Proof of Concept of Culturomics Use of Time of Care.

Sabrina Naud,Saber Khelaifia,Niokhor Dione,Didier Raoult,Jean-Christophe Lagier,Maxime Descartes Mbogning Fonkou

doi:10.3389/fcimb.2020.524769

Abstract

Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 μm and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37°C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times.

Highlights

The culturomics of the bacterial species of the digestive tract probably represents an indispensable step toward biotherapy in the coming years for colitis, especially Clostridioides difficile colitis (Amrane et al, 2019), and in the context of obesity (Everard et al, 2013), malnutrition (Tidjani Alou et al, 2017) and cancer immunotherapy (Lagier et al, 2019)
Overall, testing 4,988 colonies by MALDI-TOF MS, 121 different bacterial species were cultivated from a standard agar plate culture and two liquid enrichments in stool sample (Sample 1) from a patient requiring rapid immunotherapy treatment (Supplementary Table 1 and 2)
Of these 121 species, 1 bacterial species was previously unknown from human and 2 bacterial species were unknown to the human gut microbiota (Supplementary Table 2)

Summary

Introduction

The culturomics of the bacterial species of the digestive tract probably represents an indispensable step toward biotherapy in the coming years for colitis, especially Clostridioides difficile colitis (Amrane et al, 2019), and in the context of obesity (Everard et al, 2013), malnutrition (Tidjani Alou et al, 2017) and cancer immunotherapy (Lagier et al, 2019). In this context, the problematic aspect of culturomics is that, in its current state, it is extremely restrictive in terms of time, equipment and culture medium (Lagier et al, 2012). This allowed us to compare a method to quickly determine, before treatment was initiated, whether he had the bacterial species associated with a response to his treatment with antiPD-1 monoclonal antibody at the time of treatment (Lagier et al, 2015b; Routy et al, 2018)

Methods

Results

Conclusion