By Madeleine Ball, PhD, and Billy Boyle
Over many decades, we have built our understanding of disease mechanisms by studying organs, tissues, cell types, and their interconnectivity. Study into how these functional elements work, and work together to keep us alive has been crucial for us to learn how and why these systems break down during disease. In addition, we are now aware that the human body is colonized by various microbial species of bacteria, fungi, and archaea—known collectively as the microbiome—which interact with our bodies and each other. As a result, we realized that our understanding of disease processes was incomplete. Scientists are now researching the interaction between microbes and the tissues of the body, and crucial insights have been gained into the role that the microbiome has on our health.
The composition of the microbiome differs among the colonized tissues of the body and human hosts, and it is thought to change throughout our lives. It is particularly diverse in the gastrointestinal tract, which has one of the richest microbial ecosystems on the planet.
The gut microbiome contains species that can digest much of the fibrous material in our diet through anaerobic fermentation—material that we naturally lack the enzymes to process.1 However, our health can suffer when the gut microbiome becomes unbalanced. For example, if the upper gastrointestinal tract contains an abnormal microbial community (usually one that includes bacteria native to the colon), or if it contains an excessive number of bacteria, a condition known as small intestinal bacterial overgrowth (SIBO) can develop. SIBO symptoms include bloating, diarrhea, and nutrient malabsorption.2
The microbiome has been associated with the pathophysiology of a wide range of diseases. Specific examples include non-alcoholic steatohepatitis3 and cardiometabolic diseases.4 The microbiome has also been shown to contribute to tumor initiation and progression through both direct (antigenicity) and indirect (the secretion of metabolites leading to immunomodulation) mechanisms.5
The pharmacokinetics of drugs can be impacted by the microbiome, particularly the gut microbiome. For example, the presence of the bacterium Eggerthella lenta can metabolize the cardiac drug digoxin to an inactive metabolite, dihydrodigoxin.6 Such effects have important implications for personalized medicine, as the microbiome adds an additional layer to why individuals can have different responses to pharmaceutical interventions. The microbiome’s relevance to personalized medicine is exemplified by the well-established role the gut microbiota plays in the regulation of the immune system. It is understood that the gut microbiota can significantly impact the efficacy of immune checkpoint inhibitors, a class of drug used as an anticancer therapy.7
From VOCs of microbial origin to breath tests
There is a strong link between the microbiome and mechanisms of disease, and deciphering the activity of an individual’s microbial colonies presents the opportunity to uncover new biomarkers. Through their own unique metabolic pathways, microbes can produce volatile metabolites, which are gaseous carbon-based molecules otherwise known as volatile organic compounds (VOCs). These compounds are made by a multitude of different processes within the body, but they can also originate exogenously. VOCs of microbial origin are produced through a variety of mechanisms. In the gut for example, VOCs can be produced as waste products from the metabolism and fermentation of food.
An extensive body of published work has linked microbial metabolites to disease, as well as to their precise biological origin within the body, and many of these metabolites are volatile in nature.8 As such, a promising method to investigate the microbiome and its association with health and disease is the analysis of exhaled breath composition, which can contain informative molecular markers of microbial activity.
Breath contains thousands of VOCs, which can be produced throughout the body, travel through the circulatory system, and cross the alveolar membrane to be released into the lungs. The gut microbiome also produces hydrogen and methane, which are already measured on the breath in hydrogen and methane breath test (HMBT) kits and used for diagnostic purposes.
The specific concentrations of these gases in the breath can be used to diagnose conditions such as SIBO and carbohydrate malabsorption (varieties of the latter include lactose and fructose intolerance). However, there are many more conditions associated with the microbiome that could alter VOC concentrations in exhaled breath, and breath sampling represents an important platform to detect and study a variety of microbial biomarkers.
Advantages of breath tests
The mechanism of how the microbiome regulates human health and disease revolves around the metabolites that microbes produce. Analysis of these metabolites is often undertaken using fecal, blood, and urine samples. However, analysis of the same microbial metabolites in exhaled breath could offer a significantly improved methodology, one that takes advantage of the well-established evidence base that has already been compiled for these compounds. Breath offers an alternative, standardized, and user-friendly approach to studying volatile microbial metabolites.
There are many benefits of using breath testing: it is a virtually inexhaustible resource; samples can be preconcentrated; and its intrinsic noninvasiveness is appealing to patients, clinicians, and clinical scientists. Many diseases, especially gastrointestinal diseases, are diagnosed and monitored using invasive biopsies, such as those obtained through endoscopic or surgical procedures. These are difficult to tolerate, resulting in an unpleasant experience for the patient, and therefore lower uptake.
All forms of invasive procedures come with risks of infection or damage to the surrounding tissue, whereas breath collection is completely noninvasive, thereby removing the associated risks. Invasive tests are generally more expensive, whereas breath sampling can be much cheaper to conduct. Another benefit is that breath samples can be taken at regular, short intervals, and a large volume can be collected at any one time. This, coupled with the noninvasiveness, can allow for easy longitudinal monitoring of a range of metabolic pathways. There is also no requirement for patients to visit clinics to take breath tests. When the tests are portable, collection can happen anywhere.
These benefits are especially relevant for clinical trials, where breath analysis can provide almost real-time information about participants’ metabolic pathways in response to treatments, with the potential for compounds in exhaled breath to act as pharmacokinetic/pharmacodynamic, safety, or efficacy markers. Near-real-time information can help the managers of clinical trials adjust procedures and reduce costs. Information at this frequency can be difficult to achieve with invasive methods, as they are more expensive, more difficult as a means of repeat sampling, and more likely to encounter complications when ethical approvals are sought.
The future of breath-based medical tests
We are just beginning to understand the true scale of the impacts that the microbiome has on our health, but we are already seeing rapid progress. Many breath biomarker candidates are being generated that promise to reveal the processes behind health and disease. The insights to be gained are expected to benefit individual patients.
Current breath research is demonstrating how introducing a test substance into the body can be used to measure the production of known VOCs to provide insights into metabolic activity, such as liver function.9 A similar rationale applies in HMBT kits, where test substrates elicit the production of hydrogen and methane from intestinal microbes. This demonstrates an innovative step for breath research, and this approach could be implemented to analyze more microbial metabolic pathways.
The understanding of the relationship between VOCs and microbial metabolism is significantly advanced, with many mechanistic foundational studies already completed using fecal, blood, and urine metabolomics. Therefore, breath research is well placed to progress this field further as underlying changes in metabolic activity, including those from the microbiome, can produce patterns of VOCs characteristic of specific diseases.
Investigating the microbiome has added another dimension to our understanding of the normal function of the body as well as our understanding of disease, but there is still much to be done. The intersection between breath biomarker research and VOCs of microbial origin is a challenging, but promising field, one that has progressed significantly in recent years.
Key goals for the field include the elucidation of the precise mechanisms through which the microbiome interacts with the body during disease, and the correlation of disease states and fluctuations in VOC levels measured in breath samples. When these goals are achieved, it will be possible to develop personalized, noninvasive breath tests that can be used as diagnostic and/or monitoring tools in the clinic, or even at home.
- Cronin P, Joyce SA, O’Toole PW, O’Connor EM. Dietary Fibre Modulates the Gut Microbiota. Nutrients 2021; 13(5): 1655.
- Sachdev AH, Pimentel M. Gastrointestinal bacterial overgrowth: pathogenesis and clinical significance. Ther. Adv. Chronic Dis. 2013; 4(5): 223–231.
- Bajaj JS, Heuman DM, Hylemon PB, et al. The Cirrhosis Dysbiosis Ratio Defines Changes in the Gut Microbiome Associated with Cirrhosis and Its Complications. J. Hepatol. 2014; 60(5): 940–947.
- Aron-Wisnewsky J, Clément K. The gut microbiome, diet, and links to cardiometabolic and chronic disorders. Nat. Rev. Nephrol. 2016; 12(3): 169–181.
- Jain T, Sharma P, Are AC, et al. New Insights into the Cancer–Microbiome–Immune Axis: Decrypting a Decade of Discoveries. Front. Immunol. 2021; 12: 622064.
- Haiser HJ, Seim KL, Balskus EP, Turnbaugh PJ. Mechanistic insight into digoxin inactivation by Eggerthella lenta augments our understanding of its pharmacokinetics. Gut Microbes 2014; 5(2): 233–238.
- Li X, Zhang S, Guo G, et al. Gut microbiome in modulating immune checkpoint inhibitors. EBioMedicine 2022; 82: 104163.
- Broza YY, Mochalski P, Ruzsanyi V, et al. Hybrid Volatolomics and Disease Detection. Angew. Chem. Int. Ed. Engl. 2015; 54(38): 11036–11048.
- Ferrandino G, Orf I, Smith R, et al. Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound—Toward Improved Detection of Liver Impairment. Clin. Transl. Gastroenterol. 2020; 11(9): e00239.