A new protectant medium preserving bacterial viability after freeze drying
Authors: Jean-Christophe Lagier
Sara Bellali
Didier Raoult
Jacques Bou Khalil
Anthony Fontanini
Journal: Microbiological Research (2020)
Discipline: MICROBIOLOGY
Keywords: Bacterial viability; Freeze-drying; Gut microbiota; Preservation; Protectant medium
Affiliations: Aix Marseille Univ, IRD, AP-HM, MEPHI, IHU Méditerranée Infection, Marseille, France., IHU Méditerranée Infection, Marseille, France., Aix Marseille Univ, IRD, AP-HM, MEPHI, IHU Méditerranée Infection, Marseille, France. Electronic address: .

Abstract:  Freeze-drying technology has been widely considered for decades as a suitable technique to preserve microorganisms. However, protective agents must be added prior to freeze drying to improve the survival and storage stability of the bacteria. The objective of our study was to evaluate the effect of a new protectant medium containing sucrose (10 %), trehalose (10 %), skimmed milk (10 %) and antioxidants on the viability of gut bacteria under different storage conditions. Two strains were tested, Escherichia coli and Akkermansia muciniphila, as examples of facultative aerobic and anaerobic bacteria, respectively. We studied the cell viability and bacterial morphology in 5 fecal samples in the presence and absence of this protectant medium using plating technique, flow cytometry and scanning electron microscopy. The results of bacterial viability assessed by plating method showed that the protectant medium yielded higher survival rates for both strains whatever the storage conditions (85-93 %) compared to normal saline solution (0.36-37.50 %). It also showed its effectiveness on fecal samples, where bacterial viability after freeze-drying was 89.47 ± 7.63 % and 84.01 ± 7.44 %, as evidenced by flow cytometry analysis and plating method. However unprotected samples showed the lowest cell viability at 19.01 ± 12.88 % and 13.23 ± 9.56 %, as measured by flow cytometry and plating method. In addition, bacterial size and shape were conserved in the protectant medium. In contrast, storage without protectant medium severely damaged bacterial morphology. In conclusion, our study is the first to use morphological features as well as culture-dependant and culture-independent tests to evaluate the effectiveness of a new protectant medium. 

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 5 healthy volunteers in France. Stools have been taken. This is a prospective study with intervention on healthy volunteers. 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] ,