Alterations of Gut Microbiome in the Frontline Medical Staff Under the Stress

In December, 2019, a novel coronavirus outbreak of pneumonia emerged in Wuhan, Hubei
province, China, and has subsequently spread to more than 30 provinces in China and almost
100 countries in the world. In the fight against the 2019 novel coronavirus (2019-nCoV),
medical workers in Wuhan have been facing enormous stress, including a high risk of infection
and inadequate protection from contamination, isolation, patients with negative emotions, a
lack of contact with their families, and exhaustion. The severe situation is causing mental
health problems such as stress, anxiety, depressive symptoms, insomnia, denial, anger, and
fear. These mental health problems could also cause posttraumatic stress (PTS) symptoms in a
lasting time. A research examining the psychological impact of the 2003 outbreak of severe
acute respiratory syndrome on hospital empoyees found that about 10% of the respondents had
experienced high leves of PTS symptoms since the SARS outbreak.

Microbiome-gut-brain (MGB) axis has been validated in expanding studies, which means there
are bidirectional communication between commensal organisms within the gut and the brain. Gut
microbiota may influence brain function through neural, endocrine, and immune pathways. For
example, substances produced by the gut microbiota may be absorbed reaching the brain by the
blood stream. The brain, in turn, may influence the gut microbiota trough neuronal and
endocrine pathways. In recent years, many reseaches support the relevance of microbiota and
mental health status. Bercik et al. transplanted microbiota from adult germ-free (GF) BALB/c
mice (a high-anxiety mouse strain) into adult GF NIH Swiss mice (a low-anxiety mouse strain),
then found the behavioral profile of the donor was evident in the recipient animal, showing
that the microbiota can directly affect behavior. Moreover, preclinical studies have shown
that stress and emotions, including maternal separation and restraint, heat, and acoustic
stress, alters the composition of the gut microbiota, maybe through the release of stress
hormones or sympathetic neurotransmitters that influence gut physiology and alter the habitat
of the microbiota. In addition, researchers found that stress has the ability to increase
intestinal permeability, probably through the involvement of corticotrophin releasing factor
and its receptors (CRFR1 and CRFR2), which play a key role in stress-induced gut permeability
dysfunction. Increased intestinal permeability provides bacteria an opportunity to
translocate across the intestinal mucosa and directly access both the immune and neuronal
cells of the enteric nervous system (ENS). Stress also activates the autonomic nervous
system, which affects gastric acid, bile, and mucus secretion, as well as gut motility. Gut
motility is of particular importance since it is strongly associated with gut microbiota
composition and richness. Based on these researches, the gut microbiome increasingly deserve
attention to understand psychiatric disorders.

Therefore, the present study aim to collect the faecal samples and clinical assessments from
a part of frontline medical workers in three time points to analyse the changing profile of
gut microbiome according to outcomes of 16s rRNA sequencing. The samples from the matched
health controls will also be sequenced to compare with the exposed group in gut microbiome
community.

Methods Study design and sample collection The frontline workers in First Affiliated Hospital
of Xi'an Jiaotong University will be included if they conformed with (1) taking part in the
medical team to support Wuhan, (2) of 18 to 50 years old, (3) did not take antibiotics within
3 months before sample collection, (4) 17.5 will be selected from the staffs in First Affiliated Hospital who didn't join in the medical
team but fulfill the last three conditions above to match with the frontline staffs for age,
gender, BMI, and diet. Any participant will be excluded if he/she fulfill the following
criteria: (1) have serious cardiovascular disease, blood disease, and endocrine disease, (2)
have a history of cancer or its complications, (3) have active gastrointestinal diseases or
complications and serious systemic diseases, (4) have history of brain organic diseases or
complications and mental retardation, (5) have mental disorders such as mood disorder and
anxiety disorders, (6) pregnant or lactating, (7) drink in the past week (liquor>250ml or
beer>1bottle) or the previous day (liquor>50ml or beer>50ml). All included persons should
provide signed informed consent before sample collection, and the protocol was approved by
the Ethics Committee of First Affiliated Hospital of Xi'an Jiaotong University
(KYLLSL-2020-043). Faecal samples from each staff should be freshly collected at the hospital
and frozen at -80℃ using specified faecal collector.

Clinical assessmant PHQ-15, PHQ-9, GAD-7, PSQI, SCL-90, and IES-R will be used for the
assessment of clinical symptoms related to psychiatric disorder for exposed group in three
time point and non-exposed group.

DNA extraction and 16S rRNA sequencing The investigators will perform 16S rRNA sequencing for
all the collected faecal samples. Briefly, bacterial genomic DNA will be extracted, and the
16S rRNA whole region will be amplified by PCR, and then sequenced.

Statistical analysis The 16S rRNA sequencing data will be analysed using Quantitative
Insights Into Microbial Ecology (QIIME). The investigators then use usearch to cluster
sequences into taxonomic units (OTUs) at 97% identity and construct the OTU table. Microbial
community structure, alpha diversity, and beta diversity will be analysed. LEfSe analysis,
and random forest analysis will be used to find the biomarker between different group.
Spearman's rank correlation will be used to identify the corrolation between clinical
assessments and stress-associated microbiome.