports: Culturomics and amplicon-based metagenomic approaches for the study of fungal population in human gut microbiota] , 
 Authors isolated 38,016 bacterial colonies but succeed to u201cidentifyu201d I guess means taxonomically affiliate only 169 species by MALDI-TOFF. What about the rest? Why authors did not use a more sensitive method for microbial identification? In addition, I cannot find information about the MALDI-TOFF reference database used. Can the authors specify better in the methods section? Authors said that 9 species were newly identified in the study, on the base of what? Of the MALDI-TOFF spectra? How can we infer that a species in new (especially in bacteria) if we do not whole-genome sequence its genome? Can the authors bring proofs that are more tangible on the description of novel species associated to human gut? The MALDI-TOFF is not clearly universal as the databases associated are limited. Also, authors mentioned the use of a fraction of the 16S but again this is not sufficient to describe a new species!! Authors found differences among ethanol treated fecal samples and not-ethanol treatment but: i) they do not have technical and biological replicates of the same sample to demonstrate that the variability among the samples is not related to biases in isolating or internal variability that can be observed in a given sample. To be more clear, we can sample three and even more time in the same individual, culture the microbes and find differences among the samples because internal variability and also technical biases during the isolation. ii) Here the authors compare the effect of ethanol treatment on 8 different samples (from different donors) and they did not provide us statistical analysis to demonstrate that the effect of ethanol on species recovering is not an artefact of the internal variability present in the experimental design. Authors need multiple replicates (at least 6 or more) from the same individual, and technical isolating replicates that can be analyzed to remove the variability related to the biological sample and technical procedures to the variability related to the effect of the treatment. With the appropriate experimental design and replicates, authors can run a mixed-liner model and demonstrate the real impact of the ethanol on recovering bacterial species. Where these new species has been deposited? Are there in a collection accessible to other scientists? I ask this as any accession number related to microbial international collection has been mentioned in the manuscript. As the material and method section is very poor, I cannot understand whether the 3 frozen samples were included in the culturomics and how the authors tested the impact of conservation on the culturomics. If the samples were differently maintained the authors need to provide us statistics to infer the effect on the conservation methods on culturomics. Also, what fresh means? Did the donors were located in the IHU? How long did it take from the sample to the isolation procedure? Authors need to carefully explain this in the method section. Ethanol used for disinfection was pure? For how long and how the samples were treated? Authors cited a study but information must be clarified in this section. MALDI-TOFF was used to identify the bacteria but as I mentioned above few information is provided in this section. First, many bacteria were not classified. This is probably related to the limit of the MALDI-TOFF and the related database used. Authors did not provide information on the database. In addition, they did not provide an open access link to the MS spectra. How can we as referee judge the taxonomic affiliation of the bacteria? How can we judge u201cwhou201d is there? Second, for those bacteria that authors did not identify with MALDI-TOFF a 16S sequencing have been performed. However, here again procedures are not clearly stated and results not presented. It is hard to understand whether authors sequenced all the bacteria (38,016) or not. Most important, accession numbers of the sequences is not provided. How can we verify the data if data are missing? How can we verify the affiliation of the isolated bacteria if the sequences are not provided? Authors must to make accessible to the scientific community the obtained sequences as the raw MALDI-TOFF data. In addition, authors should perform a phylogenetic analysis to compare these species to those already reported in GenBank. Based on the results showed in the articles it is clearly impossible to estimate strain affiliations and the possible u201cdiscoveryu201d of new species. ,