Evolution of Oropharyngeal and Rectal Microbiota After Severe Traumatic Brain Injury

Overview

Modifications of the human gut microbiota have been associated with different pathological conditions such as obesity, inflammatory bowel diseases and neurodegenerative diseases. Recently the " Brain-Gut Axis ", a bidirectional communication axis between brain and gut, has been described. In recent animal studies, an acute brain injury was associated with rapid modifications of the gut microbiota. In humans, traumatic brain injury (TBI) is a leading cause of death and disability. The patterns of gut and oropharyngeal microbiota following TBI are unknown. The primary purpose of this study is to characterize gut and oropharyngeal microbiota of patients with severe TBI.

Study Protocol : Observational prospective cohort study. Patients Patients admitted to the ICU for severe trauma will be included. Two groups of patients with severe trauma will be studied: 1. Patients with isolated severe traumatic brain injury (TBI): TBI with initial Glasgow Coma Scale (GCS) ≤ 8 and AISextrahead score ≤3 2. Patients with severe trauma without TBI (AISextrahead score \> 3) A group of healthy individuals will serve as a control population. Expected total enrollment 20 patients in each group, and 10 healthy controls. Patient data collection For each patient, the following data will be collected: * Demographic data: age, sex, height, weight, ICU admission date, simplified acute physiology score II (SAPS II), injury severity score (ISS), abbreviated injury scale (AIS) at ICU admission. * Trauma-related data: number and type of trauma-related organ injuries, initial GCS, presence of mydriasis at initial management. * Factors with potential impact on microbiota: antimicrobial therapy, nutrition type, medications (proton pump inhibitors, opioids, sedations, catecholamines, steroids), surgical procedure during ICU stay. * Evolution: multidrug resistant bacteria acquisition during ICU stay, ICU acquired-infections. Mechanical ventilation duration, extrarenal epuration, ICU length of stay, neurological outcome evaluated by disability rating scale (DRS-F) at ICU discharge and at 90 days post trauma, death at ICU discharge and 90 days. Sample collection Oropharyngeal and rectal swabs will be performed for each patient within the first 24 hours after ICU admission (day 0), then 48 hours (day 2) and 7 days (day 7) after ICU admission and weekly thereafter until ICU discharge. Rectal and oropharyngeal swabs will be performed by trained paramedical staff using sterile swabs with transport medium ESwab® (Becton, Dickinson and Company, New Jersey, USA). Swabs will be stored at -80°C until DNA extraction. DNA extraction DNA extraction will be performed using QIAamp PowerFecal Pro DNA® kit (Qiagen®, Courtaboeuf, France) for rectal swabs and Extracta DNA Prep® kit (Quanta Biosciences®, Beverly, USA) for oropharyngeal swabs. DNA will be quantified by Quantit® dsDNA HighSensitivity Assay Kit (Fisher Scientific). 16S rRNA amplification and sequencing V3 and V4 regions of bacterial 16S rRNA gene sequences will be amplified by polymerase chain reaction (PCR) with universal primers (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC), following the Illumina MiSeq® System protocol (Illumina®). Amplicons will be purified and then sequenced using MiSeq® sequencing system ((Illumina®). Sequences processing Sequences processing and operational taxonomic unit (OTU) clustering will be performed using SHAMAN software (SHiny Application for Metagenomic ANalysis) based on R® software (package DESeq2), provided by Pasteur Institute. Taxonomic classification will be performed using SILVA database reference. Statistical analysis Statistical analysis will be performed using SHAMAN software (SHiny Application for Metagenomic ANalysis). Bacterial phyla, families and genera repartition will be analyzed, and relative abundance of bacterial genera will be compared between the different populations. Alpha-diversity will be analyzed using different parameters (Shannon index, Simpson's diversity index), as well as beta-diversity (principal component analysis). The different populations of patients and healthy volunteers will be compared, and the evolution of microbiota along time will be studied.