The human microbiome and COVID-19: A systematic review.

BACKGROUND: Human microbiotas are communities of microorganisms living in symbiosis with humans. They play an important role in the host immune response to respiratory viral infection. However, evidence on the human microbiome and coronavirus disease (COVID-19) relationship is insufficient. The aim of this systematic literature review was to evaluate existing evidence on the association between the microbiome and COVID-19 in humans and summarize these data in the pandemic era.
METHODS: We conducted a systematic literature review on the association between the microbiome and COVID-19 in humans by searching PubMed, Embase, and the Cochrane Library, CINAHL, and Web of Science databases for articles in English published up to October 31, 2020. The results were analyzed qualitatively. This study is registered with PROSPERO (CRD42020195982).
RESULTS: Of the 543 articles identified by searching databases, 16 in line with the research objectives were eligible for qualitative review: eight sampled the microbiome using stool, four using nasopharyngeal or throat swab, three using bronchoalveolar lavage fluid, and one using lung tissue. Fecal microbiome dysbiosis and increased opportunistic pathogens were reported in COVID-19 patients. Several studies suggested the dysbiosis in the lung microbiome of COVID-19 patients with an abundance of opportunistic pathogens using lower respiratory tract samples. The association between COVID-19 severity and the human microbiome remains uncertain.
CONCLUSION: The human fecal and respiratory tract microbiome changed in COVID-19 patients with opportunistic pathogen abundance. Further research to elucidate the effect of alternation of the human microbiome in disease pathogenesis is warranted.

Introduction

Microbiota widely colonizes the human body, and the human microbiota varies between individuals and ethnicities [1]. Although the role of the human microbiota has not been fully elucidated, the human microbiome is currently considered to be associated with several disorders, including inflammatory bowel disease [2], type 2 diabetes [3], Parkinson’s disease [4], and colorectal cancer [5]. Among them, infectious diseases such as respiratory diseases are directly or indirectly associated with specific microorganism patterns. For example, the human upper respiratory tract microbiome in influenza patients is disturbed with Pseudomonadales abundance [6].

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization on March 11, 2020. COVID-19 is a respiratory disease with a broad range of clinical manifestations, from asymptomatic or mild disease with cough and fever to severe pneumonia with multiple organ failure and acute respiratory disease syndrome (ARDS) [7]. ARDS is known to be caused by a cytokine cascade, including interleukin (IL), granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor (TNF). A previous study revealed that intensive care unit (ICU) patients with COVID-19, including ARDS, had an abundance of proinflammatory cytokines, including IL-2, IL-7, IL-10, GCSF, IP10, MCP1, MIp1A, and TNFα, compared to non-ICU patients [8]. These inflammatory cytokines were reportedly correlated with a specific pattern of the gut microbiome [9].

In the research of severe COVID-19, the interaction between the microbiome and cytokine cascade received considerable attention. However, the disparity factors of COVID-19 incidence and death among races, ethnicities, or countries remain uncertain at this moment. Hence, this systematic literature review aimed to analyze existing evidence on the association between the microbiome and COVID-19 in humans and summarize data on the microbiome in COVID-19 in the pandemic era. Moreover, in this review, the relationship between the human microbiome and COVID-19 severity has been investigated.

Methods

All procedures used in this systematic review complied with the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. This study is registered to PROSPERO (CRD42020195982).

Selection criteria and search strategy

This systematic review considered articles that were original clinical studies of any designs examining the human microbiome in COVID-19 patients. Reviews and editorial articles were manually checked for any additional relevant information. The MEDLINE/PubMed, EMBASE, Cochrane Database of Systematic Reviews, CINAHL, and Web of Science databases were systematically searched for published articles until October 31, 2020. The search terms used were as follows: (“severe acute respiratory syndrome coronavirus 2” OR “SARS-CoV-2” OR “SARS2” OR “Wuhan coronavirus” OR “coronavirus” OR “COVID-19” OR “novel coronavirus” OR “COVID19” OR “nCoV” OR “coronavirus disease 2019”) AND (“microbiota*” OR “microbiome*” OR “mycobiome” OR “virome” OR “flora”). Two authors (S.Y. and A.T.) independently reviewed titles and abstracts of the identified studies written in English, and discrepant results were resolved via a second assessment. The search strategy and method used for selecting studies are summarized in S1 File. The risk of bias was assessed using the Risk of Bias Assessment tool for Non-randomized Studies (RoBANS) [10] for each study.

Data synthesis

Data related to authors, study type, population, characteristics of patients and controls, microbiota community, microbiome characterization methods, sample collection time, key findings, treatments, and outcomes were extracted and recorded from all studies by the two authors independently. The results of this study were organized in a qualitative synthesis, including the relationship between microbiome and COVID-19 with available key study findings.

Results

Study characteristics

Our systematic search of the literature identified 543 articles for screening (Fig 1). Eighty-one full-text articles were examined for eligibility, and 16 studies that reported at least 296 patients were included in this review [11-26], including three unpublished articles, which were not peer-reviewed [21-23], and two articles [24, 25] with abstracts only (all written in English). All 16 articles were observational studies. Most of them were conducted in China (14/16 studies) [4-26], and all studies were on hospitalized adults. The microbiota community was sampled from stool (8/16) [11–14, 22, 24–26], nasopharyngeal or sputum or throat samples (4/16) [15, 16, 21, 23], bronchoalveolar lavage fluid (BALF; 3/16) [17, 19, 20], and lung tissue (1/16) [18]. No studies on skin, oral, vaginal, or urinary microbiota were reported. Eight studies [11–13, 16, 17, 19–21] used whole-genome sequencing to analyze the microbiome, and five studies [14, 15, 18, 23, 26] used 16S rRNA sequencing. Three articles (one unpublished article [22] and two abstracts [24, 25]) had no sequencing information. The findings of these studies are summarized in Table 1. The time point at sample collection depended on each study. Most respiratory samples were collected as part of clinical management, such as diagnostic tests; stool samples were collected at admission, while other studies collected samples during hospitalization.

Fig 1

PRISMA flow diagram.

Table 1

The characteristics of all included studies.

Study	Population and characteristics	Control	Samples or Microbiota community	Timepoint at sample collection	Methods of microbiome characterization	Change in microbiota	Treatment/Outcome	Other key findings
Gu et al. (2020)	30 COVID-19 patients, China	(1) 24 H1N1 flu patients	Fecal samples	Samples were collected at admission.	V3-4 16S rRNA gene sequencing	Patients with COVID-19 or influenza had reduced bacterial diversity compared with controls. Stool samples of COVID-19 patients had an abundance of opportunistic pathogens, such as Streptococcus, Rothia, Veillonella, and Actinomyces. In contrast, Ruminococcaceae family and several genera from the family Lachnospiraceae were reduced in COVID-19 patients.	No outcome data	Richness, diversity, and structure of the gut microbiota were not significantly different between general COVID-19 and severe COVID-19.
	13 were women	(2) 30 healthy controls (matched for age, sex, body mass index)
	The median age was 55 (Range 29–70) years
	10 patients had comorbidities, including hypertension (30%)
Zuo et al. (2020a)	15 COVID-19 patients, China	15 healthy controls	Stool samples	2–3 times per week during hospitalization	Whole-genome sequencing	COVID-19 patients who were antibiotic naïve had increased opportunistic pathogens compared to controls.	Among 15 COVID-19 patients, 7 were antibiotic-naive, 8 received empiric antibiotics.	Clostridium ramosum and C. hathewayi were positively associated with severe COVID-19. In contrast, Alistipes onderdonkii and Faecalibacterium prausnitzii were negatively correlated with severe COVID-19.
	7 were males,	6 community-acquired pneumonia
	The median age was 55 (22–71) years
	11 patients had moderate-severe COVID-19
	Comorbidities: Hypertension (4), Hyperlipidemia (4), Diabetes (2), etc.
Zuo et al. (2020b)	30 COVID-19 patients, China	30 healthy controls	Stool samples (Fungal microbiome)	2–3 times per week during hospitalization	Whole-genome sequencing	The mycobiome in most patients (22 of 30 COVID-19 patients) was similar to that in healthy controls. In contrast, gut microbiome in COVID-19 patients (8 of 30) had alterations, with enrichment of Candida albicans and heterogeneous composition.	No treatment data	COVID-19 patients had increased proportions of opportunistic pathogens (Candida albicans, C. auris, Aspergillus flavus) during hospitalization.
	16 were males,	9 community-acquired pneumonia
	The median age was 46 (15–71) years
	11 patients had comorbidities
Zuo et al. (2020c)	Same population as Zuo et al. (2020a)	None	Stool samples	2–3 times per week during hospitalization	Whole-genome sequencing	Stool with high SARS-CoV2 infectivity had a higher abundance of bacterial species, including Collinsella aerofaciens, Collinsella tanakaei, Streptococcus infantis, and Morganella morganii.	Same treatment as Zuo et al. (2020a)	Stool with low to no SARS-CoV-2 infectivity had higher abundances of Parabacteroides merdae, Bacteroides stercoris, Alistipes onderdonkii, and Lachnospiraceae bacterium.
Zhang et al. (2020)	24 COVID-19 (nasopharyngeal swab) patients	Pneumonia cases	Nasopharyngeal swab, sputum samples	Samples were collected during inclusion in the cohort.	Whole-genome sequencing	COVID-19 patients had reduced alpha diversity in the airway microbiome. Opportunistic pathogens, including Candida albicans and human alphaherpesvirus 1, were frequently detected.	No treatment data	None
		36 non-COVID-19 (nasopharyngeal swab)
	14 COVID-19 (sputum) patients, China
		39 non-COVID-19 (sputum)
	37% were females
	The median age was 40.5 (25–82) years
De Maio et al. (2020)	18 COVID-19 patients, Italy	22 healthy controls	Nasopharyngeal swab samples	Samples were collected because of suspected COVID-19.	Sequencing of V5-V6 hypervariable region of bacterial 16S rRNA	The microbiota of the nasopharynx was not different in COVID-19 patients compared with that in controls.	No treatment data	None
	All patients had non-critical COVID-19
Shen et al. (2020)	8 COVID-19 pneumonia patients, China	25 community-acquired pneumonia	Bronchoalveolar lavage fluid samples	Samples were collected at bronchoscopic examination in clinical management.	Whole-genome sequencing	The BALF microbiota in COVID-19 patients was similar to that in patients with community-acquired pneumonia. The microbiome had an abundance of oral and upper respiratory commensal bacteria.	No treatment data	None
		20 healthy controls
Chen et al. (2020)	2 patients with COVID-19, China	None	Bronchoalveolar lavage fluid samples	Samples were collected at bronchoscopic examination for a diagnostic test.	Microbial next-generation sequencing analysis	Authors found Capnocytophaga species and Veillonella species in BALF samples.	Antiviral and anti-infectious treatment	None
	A 39-year-old male and a 21-year-old female
Ren et al. (2020)	5 patients with COVID-19, China	None	Bronchoalveolar lavage fluid samples	Samples were collected at bronchoscopic examination during hospitalization.	Microbial next-generation sequencing analysis	Most genome reads were viral (betacoronavirus), with bacterial pathogens such as Acinetobacter, Pseudomonas, Chryseobacterium, Escherichia, Streptococcus, Enterococcus, Rothia, and Lactobacillus.	5 patients received antibiotic therapy. Outcomes are recovered (1), hospitalized (3), and died (1).	None
	The median age was 52 (41–65) years
	2 patients were female
	All had moderate to severe COVID-19
	Comorbidities: hypertension, chronic liver disease
Fan et al. (2020)	20 deceased COVID-19 patients, China	None	Lung tissue samples	Samples were collected from deceased patients.	Sequencing of V3-V4 regions of bacterial 16S rRNA, ITS gene	Acinetobacter was the most common bacterial genus, followed by Chryseobacterium, Burkholderia, Brevundimonas, Sphingobium, and Enterobacterales. Cryptococcus was the most prevalent fungus, along with Issatchenkia, Wallemia, Cladosporium, and Alternaria.	All patients received antibacterial and antiviral therapy.	None
	14 were males
	The median age was 66 years
	Comorbidities: cardiovascular disease (10), hypertension (9), malignancy (7), diabetes (2), chronic kidney disease (2), chronic lung diseases (1)
Tao et al. (2020)	62 patients with COVID-19, China	33 seasonal influenza patients	Fecal samples	Samples were collected at first time of visit to the hospital.	Next-generation sequencing of V4 region of the 16S rRNA	COVID-19 patients had an abundance of Streptococcus, Clostridium, Lactobacillus, and Bifidobacterium in gut microbiota. In contrast, lower levels of Bacteroides, Roseburia, Faecalibacterium, Coprococcus, Parabacteroides were found.	No data	Alpha-diversity of gut microbiome decreased in COVID-19 compared with that in healthy control, flu patients.
		40 healthy controls
Yu et al. (unpublished)	2 male patients with COVID-19, China	22 healthy cohort (Data imported from Arumugam M, Raes J, Pelletier E, et al. Enterotypes of the human gut microbiome. Nature. 2011;473(7346):174–80.)	Anal swab samples	No data	No data	The proportion of gut microbiota, including Corynebacterium and Ruthenibacterium, was increased.	Both patients received antiviral and antibacterial agents. and both patients died.	None
	2 patients with COVID-19, China
	65 years old male and 78 years old male
	Comorbidities: prostatic hyperplasia, chronic bronchitis
Ai et al. (unpublished)	20 patients with COVID-19, China	33 pneumonia without COVID-19	Nasopharyngeal swab samples	Admission day	Multiplex RT-PCR assays	More than half of patients had co-infection with COVID-19 and another virus, such as influenza A/B, rhino- or enteroviruses, or respiratory syncytial virus.	No data	None
	10 were female				next-generation sequencing
	The median age was 37 years
Budding et al. (unpublished)	46 patients with COVID-19, Netherlands	89 SARS-CoV2 (-) patients	Throat swab samples	Samples were collected for routine diagnostic tests.	16S rDNA sequencing	Haemophilus parainfluenzae, Neisseria cinerea,	No data	None
						S. mitis, S. bovis, Leptotrichia buccalis, and Rothia mucilaginosa were the main composition of throat microbiome.
Xu et al. (a) (abstract only)	No data, China	No data	Intestinal microbiome	No data	No data	COVID-19 patients had intestinal dysbiosis with decreased Lactobacillus and Bifidobacterium.	No data	None
Xu et al. (b) (abstract only)	No data, China	No data	Gut microbiome	No data	No data	Decreased proportions of Lactobacillus and Bifidobacterium were observed in COVID-19 patients.	No data	None

Quality of studies

The quality assessment results are summarized in S1 Table. Most studies were generally unclear and of low quality, according to RoBANS.

Dysbiosis in the fecal microbiome of COVID-19 patients

In five observational studies [11–14, 26], COVID-19 patients had altered intestinal microbiomes compared with the control groups. Three studies by Zuo et al. [11−13] using shotgun metagenomic sequencing of fecal samples described dysbiosis in the bacterial microbiome and mycobiome in COVID-19 patients when compared with those in healthy controls. Notably, COVID-19 patients mainly had increased numbers of opportunistic pathogens (OPs), a part of commensal microbiota that may become pathogenic in the event of host perturbation, such as dysbiosis or impaired immune system (immunocompromised host) [27]. In these reports, the OPs included Clostridium hathewayi, Actinomyces viscosus, and Bacteroides nordii at the time of hospitalization, and gut bacterial dysbiosis persisted even after SARS-CoV-2 tests using nasopharyngeal swabs or saliva samples became negative. In another report, SARS-CoV-2 RNA was detected in 46.7% of stool samples regardless of the gastrointestinal symptoms [13]. That report also showed that the numbers of specific OPs (e.g., Collinsella aerofaciens and Morganella morganii spp.) were increased in fecal samples with high SARS-CoV-2 active viral transcription and replication in vitro (infectivity) compared with those in fecal samples with low to no SARS-CoV-2 infectivity [13]. Conversely, fecal samples with low or no SARS-CoV-2 infectivity had increased levels of bacteria belonging to Parabacteroides, Bacteroides, and Lachnospiraceae, which produce short-chain fatty acids (especially butyric acid). Short-chain fatty acids are known to play an important role in boosting the host immunity; thus, these data suggest that OPs pose threats of both reduced host immunity and opportunistic infections in proportion to the load of SARS-CoV-2. In another cohort, reduction in bacterial diversity was described in fecal samples from patients with COVID-19 or influenza compared with that in healthy controls (matched for age, sex, and body mass index) by analyzing the V3–V4 region of the 16S rRNA gene [14]. The study also revealed an increase in the number of OPs such as Streptococcus, Rothia, Veillonella, and Actinomyces among COVID-19 patients. Moreover, C-reactive protein, an indicator of bacterial infection, was positively correlated with the bacterial taxa [14].

One gut microbiome study based on 16S rRNA in COVID-19 patients [26] found that the alpha diversity in these patients was lower than that in healthy controls and influenza patients. The abundance of four genera, Streptococcus, Clostridium, Lactobacillus, and Bifidobacterium, tended to increase. Conversely, five other genera, Bacteroides, Roseburia, Faecalibacterium, Coprococcus, and Parabacteroides, showed lower abundance in COVID-19 patients than in control subjects. Furthermore, the study showed that the levels of IL-18, a pro-inflammatory cytokine produced by multiple intestinal cells and the intestinal nervous system, were increased in the sera of COVID-19 patients compared with those in influenza patients and healthy individuals. We also identified one unpublished, non-peer-reviewed article concerning the intestinal microbiome [22]. The study showed that the proportion of pathogenic bacteria in the gut, including Corynebacterium and Ruthenibacterium, increased in COVID-19 patients compared with another historical cohort data previously reported [22]. In contrast, the abundance of Bifidobacterium, Lactobacillus, and Eubacterium decreased in COVID-19 patients. Moreover, two Chinese articles with English abstracts described dysbiosis with decreased Lactobacillus and Bifidobacterium levels in some COVID-19 patients [24, 25].

Further, one study reported the fungal microbiome in COVID-19 patients. It showed that the composition of the fecal mycobiome in 30 hospitalized COVID-19 patients was heterogeneous; however, some were enriched in fungal pathogens of Candida and Aspergillus spp. compared with control subjects. In addition, in some of the COVID-19 patients, intestinal mycobiome instability and prolonged dysbiosis persisted for up to 12 days after the disappearance of SARS-CoV-2 from the nasopharynx [12].

Severity of COVID-19 and the intestinal microbiome

The association between the severity of COVID-19 and the gut microbiome was mentioned in two studies [11, 14]. The classification of COVID-19 severity in each study are summarized in the S2 File. One study showed the potential importance of Firmicutes spp. in the severity of SARS-CoV-2 infection [11]. To understand the association between the gut microbiome and COVID-19 severity, the study assessed the association between the fecal microbiome and COVID-19 severity (mild, moderate, severe) in seven antibiotic-free COVID-19 patients. A total of 23 bacterial taxa were significantly associated with the severity of COVID-19, and most (15 of 23) were in the phylum Firmicutes. Of these, eight classes were positively correlated with disease severity, and seven were negatively correlated. Firmicutes spp. were diversely affected in COVID-19 patients. In particular, Coprobacillus, Clostridium ramosum, and C. hathewayi in the Firmicutes phylum were the top bacteria showing a positive correlation with the severity of COVID-19.

The other paper reported that COVID-19 patients have lower lymphocyte counts and increased interleukin (e.g., IL-6) and TNF-α levels compared with the healthy cohort [14]. In their study, the differences in gut microbiota abundance, diversity, and structure were not significantly different between patients with mild and severe COVID-19.

Alterations in the upper respiratory tract microbiome of COVID-19 patients

The upper respiratory microbiome in COVID-19 patients was analyzed in two observational studies (one using nasopharyngeal swabs and the other using nasopharyngeal swabs and sputum) [15, 16]. One study found that the nasopharyngeal microbiome was not significantly different between 18 acute-phase COVID-19 patients and 12 control individuals (SARS-CoV-2 negative) upon analyzing the V5–V6 regions of the 16S rRNA gene in the nasopharyngeal swab samples [15]. The other study performed a metatranscriptomic analysis in a cohort of 113 patients (38 patients with COVID-19 and 74 patients with non-COVID-19 pneumonia). The Shannon index of nasopharyngeal swab specimens showed little difference between both groups. In contrast, the Shannon index of sputum specimens was significantly lower in COVID-19 patients, suggesting that they may have an altered middle pharynx microbiome compared with that in the other patients with non-COVID-19 pneumonia. In that report, co-infection analysis also identified 24 potentially pathogenic microorganisms in 18 of the 38 (47.4%) COVID-19 patients. Among them, 16 different microorganisms were detected, with the OPs Candida albicans and human alpha-herpesvirus 1 being the most abundant. Moreover, the remaining eight microorganisms were viral pathogens, including the human influenza virus and respiratory syncytial virus [16]. We identified two unpublished (non-peer-reviewed) articles examining the upper respiratory tract microbiome [21, 23]. Using next-generation sequencing of nasopharyngeal swabs, one study found that 11 of 20 patients had co-infection with COVID-19 and another virus, including influenza A/B, rhino- or enteroviruses, or respiratory syncytial virus [21]. The other study described the main microbiome composition of COVID-19 throat samples to include Haemophilus parainfluenzae, Neisseria cinerea, Streptococcus mitis, Streptococcus bovis, Leptotrichia buccalis, and Rothia mucilaginosa. The authors also observed that the pharyngeal microbiome diversity of all examined bacterial phyla (e.g., Bacteroidetes, Proteobacteria) decreased in older adults than younger adults, both having COVID-19, which could explain the differences in severity [23].

Alterations in the lower respiratory tract microbiome of COVID-19 patients

One study on whole-genome sequencing of BALF samples of COVID-19 patients, community-acquired pneumonia (CAP) patients, and healthy controls found that the microbiota in COVID-19 patients was similar to that in CAP patients. However, the microbiomes of both the COVID-19 and CAP groups were significantly different from those of healthy controls, who demonstrated enrichment with known oral and upper respiratory commensal bacteria, suggesting dysbiosis in the lung biota of COVID-19 patients [17]. Another study based on 16S rRNA sequences found that Acinetobacter was the most common bacterial genus, followed by Chryseobacterium, Burkholderia, Brevundimonas, Sphingobium, and Enterobacterales in lung tissues of deceased COVID-19 patients [18]. Moreover, the study identified Cryptococcus as a prevalent fungus, along with Issatchenkia, Wallemia, Cladosporium, and Alternaria. A case study using whole-genome sequencing reported co-infection of Capnocytophaga and Veillonella species in the BALF samples of one out of two COVID-19 patients, but the pathogenicity was unknown [19]. Another case series based on whole-genome sequencing of samples from five COVID-19 patients found that most non-human genomic reads from BALF samples were from a beta coronavirus genome. A few bacterial genomes, including those of Acinetobacter, Pseudomonas, Chryseobacterium, Escherichia, Streptococcus, Enterococcus, Rothia, and Lactobacillus, were also detected [20].

Discussion

Our systematic review accumulated the evidence in the intestinal and respiratory microbiome in hospitalized COVID-19 patients. Several studies indicated the fecal, respiratory microbiome dysbiosis with increasing OPs [11–13, 16, 17]. One observational study showed that COVID-19 severity could be associated with the baseline fecal microbiome [11]. However, the extent to which the corresponding human microbiome contributes to COVID-19 remains unclear. Further microbiome research, including longitudinal data (e.g., post-hospitalization), studies from various countries and ethnicities, and evidence of severity is needed.

The common intestinal microbiota comprises four domains, including Firmicutes (i.e., Clostridium, Bacillus, and Lactobacillus), Bacteroides, Actinobacteria (i.e., Bifidobacterium), and Proteobacteria [27, 28]. The dysbiosis of the intestinal flora is currently considered to be associated with the host antiviral immune response. Previous in vivo and in vitro studies have revealed that the intestinal microbiome could affect innate and adaptive immune responses to viral infections such as influenza [29-33]. Results of two independent studies revealed an increase in OPs in the gut of COVID-19 patients with prolonged dysbiosis [11, 14]. Similarly, we showed that about 20 Japanese COVID-19 patients also had dysbiosis associated with increased OPs compared with healthy cohorts by 16S rRNA analysis (unpublished data). Although the findings suggested a relationship between OPs and the intestinal microbiome, the correct understanding of the enrichment and pathogenicity of OPs remains uncertain. The role of these OPs may be connected to potential secondary bacterial infection when the host immune system is impaired [11]. Further research is needed to address the mechanism of harm by these OPs better.

Disruption of the intestinal microbiome is also reported to be a predisposing factor for pro-inflammatory conditions (e.g., sepsis) [34], although the mechanism of intestinal microbiome change underlying severe conditions is uncertain. A previous study related to inflammation showed that patients with systemic inflammatory response syndrome had a low abundance of gut anaerobes such as Bifidobacterium and Lactobacillus and high OP abundance [35]. Similarly, the gut microbiome dysbiosis with an abundance of OPs occurred in patients with COVID-19 [11–14, 26]. One study showed that Faecalibacterium was negatively correlated with severe COVID-19 [11], although Faecalibacterium has been reported to be associated with a pathway to reduce intestinal inflammation [34]. In this review of the microbiome in COVID-19, few studies reported intestinal microbiota alteration and severe COVID-19. Additional comparative studies on COVID-19 severity, including the convalescent phase of COVID-19 and the phase of antibody production against SARS-CoV-2, are needed. The dysbiosis of human microbiotas could be one possible host factor underlying severe COVID-19.

Microbiomes of the upper and lower respiratory tracts have been reported to have similar microbiological characteristics [28]. The upper respiratory microbiome influences respiratory viral infection, including disease severity [36], vaccine response, and secondary bacterial infection [37]. Similarly, the lower respiratory microbiome, including the lung microbiome, shows decreased diversity in ARDS patients without COVID-19, with increased abundance of Staphylococcus, Streptococcus, and Enterobacterales species and increased IL-6 levels [38]. Our review showed that the airway microbiome of COVID-19 patients was different from that of healthy controls. There was no difference between the microbiomes of COVID-19 and CAP patients, but this observation may be attributed to the use of antibiotics for these patients, affecting the composition of their microflora in the same way. In contrast, the nasopharyngeal microbiome may be less affected by acute respiratory virus infection. A previous study revealed that the nasopharyngeal microbiome was not different between patients with influenza and control individuals [39]. Two independent studies [15, 16] on nasopharyngeal swabs showed no significant difference in the microbiomes of COVID-19 patients and control subjects. In the future, more data on the upper and lower airway microbiomes would provide a detailed understanding of the relationship between respiratory virus infection, including COVID-19, and the microbiome.

Currently, little is known about the relationship between human microbiota and COVID-19. The review revealed several necessary points that warrant future research on the microbiome in COVID-19. First, longitudinal data, including post-hospitalization information, are lacking. Besides the poor understanding of the pathogenesis of COVID-19, there is limited knowledge about the clinical sequelae that may persist after viral clearance because COVID-19 is a new disease. It is important to establish a longitudinal cohort that has recovered from COVID-19 and investigate the association between human microbiota and clinical features of acute infection of the respiratory tract. Second, reports from China dominate this review. Data from other countries are needed, as ethnicity is known to be a significant factor in the diversity of the microbiome. Our results have a population bias and may not be generalizable. Third, analysis of the human microbiome of non-intestinal organs, such as the respiratory tract, oral cavity, skin, or urinary tract, will prove fruitful in understanding the microbiota in COVID-19 patients. Fourth, additional microbiome data regarding their correlation to COVID-19 severity are essential. If the gut microbiota profile for each disease severity becomes clear in the future. The microbiome may become a prognostic marker for disease progression. Furthermore, in that case, early control of the gut microbiota (e.g., by symbiotics, probiotics, fermented food, and fecal transplant) would be effective in the aspect of prevention or therapeutics. Several trials, including randomized controlled trials for the efficacy of probiotics for COVID-19 (Cochrane Central Register of Controlled Trials: ChiCTR2000029974, NCT04390477), are ongoing [40]. To date, the effects of altering the gut microbiome remain uncertain, and the results of the ongoing trials are needed.

This review has several limitations. First, only English publications were searched; literature in other languages, including Chinese, was not included. Second, this review did not consider antibiotic and probiotic use. Not all studies documented antibiotic and probiotic use, which could be a potential factor affecting the microbiome findings. Third, various approaches, such as whole-genome sequencing and 16S rRNA sequencing, were used to assess the microbiota, and the spectrum of detected microorganisms (bacteria, fungi, viruses) differed for each study. Fourth, the causality between the microbiome and disorder is uncertain. The question of which came first, the chicken or the egg, is a classic problem of the microbiome. Therefore, longitudinal data on the microbiome in COVID-19 patients are needed. Fifth, our review lacked metabolomics studies. Although it is unclear whether changes in the bacterial flora are a cause or consequence, changes in secondary metabolites secreted by bacteria may lead to changes in the gut environment, which may be related to the pathogenesis of the disease by leading to some inflammatory stimuli. Further elucidation of the correlation between metabolomics and human microbiome due to SARS-CoV-2 infection is expected.

In conclusion, limited evidence implies that COVID-19 patients had altered gut and respiratory tract microbiomes along with an increased abundance of OPs.

The results of database search.

(DOCX)

Click here for additional data file.

The classification criteria of COVID-19 severity.

(DOCX)

Click here for additional data file.

Risk of bias for each study.

(DOCX)

Click here for additional data file.

PRISMA_2009_checklist.

(DOC)

Click here for additional data file.

12 Apr 2021

PONE-D-21-06559

The human microbiome and COVID-19: A systematic review

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Even if it seems interesting to study the potential association between the dysbiosis and the immune response to the SARS_CoV_2, it should have been done with more well defined methodology. What is the real scope of the review ? Association between severity of the disease and ? With which criterias ? How the biais are controlled in the studies selected ?

The methodology of the studies retained are not well enough detailed. So it is impossible for the reader to assess the validity of the association described.

Reviewer #2: Yamamoto et al. conducted a systematic literature review on the association between the

microbiome and COVID-19 in humans. They shown that human fecal and respiratory tract microbiome changed in COVID-19 patients with opportunistic pathogen abundance. The quality of the process to conducted this study is excellent. Please find my comments.

- The conclusion need to be more shorter with a highlight in terms of Microbiome dysbiosis.

- Do you think that there is non supplementation with probiotics or prebiotics or fermented food in patients ?

- Can you added your recommendations or can you speculate if patients can be supplemented with probiotics ?

- Can microbiota dysbiosis be uses as a signature of COVID-19 severity (as a biomarker)?

- The aspect of microbiota or microbiome modulation in patients can be discussed.

- The prevention or therapeutic aspects : can you comments please?

- One of the limitation may be the lack of metabolomics studies, can you comments on this point.

Reviewer #3: In this systematic review, the authors collected evidence on alterations in the gut, upper and lower respiratory microbiota in Covid-19 infection.

The study is well conducted and written, offering a good perspective on the work in literature.

The evidence collected is unfortunately of low grade. All the studies collected had an observational design, 14/16 are conducted in China and consider only hospitalized patients. For these reasons, many confounding factors may have altered the results. Moreover, different types of samples and different methods of analysis are used.

Major revision

- Clarify whether unpublished studies were reviewed by a third-party researcher not involved in the study.

Minor revision

- The design and characteristics of the included studies are well described in Table 1. However, they could be reported more clearly in the text.

- In the "Selection criteria and search strategy" paragraph, words used for the research could be placed in quotation marks rather than in parentheses.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Marvin Edeas, MD, PhD

Professor of Medicine

Université de Paris, INSERM U1016

Institut Cochin

Department Endocrinology, Metabolism

and Diabetes

Faculté de médecine Cochin-Port Royal

24 rue du Faubourg St Jacques,

75014 Paris-France

Reviewer #3: No

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Submitted filename: Rev. Microbiota and Covid-19.docx

Click here for additional data file.

17 May 2021

Reviewer #1: Even if it seems interesting to study the potential association between the dysbiosis and the immune response to the SARS_CoV_2, it should have been done with more well defined methodology. What is the real scope of the review ? Association between severity of the disease and ? With which criterias ? How the biais are controlled in the studies selected ?

The methodology of the studies retained are not well enough detailed. So it is impossible for the reader to assess the validity of the association described.

Response: Thank you for your comment. All of your suggestions have been incorporated into our revised manuscript. The scope of review has been provided in the Introduction section (Page 4, line 72−74)” Moreover, in this review, the relationship between the human microbiome and COVID-19 severity has been investigated”. The severity criteria have been added to the Result (Page 8,9 line 189-190) and Supplementary sections. We assessed the risk of bias in each study (Supplementary Table 1).

Reviewer #2: Yamamoto et al. conducted a systematic literature review on the association between the microbiome and COVID-19 in humans. They shown that human fecal and respiratory tract microbiome changed in COVID-19 patients with opportunistic pathogen abundance. The quality of the process to conducted this study is excellent. Please find my comments.

- The conclusion need to be more shorter with a highlight in terms of Microbiome dysbiosis.

Response: Thank you for your comment. The conclusion section has been shortened as suggested. Page 15. line 354−355. “In conclusion, limited evidence implies that COVID-19 patients had altered gut and respiratory tract microbiomes along with an increased abundance of OPs.”

- Do you think that there is non supplementation with probiotics or prebiotics or fermented food in patients ?

- Can you added your recommendations or can you speculate if patients can be supplemented with probiotics ?

- Can microbiota dysbiosis be uses as a signature of COVID-19 severity (as a biomarker)?

- The aspect of microbiota or microbiome modulation in patients can be discussed.

- The prevention or therapeutic aspects : can you comments please?

Response: Thank you for your comment. All of your suggestions have been incorporated into our revised manuscript. Information on probiotics, prebiotics, or fermented food and microbiome modulation and clinical use of microbiome have been provided in the discussion section. Page 14, line 329-332. “If the gut microbiota profile for each disease severity becomes clear in the future. The microbiome may become a prognostic marker for disease progression. Furthermore, in that case, early control of the gut microbiota (e.g., by symbiotics, probiotics, fermented food, and fecal transplant) would be effective in the aspect of prevention or therapeutics..”

- One of the limitation may be the lack of metabolomics studies, can you comments on this point.

Response: Thank you. A relevant text has been added to the limitation paragraph of the Discussion section. Page 15, line 347−352. “Fifth, our review lacked metabolomics studies. Although it is unclear whether changes in the bacterial flora are a cause or consequence, changes in secondary metabolites secreted by bacteria may lead to changes in the gut environment, which may be related to the pathogenesis of the disease by leading to some inflammatory stimuli. Further elucidation of the correlation between metabolomics and human microbiome due to SARS-CoV-2 infection is expected..”

Reviewer #3: In this systematic review, the authors collected evidence on alterations in the gut, upper and lower respiratory microbiota in Covid-19 infection.

The study is well conducted and written, offering a good perspective on the work in literature.

The evidence collected is unfortunately of low grade. All the studies collected had an observational design, 14/16 are conducted in China and consider only hospitalized patients. For these reasons, many confounding factors may have altered the results. Moreover, different types of samples and different methods of analysis are used.

Major revision

- Clarify whether unpublished studies were reviewed by a third-party researcher not involved in the study.

Response: Thank you for your suggestions. The unpublished studies (Yu et al. 2020, Budding AE, et al. 2020, Ai JW, et al.2020) reported were not peer-reviewed by a third-party researcher. This information has been provided in the revised manuscript. Page 5, line 114.

Minor revision

- The design and characteristics of the included studies are well described in Table 1. However, they could be reported more clearly in the text.

Response: Thank you for your comment. Revisions describing the included studies have been made to the revised manuscript (Page 7, line 143−144, 145, 148−149, 157,160-161. Page 8, line 174-175; Page 9, line 196, 212; Page 11, line 244-245).

- In the "Selection criteria and search strategy" paragraph, words used for the research could be placed in quotation marks rather than in parentheses

Response: Thank you for your suggestion. Each searched terms were placed in quotation marks as suggested, and some round brackets that are needed as an operator for the search condition were retained.

Submitted filename: Response_to_reviewers_MS_SY_MS.docx

Click here for additional data file.

2 Jun 2021

The human microbiome and COVID-19: A systematic review

PONE-D-21-06559R1

Dear Dr. Mizutani,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Francois Blachier, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors are very clear with the fact that the article is exploratory. It is still quite difficult to extrapolate the observations reported in the review and more, it is not simple to imagine any clinical implications of these observations. Nevertheless, it is interesting to observe that, like for other diseases, dysbiosis is associated with covid19. All the results of the articles included in the review are well reported.

Reviewer #2: Thanks for all your comments and the clarifications. The paper is excellent and will provide a strong contribution to this field.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Marvin EDEAS

14 Jun 2021

PONE-D-21-06559R1

The human microbiome and COVID-19: A systematic review

Dear Dr. Mizutani:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Francois Blachier

Academic Editor

PLOS ONE