In part 1 of this feature, we discussed how humans are ‘more microbe than man’, with trillions of these microscopic organisms living within our bodies. These microbial genomes confer a significant genetic payload, containing over 2 million genes, compared to our own genomes, which are composed of roughly 20,000 genes.
Since the discovery that our microbes are mostly friend rather than foe, wellness initiatives have highlighted the important task of maintaining our microbiome’s balance for our overall health. Furthermore, recent microbiome research has linked imbalances in our microbiomes to the development of a wide range of diseases. This provides opportunities to harness the power of our microbiome to treat these illnesses, including through the use of personalised and precision medicine.
In part 2 of this feature, we discuss how the microbiome is established from birth, how it evolves throughout our lifetimes and how a bacterial imbalance can lead to disease development. We also examine how microbiome-specific precision medicine is rebalancing the microbiome and advancing the field of personalised medicine, providing hope for patients with antibiotic- and immunotherapy-resistant diseases.
Anatomical differences
The human intestine is a complex and dynamic ecosystem composed of a complex and diversified set of microorganisms, known as microbiota or the microbiome. Although these two terms are often interchangeable, there are certain differences to remember. Microbiota describes the living microorganisms within a defined environment, such as the oral and gut microbiota. The term microbiome, however, refers to the collection of genomes from all microorganisms within the environment, including not only the community of the microorganisms but also the microbial structural elements, metabolites and the environmental conditions. In this regard, the term microbiome encompasses a broader spectrum than that of microbiota.
In humans, several microbiomes exist, including those of the skin, mouth, GI tract, lungs and reproductive tract. These miniscule organisms are essential for a complex range of functions to maintain the body’s overall homeostasis and can even influence our behaviour.
The microbiome composition varies between the body sites, as shown in Figure 1. However, the gut microbiota is considered the most important for overall health and serves several functions, including the fermentation of food, protection against pathogens, stimulating immune response and production of vitamins. The gut microbiota is composed of six phyla including Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia, out of which Firmicutes and Bacteroidetes are the key types. The gut microbiome also includes fungi, known as mycobiota, of which the most well classified are Candida, Saccharomyces, Malassezia and Cladosporium.
Figure 1. Human microbiota composition in different anatomical locations. Key bacterial genera are highlighted. Taken from Hou et al. 2022.
The body provides the microbiome’s environment and nutrients, and in return receives beneficial metabolic, immunological and trophic functions that help sustain overall health. Therefore, it is unsurprising that an imbalance, or dysbiosis, in the microbial community can lead to a decline of a person’s health.
Gut microbiota, in particular, has been implicated in the pathogenesis of many diseases, including autoimmune diseases, chronic intestinal inflammation, neurological disorders, cancer, diabetes and obesity. Therefore, understanding how to keep our microbiome balanced and healthy is becoming a key health priority from birth, while also providing unique opportunities to target and rebalance the microbiome to effectively treat a wide range of diseases.
Friends since birth
Development of this complex community of microorganisms begins in utero and continues during birth as the baby travels along the birth canal. During this process, “seeding” or transfer of microbes from the mother to newborn occurs, which is a key event in the establishment of the baby’s immune system. Disruption of this process can significantly affect the long-term health of infants.
A second key microbiome development period occurs during breast feeding, which further improves the baby’s immune and digestive systems, helping to prevent infection and even helping with brain development. Therefore, the health of the mother’s microbiome is essential for the healthy development of the baby’s microbiome, and dysbiosis can result in a range of developmental issues. Antibiotic use can also affect the microbiome, with use during pregnancy associated with lower levels of protective bacteria including Lactobacillus and Bifidobacteria in the newborn’s gut microbiome.
The way a baby is delivered can significantly alter the development of the microbiome. Microbiome profiles of newborns born vaginally compared to those born by caesarean have been shown to be distinctly different, which could result in development problems in the baby’s immune and digestive systems.
Babies born through caesarean have been shown to have microbiomes less like that of the mother and are more likely to contain unexpected skin and oral microbes, including those found in the delivery or operating room. As adults, this group has been shown to have distinctly different gut microbiomes compared to those who were born vaginally. Babies born through caesarean have also been shown to be more likely to develop immune-related disorders as adults including asthma, allergies and inflammatory bowel disease. These adults are also more prone to obesity.
Research into the development of the microbiome from birth is of great interest as it appears that the microbiome we are born with remains with us throughout our lives. It is hoped that we will soon be able to use microbiome information to predict which diseases people may be predisposed to, and to target the microbiome directly to treat a wide range of diseases.
It takes all sorts
The intestinal microbiota is the most populous community in the human body. The stomach contains around 103-104 bacteria, the duodenum 105-106 and the terminal ileum 108-109 bacteria per gram of tissue. However, the largest and most diverse population is found in the large intestine, which contains around 1012-14 bacteria per gram of tissue.
It is estimated that 400-500 different genera of microorganisms make up the human intestinal microbiota, 90% of which are predominantly anaerobic. Most of them belong to two key genera, Bacteroidota and Bacillota, while other less common populations include Pseudomonadota, Actinomycetota, Fusobacteria and Verrucomicrobia.
Studying this many different types of bacteria presents its own set of challenges, and so the methodology used for microbiome research generally depends on the research question being asked. If the goal is to determine what types of bacteria are present in a particular microbiome, 16s rRNA sequencing is commonly used to identify the order of the bacteria in a sample. Shotgun metagenomics approaches measure DNA sequences but utilize more targeted sequencing approaches.
If the focus of the research question is to determine overall mRNA expression levels in the microbiome, then metatranscriptomics approaches can be used. All of these techniques employ next generation sequencing (NGS) methods but utilise different software packages to analyse the sequences. Metaproteomic approaches utilise liquid chromatography to measure proteins present in a microbiome, while metabolomic approaches use mass spectrometry to measure metabolites within a sample. These techniques are illustrated in Figure 2.
Figure 2. Microbiome analysis techniques.
Adapted from https://www.labome.com/method/Microbiome-Research-Methodologies.html.
Traditional microbe studies involve culturing bacterial strains and transferring to isolated cells, tissues, or whole animals for further studies. However, unfortunately, an estimated 70% of the gut’s bacteria cannot be cultured with conventional microbiological lab techniques. This means most microbiome studies must use samples taken directly from a host or environment and proceed with DNA isolation without culturing. These culturing difficulties have previously slowed microbiome research, meaning we still do not currently have the full picture of how our microbiome works. However, recent advances in genetic sequencing techniques and bioinformatics approaches for the analysis of large datasets have significantly increased our capacity to question the relationship between the microbiome and human health.
Many clinical diagnostic and therapeutic applications for microbiome analysis are currently in development. Falling under the umbrella of precision medicine, microbiome analysis can tell us a significant amount of highly specific information in relation to a person’s overall health. This may allow for personalised treatment decisions to be made based on an individual’s specific microbial community, including nutritional recommendations.
Precision nutrition
Scientists are now using cutting-edge methods to advance precision nutrition, a research area which falls under the umbrella of precision medicine. These studies hope to establish differences in metabolism and nutritional needs among individuals, with an ultimate goal to develop targeted nutritional interventions to improve health on a personalised level. While this may sound simple enough, this area is highly complex as multiple individual factors can affect our response to diet, including genetics, epigenetics, metabolic processes and environmental exposures, medications, drug use and tobacco. However, this exciting area could revolutionise treatment for many diseases through diet alteration including inflammatory bowel diseases, autoimmune diseases and even Parkinson’s disease, reducing the need for pharmaceutical intervention.
Precision medicine
Although the ‘healthy’ microbiome has not yet been fully characterised, distinct ‘dysbiotic’ microbial signatures associated with disease states are being characterised using high throughput sequencing techniques (Figure 3). This allows for the development of disease signatures, distinct from those of healthy individuals, that can be used as biomarkers, for example in hepatocellular carcinoma. Furthermore, due to advancements in sequencing methods, research into diseases such as inflammatory bowel disease have transitioned from just mapping microbiome compositions to the development of specific microbial based therapies.
Figure 3. Diseases associated with microbiome dysbiosis. Taken from Hou et al. 2022.
It is hoped that recent developments in microbiome sequencing will allow for advances in the diagnosis and treatment of many common diseases, allowing for personalised treatment decisions to be based on this analysis. Success has already been found in clinical trials for several diseases using microbiome sequencing not only for diagnostics, but also to modulate the microbiome to improve treatment responses. Key successes in these fields have been described below.
Cancer
An area where the immense implications of the microbiome have most clearly been demonstrated is tumour biology. Over the past few years, research has suggested that there are close links between cancer and the microbiome, with many microbes living within or around tumour tissue, creating the cancer microbiome. This microbe community is thought to influence how the immune system and/or therapeutics interact with the tumour, significantly affecting cancer treatment effectiveness, drug resistance and overall patient outcomes.
The cancer microbiome affects tumour development at all stages from initiation to establishment and further progression, either directly through effects on the tumour cells themselves, or indirectly through manipulation of the immune system.
Over the past few decades, some individual microbe species have been linked to the development of specific cancers, such as human papillomavirus (HPV), which is linked to cervical cancer, or Helicobacter pylori, which is linked to stomach cancer. More recently, links between levels of specific bacteria and cancer patient outcomes have also been made. For example, in colorectal cancer patients following surgery, higher levels of Fusobacterium nucleatum adherent to the tumour tissue were associated with worse patient outcomes, increased mortality and decreased disease-free and overall survival rates.
Recent research has also highlighted the key role of the gut microbiome in mediating tumour responses to immunotherapies, such as immune checkpoint inhibitors (ICIs) targeting PD-L1 or cytotoxic T lymphocyte–associated protein 4 (CTLA-4). It is thought that dysbiosis associated with malignant disease or concomitant antibiotic use could influence primary resistance to immunotherapy in cancer patients. Modulation of the microbiome using faecal microbiota transplantation (FMT) has been shown to increase the efficacy of immunotherapies and decrease their toxicity. This therapy positively affected how anti-PD-1 refractory metastatic melanoma patients responded to anti–PD-1 immunotherapy, resulting in increased abundance of microbes and activated CD8+ T cells, and decreased frequency of immunosuppressive immune cells. This effect has also been observed in other epithelial tumours.
In pancreatic cancer patients, substantial levels of tumour microbial diversity are associated with better outcomes. Patients with higher levels of diversity (alpha diversity) were found to have more favourable responses to treatment and significantly longer overall survival outcomes. It is therefore thought that alpha microbiome diversity could be a predictive marker for overall survival in resected pancreatic cancer patients.
Microbiome research is now taking on one of the key challenges in cancer diagnosis: early detection. Microbiome-based oncology diagnostic tools are in development, using workflows of machine learning methods to analyse whole genome and RNAseq cancer microbiome data for different cancer types from thousands of treatment-naive patients. This research aims to generate a set of diagnostic classifiers capable of distinguishing between healthy and tumour tissue. It is hoped that this technology could even be used to distinguish between cancer types and determine the cancer stage.
Overall, understanding how widely host–microbe associations are maintained across populations may reveal individualised host–microbiome phenotypes. This information can then be integrated with other ‘omics’ data sets to enhance precision medicine capabilities in cancer.
Parkinson’s disease
The microbiome has recently been recognised as an important modulator of neurogenerative diseases, including Parkinson’s disease, which is often preceded by gastrointestinal symptoms. The acceptance of a gut-origin hypothesis (Figure 4) has highlighted the importance of gut microbiota in the prodromal stage of the disease, which is often 10–20 years before the onset of motor symptoms. Although direct cause-effect links between gut microbiota and the development of Parkinson’s disease are currently unclear, research using animal models supports the idea that dysbiotic gut microbiota can aggravate Parkinson’s disease pathology. Excitingly, re-establishment of the gut microbiota has also been shown to delay or correct the onset of Parkinson’s disease.
Figure 4. Schematic representation of gut microbiota dysbiosis associated with Parkinson’s disease.
Microbiota dysbiosis plays vital roles in the occurrence and development of Parkinson’s disease and is associated with increased intestinal permeability, aggravated neuroinflammation, abnormal aggregation of α-synuclein fibrils, oxidative stress, and decreased neurotransmitter production. Microbiota-targeted therapies could be used to modify the composition of the gut microbiota to revert a dysbiotic condition. Taken from Zhu et al. 2022.
Enrichment of the genera Lactobacillus, Akkermansia, and Bifidobacterium, and depletion of bacteria belonging to the Lachnospiraceae family and the Faecalibacterium genus, both important short-chain fatty acids producers, have been reported as the most consistent Parkinson’s disease gut microbiome alterations. This dysbiosis may result in a pro-inflammatory status, which could be linked to the recurrent gastrointestinal symptoms affecting Parkinson’s disease patients and development of the disease.
It is hoped that the gut microbiome therefore represents a promising therapeutic target for Parkinson’s disease, with many treatments now being researched including probiotics, psychobiotics, prebiotics, synbiotics, postbiotics, FMT, diet modifications and even traditional Chinese medicine.
Inflammatory bowel disease
Inflammatory bowel disease (IBD) is an umbrella term for chronic inflammatory conditions affecting the GI tract, including Crohn’s disease, characterised by lesions in various regions of the digestive tract, and ulcerative colitis, characterised by continuous and superficial inflammation of the colon. Although IBD development is related to a number of complex and multifactorial mechanisms, several risk factors have now been well documented, including a dysregulated immune response, genetic mutations and environmental factors.
Studies have shown that microbiota can interact directly with the intestinal barrier. Dysbiosis of gut microbiota is therefore highly associated with the development of IBD and chronic intestinal inflammation, which can ultimately lead to the development of several other conditions including cancer, diabetes and heart diseases.
Several studies have determined that the composition of gut microbiota is different between IBD patients and healthy controls. In IBD patients, the ratio of Bacteriodetes to Firmicutes has been shown to be decreased, while the abundance of gammaproteobacterial is increased. The levels of normal, protective bacteria, Bacteroides, Eubacterium, and Lactobacillus are also significantly reduced in IBD patients. However, it is possible that microbiota dysbiosis can be considered a response to environmental changes resulting from intestinal inflammation. The possible involvement of fungi and viruses in IBD are also being studied, but no link has been established so far.
Currently, a multi-faceted evaluation system of clinical, biochemical and endoscopic techniques is used to diagnose and evaluate IBD patients with the aim of predicting drug response. It is hoped that in the future, the analysis of gut microbiota and metabolites could more reliably predict drug responses and enable the use of personalised treatments to eliminate debilitating IBD symptoms.
COVID-19
Studying the effects of COVID-19 on the microbiota is a relatively new area of research. However, there is accumulating evidence that the microbiome is significantly altered during both active COVID-19 infection and in post-acute COVID-19 syndrome (PACS).
Common COVID-19 symptoms relating to respiratory infection include shortness of breath, cough, fever, fatigue and abnormal chest x-ray. COVID-19 related GI symptoms include diarrhoea, nausea, vomiting and abdominal pain. Clinical studies have therefore suggested that COVID-19 infection results in disruption of the respiratory and GI microbiota and make patients vulnerable to secondary pathogen infections. Combined, this may be responsible for much of the morbidity and mortality associated with the COVID-19 pandemic.
In severe to critical patients, as the COVID-19 infection progresses, the symptoms affecting the respiratory systems generally worsen, resulting in further complications including acute respiratory distress syndrome (ARDS), secondary pathogen pneumonia, sepsis and end-stage organ failure. As microbiota are key for the maintenance of GI and respiratory homeostasis and overall health, these symptoms strongly link COVID-19 symptoms and their severity with disruption of the microbiota.
GI symptoms during COVID-19 infection are associated with a higher risk of hospitalisation and/or greater disease severity. Several studies have detected viral SARS-CoV-2 RNA in the stool samples of COVID-19 patients, while further studies have shown that ACE2 and TMPRSS2, important functional proteins expressed in the GI tract, are targeted by SARS-CoV-2. Endoscopy investigations in these patients also revealed colon damage caused by the infection.
A shotgun metagenomics analysis of 15 hospitalised COVID-19 patients disclosed that their faecal microbiomes were deficient in beneficial bacteria and abundant in opportunistic pathogens. Compared to healthy samples, COVID-19 patients were found to have low levels of the anti-inflammatory bacteria Lachnospiraceae, Roseburia, Eubacterium, and Faecalibacterium prausnitzii and high levels of pathogens known to cause bacteremia such as Clostridium hathewayi, Enterobacteriaceae, Enterococcus, Actinomyces viscosus, and Bacteroides nordii. This dysbiosis was found to persist even after clearance and recovery from SARS-CoV-2 infection. A further study found increased proportions of opportunistic fungal pathogens including Candida albicans, C. auris and Aspergillus flavus in the faeces of COVID-19 patients, which have been associated with the development of pneumonia and other respiratory infections. Therefore, gut fungi dysbiosis may predispose COVID-19 patients to fungal infections and/or secondary fungal infections.
How clean is too clean?
In the wake of the COVID-19 pandemic, we have never paid so much attention to the cleanliness of our hands, surfaces and general environment. But how is this increased hand washing and sanitiser use affecting our natural microbiomes? Through putting ourselves into lockdowns and social isolation, have we permanently altered our natural defence mechanisms?
Throughout history, improved sanitation and medicinal developments have reduced the toll of infectious diseases. However, experts warn that antimicrobials are a double-edged sword. While killing the microbes that cause disease, we are also affecting our microbiome, which could be detrimental to our health.
It is thought that increasing urbanisation and significant environmental changes are also leading to decreased microbial diversity, which in turn could lead to the development of untreatable, chronic diseases. To help lessen these risks, projects such as the Microbiota Vault hope to preserve the biodiversity of human-associated microbiota and ensure the safe storage and preservation of microbiota samples and collections for collaborative use with research groups worldwide.
Future research
There are still many unclear areas surrounding the connection between the microbiome, health and disease. Research priorities include understanding what information we need to collect about how the microbiome responds to environmental exposures, how we can use this information to prevent issues such as antibiotic resistance and discovering what strategies could be used to understand the cause and consequences of disorders with a lifelong pathogenesis. Further, we still have a lot more to learn about the microbiome’s relationship with other key body systems, including the nervous and endocrine systems. With time it is hoped that the bigger picture will become clear, paving the way for a new era of personalised medicine.
An interesting developing area of research is the study of the microbiomes of native people, in particular those living in uncontacted populations in remote and isolated locations. This research has already shown that people living in Western, industrialised countries have reduced gut microbiome diversity compared to those living a traditional lifestyle. Compared to these isolated populations, those with a Westernised lifestyle have a substantially lower level of faecal bacterial diversity. This is thought to be due to a wide number of lifestyle factors that have not yet been fully characterised. However, dietary factors, antibiotic use and caesarean birth routes are thought to significantly contribute to these differences.
These microbiome alterations are thought to be linked to many diseases that are commonly found in Westernised societies such as obesity, asthma, allergies, coeliac disease and diabetes. Through the study of the microbial makeup of these non-Westernised populations, it is hoped that a reservoir of microbiota could be developed to replace the protective microbes that we are currently missing. There is, however, an ethical issue surrounding this subject, with a need to ensure the participants understand the needs and requirements for this work. This is alongside the need to further research the required microbial strains, their cultivation and, importantly, the safety of their use.
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