The microbiome challenges what it means to be an individual.
Our understanding of the factors that contribute to health and disease has been revolutionized by the discovery that the human body is colonized by an entire ecosystem of microbes – referred to as the microbiome. Although once considered to be much higher, calculations put the ratio of microbial to human cells close to 1:1, which remains a staggering estimate and contextualizes the power that the microbiome can exert on the human host (1). The composition of microbial communities is different depending on location within the body, with the microbial colony in the gastrointestinal tract representing one of the richest and most dynamic microbial ecosystems on the planet (2). The scale which we interact with microbes reframes our body as a system of many interacting organisms, and changes in the microbiome composition have been linked to a range of different diseases, including inflammatory bowel disease (IBD) (3,4), alcoholic and non-alcoholic fatty liver disease (NAFLD) (5), non-alcoholic steatohepatitis (NASH) (6), cardiometabolic diseases (7), and cancer (8,9). The microbiome has beneficial effects for the human host, with roles in nutrient metabolism, protection against pathogenic microbiota, and immunomodulation (10). Microbe-human interactions are complex and multifactorial, and so a major focus of biomedical research involves investigating the mechanisms through which the microbial inhabitants of our body can help and hinder us.
Examining the composition of blood, mucus, and waste products has been utilized for centuries to gain insight into the inner workings of the body. These substances contain biomarkers, quantifiable compounds that pertain to biological processes and can be used to measure various states of health and disease. Two methods of studying the microbiota are the use of metabolomic methods to quantify microbial metabolites and DNA-sequencing for the identification of species (usually through 16S ribosomal RNA genes) or functional analysis through metagenomics (11). These methods are usually undertaken on fecal samples, however, a waste product of the human body that has gone underutilized is exhaled breath. The earliest known reference to the link between the breath and disease dates to ancient Greece, with Hippocrates in his treatise on breath aroma and disease attributing a distinctive breath odor to liver failure, which he referred to as fetor hepaticus (12). Although rudimentary, this establishes two fundamental principles: that breath contains compounds originating from deeper within the body than just the respiratory system, and that alterations in the levels of these informative compounds can potentially alert to disease states within the body.
Exhaling clues to the activity of the microbiome
Breath is a virtually inexhaustible resource, comprised of a rich matrix of compounds originating from both outside and inside of the body. Specifically, breath is enriched for volatile metabolites, also known as volatile organic compounds (VOCs). These gaseous, carbon-based molecules travel systemically through the circulatory system. The vascular surface of the alveolar membrane serves as a launch pad from which VOCs in the blood can take flight and volatilize into the air. This air contains many potential biomarkers from metabolic processes in the body and is exhaled through breath. One of the most significant producers of breath VOCs is the bacterial, archaeal and fungal metabolic pathways of the gut microbiome, particularly surrounding the fermentation of dietary fibers that the human body cannot metabolize (2). Fermentation of fiber produces volatile short-chain fatty acids (SCFAs) and alcohols, but many other volatile compounds are generated from other microbial metabolic pathways such as aromatic amino acid catabolism that produces phenol, phenyl acetaldehyde, and p-cresol (13).
Different species of microbes have their own forms of metabolism that produce specific by-products, and therefore the composition of breath depends on the types of microbes present. For example, methane gas in the breath indicates methanogenic archaea in the gut (14–16). Normally, the gastrointestinal tract is dominated by bacterial species from the phyla Bacteriodes and Firmicutes, with their combined presence accounting for approximately 95% of gut microbial communities (17,18). In disease states, other phyla can dominate such as Proteobacteria (containing E. coli), Actinobacteria, Fusobacteria, or Verrucomicrobia (18), all with their own characteristic metabolic signatures, which can produce a change in the levels of VOCs detectable. Therefore, changes in breath VOCs could imply specific microbiota composition changes, laying the groundwork for the discovery of breath biomarkers for the numerous microbiome-associated drivers of health and disease status.
Breath, microbiome, and disease.
Breath analysis is already being incorporated in microbiome research with great success, as many of the well-studied metabolites are volatile. One of the greatest challenges in establishing breath-based VOCs as biomarkers is understanding the mechanistic origin of the VOC, however, many microbial metabolites already have strong links to specific species, pathways, and processes (4,9,19–22). The next step of linking fecal microbiota composition to exhaled VOCs and disease states has already begun, with one recent example demonstrating the correlation between gut microbiota changes, breath VOC changes, and active or inactive Crohn’s disease (CD) status (23).
In this study, the SCFAs acetate and propionate in breath significantly correlated with Bifidobacteria and certain species within the Firmicutes phylum in both CD states. The relative abundances of VOCs and the correlated microbial strains both decreased in active disease subjects. Other findings from the study included significant correlations between inflammation-related VOCs like pentane and octane, and Bacteroides fragilis and Ruminococcus gnavus in active disease state subjects. Although these compounds might be indirectly associated with microbial activity, these results contribute towards a comprehensive view of the processes involved in changing VOC levels on the breath.
Another example of where breath analysis has the potential to revolutionize diagnostic and monitoring technology in the microbiome space is in liver disease. It has been hypothesized that NAFLD and NASH may share common physiological causes with alcoholic fatty liver disease. Ethanol exposure may not just arise from alcohol intake but also excessive bacterial metabolism producing ethanol in the gut. Recent studies have shown that microbial dysbiosis may indeed be linked to the development of NAFLD and NASH, with bacterial species such as Klebsiella pneumoniae capable of producing high amounts of ethanol (6,24). This is crucial, as bacterial-derived ethanol can result in exceeding the daily recommended dose of alcohol without any intake of alcoholic drinks. Ethanol is metabolized by hepatic alcohol dehydrogenase before it reaches the peripheral circulation, blood concentrations of ethanol are negligible regardless of the amount produced by the microbiota. As ethanol is volatile, bacterial-derived ethanol can be detected in exhaled breath, albeit at low concentrations; less than 20 ppb (parts per billion), which is below the current capabilities of commercially available breathalyzers. Alcohol has the potential to counteract the beneficial effects of NASH drugs, including many currently in clinical trials, and highlighting the importance of developing a test for microbially-generated alcohol. Owlstone Medical has developed a breath analysis device capable of detecting ethanol in the ppq (parts per quadrillion) range, and has the potential to be utilized for the identification of patients at risk of high ethanol exposure before clinical visits, enabling better patient selection for drug trials and treatment.
Breath analysis can transform microbiome research.
Breath has numerous advantages that go beyond the added comfort for participants. The current method of choice for many microbiome studies is fecal analysis, which despite containing informative compounds, reflects a broad period of digestive passage as the average time for whole gut transit in a healthy person can range from 10 to 73 hours (25). Mucosally adherent microbes, as well as those present higher up in the small intestine are commonly missed in fecal analysis, and the storage of fecal matter in the rectum causes dehydration and other environmental changes that may lead to misrepresentative microbial composition (18). This is important when considering location-specific changes, for example in conditions such as small intestinal bacterial overgrowth. Informative compounds that are produced by the microbiota such as butyrate are the main energy source for enterocytes, and stool analysis may be confounded by the metabolism of compounds. Although many of the microbial metabolites of interest happen to be volatile, often they are monitored in liquid samples of urine or feces, but sample variability can be introduced during handing due to the loss of highly volatile compounds into the air away from detection equipment.
Breath can detect the same microbial-originating VOCs that are studied in fecal matter, with the bonus that a large volume of breath can be taken regularly, allowing for detailed longitudinal monitoring of a wide range of metabolic processes. The area in which these methodological benefits could be best utilized is in clinical trials, where biomarkers for metabolic pathways could be monitored in real-time in response to treatments. Breath VOCs could serve as pharmacokinetic/pharmacodynamic, safety, or potentially even efficacy markers, allowing for faster endpoint decisions and minimizing costs of lengthy clinical studies, as well as reducing the need for expensive invasive monitoring. Microbial VOCs could also be used for the testing and research of the impact of food ingredients such as fibers, pro- and pre-biotics, and fermented foods on digestive health, as well as the response to different therapeutics.
How can Owlstone Medical help.
Breath is an underutilised methodology for study into microbial metabolites that has significant benefits over other sampling methods. Owlstone Medical is a world-leader in breath research, utilising breath as a revolutionary biomarker detection and analysis technique in an ever-expanding number of fields, including in the microbiome space. We have collaborated with many industrial and academic partners, giving expert advice and supplying specialist equipment such as our ReCIVA Breath Sampler, which has been utilised in several recent clinical studies (26–28). A recent example includes the EMBER consortium publication in Science Translational Medicine, whereby machine learning was used to identify VOC signals in breath capable of distinguishing between healthy participants and those suffering from acute breathlessness. In this study, breath VOCs were demonstrated to have the potential to subtype patients with different underlying breathlessness causes (such as pneumonia, heart failure and chronic obstructive pulmonary disease) (26). If you are interested in incorporating breath analysis to your microbiome research, Owlstone Medical can provide all the expertise, and tools you need.
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