Ever since the World Health Organisation declared the COVID-19 pandemic to be a “public health emergency of international concern” in January 2020 (1), the lives of billions of people have been affected as the novel coronavirus spread across the world and countries implemented measures to contain the virus. Currently, over 150 million cases have been recorded and 3 million deaths. COVID-19 is primarily a respiratory infection, with fatal outcomes occurring mainly due to acute respiratory distress (2). While the main risk factors for infection and illness from the virus are well-known, researchers have also been looking into how the coronavirus interacts with the microbial world within the human body, both directly and indirectly. Through the use of cutting-edge genomic technologies, scientists have investigated important questions that could affect infection, disease progression and our wellbeing on the intersection between coronavirus and the human microbiome.
The two most important interfaces between humans and their environment are the gut and the lung. Both are absorptive organs with a high surface area that are constantly exposed to microorganisms from the environment. And the two systems are linked: via the immune system, signals originating from one organ interact with the other (3).
The lung microbiome has several key differences from the gut microbiome. It has a much lower biomass content, with roughly 10 to 100 bacteria per 1000 human cells (3). The lung microbiome composition is affected by the local lung environment. In particular, the host immune system, the pH of the lung lining, mucus production and oxygen concentrations in the lung affect how readily microbes can survive in the lung. Diseases of the lung generally cause drastic changes to the lung environment, which changes its microbial composition. The healthy lung microbiome boosts both specific and systemic immunity to pathogens, and can out-compete pathogens from the environment. The development of normal lung function in infants requires exposure to an array of microbes from the environment (4).
In terms of the bacterial microbiome composition of the lung in healthy subjects, the main species that dominate are Firmicutes and Bacteroidetes, followed by Proteobacteria and Actinobacteria. This is similar to the gut. Fungi that colonise the lung generally originate from the environment and are usually ascomycetes and microsporidia. A healthy lung is generally considered to have a low microbial density and high microbial diversity, while diseased lungs have high microbial density with low diversity (4). Studies of the lung microbiome will ideally look across microbial kingdoms. Although the lung microbiome is not widely studied, it may affect both risk of infection and disease severity.
Sampling the lung microbiome offers its own challenges, particularly due to the low biomass, semi-invasive location of the lungs and risk of contamination from the nose or mouth during sampling (5). Sputum, the mucus found in the lungs and respiratory tract, contains a mixture of upper- and lower respiratory tract microorganisms. Sputum samples are readily obtained in healthy subjects or patients. To collect a sputum sample, subjects need to force a cough to expel sputum through the mouth, therefore there will always be residual contamination with saliva. Bronchoscopy can be used to sample the lung microbiome by expelling and suctioning a small volume of water, or taking brush samples of the lung tissue. Targeted suctioning through the trachea is a possibility for intubated patients. Whatever method is used, consideration of contamination risk and standardized procedures will assist in obtaining representative samples (5).
Tracking the effect of COVID-19 infection on the lung microbiome is an important step in assessing whether changes in the microbiome can affect disease severity. Currently, few studies have investigated the lung microbiome’s response to COVID-19 infection.
In one study conducted in 8 COVID-19 patients in Wuhan, China, bronchoalveolar lavage fluid was analysed via metatranscriptome sequencing and compared to samples from 25 pneumonia patients and 20 healthy controls. This allowed transcriptionally-active microbes to be profiled. The authors found distinct differences between the microbiota of healthy subjects compared to the COVID-19 and pneumonia patients. The healthy controls were more likely to have a higher microbial diversity than the two disease groups (6). The researchers were also able to look at co-infection with other common respiratory viruses, and genetic variation within the coronavirus in patients using the same genomic technology (6). Functional profiles were prepared using this dataset, using the KEGG Enzyme Nomenclature reference hierarchy to annotate the reads. A functional signature was found for COVID-19 patients: decreased potential for lipid metabolism and glycan biosynthesis, with increased potential for carbohydrate metabolism (7).
In another study conducted in Wuhan, the lung microbiota of 20 deceased COVID-19 patients was investigated from lung tissue samples. 16S shotgun sequencing was used to identify bacterial OTUs in these patients, while ITS gene sequencing was used for fungi. Over 80% of the total bacterial sequences were Acinetobacter, with other genera making up less than 4% each of total reads. The fungal communities found were represented by Cryptococcus (28%), followed by Issatchenkia (8%) and Wallemia, Cladosporium, Alternaria, Dipodascus making up 4-5% of reads. Most patients had mixed bacterial and fungal infections. The species found were associated with multi-drug resistant infections and opportunistic infections, indicating profound dysbiosis in the lung. These patients had all been treated with antibiotics and antiviral agents, therefore their lung microbiota may not be a true representation of what happens during infection (8).
Although the research base is currently limited, various techniques have been used to investigate the bacterial, fungal and viral lung microbiome, as well as the functional capacity of the microbiome. These show the impact of COVID-19 infection in the lung environment.
How does COVID infection affect the gut microbiome?Although primarily considered a respiratory disease, COVID-19 has certain gastrointestinal manifestations that could be responsible for changes in the gut microbiome seen in COVID patients. Digestive symptoms consistently found in COVID-19 patients include diarrhoea, vomiting and nausea, and the virus is shed in the stool (9-11). Increases in faecal calprotectin, a marker of inflammation in the intestines, have also been seen in COVID-19 patients, indicating an immune response in the gut to infection (11). Furthermore, conditions such as obesity that are associated with gut microbiota dysbiosis appear to have more severe COVID-19 disease progression (12).
A limited number of studies have looked at how the gut microbiome changes during COVID-19 infection. A common pattern seen across studies conducted is the increase in opportunistic bacterial and fungal pathogens such as Streptococcus, Veillonella, Aspergillus and Candida, with a decrease in commensals such as Faecalibacterium (11, 13).
The microbiome affects the innate immune system and the response to infection in general, so it stands to reason that there may be effects from the gut microbiome on disease risk or severity. Research has currently focused on two main areas. Firstly, the cell location that is targeted by the coronavirus for initial entry into the cell and the inflammatory response to infection, the ACE2 receptor, is also located in the lining of the intestines, and the coronavirus is also excreted in the stool. Therefore, modulation of the immune response via the ACE2 receptor in the gut may influence resistance to infection and the immune response. Secondly, the gut microbiome influences systemic inflammation via intestinal cell permeability and the production of pro-inflammatory substances such as lipopolysaccharides. The production of short chain fatty acids by the gut microbiome may suppress inflammation by contributing to the barrier function of intestinal cells and reducing the production of cytokines (14). COVID-19 disease severity is related to a pro-inflammatory cytokine storm that leads to organ failure and death (15).
Certain bacterial genera or species have been found to be elevated in COVID-19 patients and produce systemic pro-inflammatory effects. For example, Streptococcus induces the secretion of pro-inflammatory cytokines, and Actinomyces viscosusella induces inflammatory lesions (16). A study conducted in COVID-19 patients in Wuhan, China, investigated whether there were gut microbiome shifts according to three levels of disease severity. Q-PCR was used to compare changes in the 10 predominant bacterial groups. Acute-phase inflammation markers such as C-reactive protein and interleukin-6 were elevated in the most severe disease group. Anti-inflammatory and commensal bacteria Faecalibacterium, Lactobacillus and Bifidobacterium decreased in all groups while opportunistic pathogens Enterococcus and Enterobacteriaceae increased in the most critically ill patients (17).
In a similar study conducted in Hong Kong, shotgun sequencing of the microbiome from the stool of 100 COVID-19 patients and 78 non-COVID controls showed gut microbiota changes related to disease severity and markers of inflammation (18). Likewise, commensals such as Faecalibacterium and Bifidobacterium were suppressed in COVID-19 patients. Principle Component Analysis indicated that the gut microbiota was stratified by disease severity. Inflammation markers and inflammatory cytokine concentrations were concordant with these differences. In addition, stool samples collected up to one month after a negative COVID-19 test showed that dysbiosis persisted (18).
Further to changes in the microbiome due to coronavirus infection, these studies show that increasing microbial dysbiosis occurs with increases in disease severity. Inflammation may be the mediating factor. Prospective studies would help strengthen the research base.
How does COVID-preventive handwashing affect the skin microbiome?A key strategy in preventing infection since the start of the pandemic has been frequent handwashing or using hand sanitisers. Both these products are effective in washing off microbes from the hands or killing them. However, they also affect the microbiome of the skin. Could handwashing practises in the pandemic affect the skin microbiome?
A recent article compared both the effect of different handwashing techniques, sampling methods and analysis methods on the skin microbiome (19). Much of the skin disinfection literature is focused on culturable bacteria, however culture-independent techniques allow cross-kingdom skin ecologies to be measured, and can capture a broader microbial diversity. In this study, 16S rRNA sequencing showed that there were no long-term effects from a single washing treatment on microbial diversity (19). This may not be the case with frequent handwashing. For health-care workers, damaged skin due to handwashing actually increased the skin’s bacterial load overall, including skin pathogens such as Staphylococcus aureus and antibiotic-resistant microbes (20). Handwashing may therefore actually contribute to spreading pathogens if it causes skin damage.
In response to the pandemic, most countries have implemented restrictions to reduce the number of in-person social interactions and control the spread of the virus. These include restricting travel, advising that people work from home, closing restaurants and shops, limiting visiting other people, and shutting down recreational and sporting locations. Depending on the effect these restrictions have on individuals’ lifestyles, the gut microbiome will be affected.
The pandemic, concerns about contracting the disease, and impacts on our social support and lifestyles can manifest themselves as psychological stress. A 21-country comparison of the effect of the COVID-19 on psychological wellbeing found increases in depression, stress and anxiety (24). There is a two-way relationship between the gut microbiome and stress, with stress impacting the gut microbiome, and certain microbial patterns associated with stress resilience (25). Potentially, systemic inflammation or gut permeability may be responsible for stress resilience and how the gut microbiome responds to stress.
The current evidence base only scratches the surface of the relationship between COVID-19 and the microbiome. Even though some nations have already been able to reduce the impact of the pandemic through vaccination – and more countries will do so in the coming months – it’s likely that the novel coronavirus is now established and we may have to handle successive epidemics, as we do with influenza. As there are links between COVID-19 infection, microbial dysbiosis and disease severity, the use of prebiotics, probiotics or synbiotics may be useful strategies going forward. Clearly, more research using genomic technologies is necessary in order to develop safe and effective therapies.
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