May 1, 2023
Stool testing provides markers for various gut bacteria, parasites, viruses and fungi. Additional markers provide insight into maldigestion and malabsorption (pancreatic elastase, steatocrit), inflammation (calprotectin), immune function and intestinal barrier integrity (sIgA and zonulin) (Khaki-Khatibi et al., 2020; Malinen et al., 2005). Imbalanced microbial levels suggest dysbiosis which is associated with numerous pathologies.
Stool tests such as Diagnostic Solutions Gastrointestinal Microbial Assay Plus (GI Map) are utilized by healthcare practitioners to guide protocols to restore balance to clients’ health and correct disturbances in digestive, hormonal, immune, neurological functions and more.
Although there is limited public research specifically assessing GI Map accuracy, there is evidence highlighting the accuracy of the quantitative Polymerase Chain Reaction (qPCR) method used by this test. For example, a meta-analysis found qPCR accurately detected Clostridium difficile with 90% sensitivity and 96% specificity (Deshpande et al., 2011). GI Map is Clinical Laboratory Improvement Amendments (CLIA) approved; It adheres to quality standards to ensure accuracy.
Confounders can impact test results, including improper stool sample handling, use of probiotics or medications such as antibiotics, or dietary changes prior to stool sample collection.
Here, we focus on clinical implications of low Roseburia spp.. Roseburia promotes healthy: gastrointestinal motility, neurological function, immune function, and inflammatory response, largely through Short Chain Fatty Acid, butyrate, production. To do so, Roseburia must feed on resistant starch, soluble fibers (β-mannan, xylan and inulin type fructans), and polyphenols. Regular consumption of potatoes, bananas, rice, oats, aloe, konjac flour, leeks, garlic, chicory and asparagus can increase low Roseburia. Roseburia is hydrogen producing and inhibited by excess hydrogen; It may be inhibited by overgrowth of other hydrogen producing strains and low levels of hydrogen consuming bacteria. Thus, ensuring balanced gut microbes through strategies discussed below is key for healthy Roseburia levels.
Stool testing allows for the development and implementation of strategies that improve one’s gut health which can have far-reaching implications on all body systems and processes, including improved neurological health, detoxification, inflammatory response, immune health and more. Those seeking answers to such underlying health conditions will find this information most relevant, although any individual attempting to improve overall health may also benefit. While microbial levels that are too low or too high indicate dysbiosis, in this paper we discuss how low Roseburia spp. can cause gastrointestinal and neurological disturbances.
Stool testing can give valuable insight to individuals suffering from any of the following: bloating, gas, diarrhea or constipation, weight gain or loss, abdominal pain, acid reflux, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), allergies, food sensitivities, anemia, fatigue, depression, anxiety, acne, immune system dysregulation conditions such as autoimmune, or immunodeficiency (Carabotti et al., 2015; Clapp et al., 2017; Suganya & Koo, 2020). Given the interconnectedness of bodily systems, individuals may also find that the results of their stool test aid in devising nutritional and lifestyle changes that help with overall health.
Stool testing provides great insight into the potential causes of the aforementioned issues by providing markers related to the quantity of specific bacteria, viruses, parasites, and fungi inhabiting the colon. Additional markers for maldigestion, malabsorption, inflammation, detoxification and immune function indicators further aid in such (Diagnostic Solutions, 2023).
The GI Map by the Diagnostic Solutions is a comprehensive stool test that analyzes the DNA of various gastrointestinal microorganisms and other fecal biomarkers through a quantitative real-time Polymerase Chain Reaction (qPCR) technique, which is often the preferred analysis method. Other stool testing may only indicate whether a bacteria is present or absent, however, the quantification of the bacterial species is key, as dysbiosis is determined not only by the types of bacteria, but more importantly, by the levels of microbes, making qPCR a preferred method in many instances (Diagnostic Solutions, 2023).
Given the types and amounts of microbes that reside in our gut have far reaching implications on every system in the body, restoring microbial and digestive balance has the potential to reverse numerous pathologies ranging from digestive to neurological to endocrine. The explanation is multifaceted, ranging from the gut brain connection largely influenced by the vagus nerve, to the Short Chain Fatty Acid (SCFA) metabolites produced by the various gut bacteria serving as signaling molecules in other areas of the body, especially the brain (Carabotti et al., 2015; Clapp et al., 2017; Suganya & Koo, 2020).
The GI-MAP stool test provides quantitative measurements of gut commensal bacteria (Bacteroides fragilis, Bifidobacterium spp., Lactobacillus spp., Akkermansia muciniphila, Faecalibacterium prausnitzii, Enterococcus spp., Roseburia spp., Desulfovibrio spp., Methanobacteriaceae spp., Fusobacterium spp., Prevotella spp., Enterobacter spp., Escherichia spp., etc.); opportunistic bacteria (Clostridium difficile, Klebsiella pneumonia, Pseudomonas aeruginosa, Citrobacter freundii, Proteus mirabilis); protozoa parasites (Blastocystis hominis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium spp., Entamoeba histolytica, Cyclospora cayetanensis); viruses may be included (Epstein-Barr Virus, Cytomegalovirus, Norovirus, etc.); fungi/yeast (Candida spp., Geotrichum spp.) and worms (Ascaris lumbricoides, Trichuris trichiura, etc.) (Diagnostic Solutions, 2023).
Low commensal bacteria levels suggest suboptimal fiber intake, low digestive capacity, and increased risk of constipation (Zhao & Yu, 2016). Below, we will discuss specifically how low levels of Roseburia is associated with inflammatory conditions such as Ulcerative Colitis (UC), neurological dysfunction and digestive issues such as constipation.
It is important to note that isolated commensal bacteria markers typically do not indicate health or disease. Rather, it is the level in context with one’s overall microbial balance and diversity, their environment, diet, lifestyle and genes that is key in determining health outcomes. Nonetheless, ensuring balanced levels of the individual microbe levels is an important piece to improving one’s health outcomes. This is especially the case with the “Bellwether Targets”, a group of commensal bacteria that are indicative of overall health of the microbiome as they each play critical roles in contributing to one’s overall health. While the Bellwether Targets are typically considered to be M. smithii, D. piger, and A. muciniphila, Roseburia may be considered among this group in light of the critical roles it plays, as discussed below (Lord, 2021).
GI-Map also provides markers for digestive function (pancreatic elastase-1, steatocrit and various microbial levels), intestinal inflammation (calprotectin), immune function and intestinal barrier integrity (sIgA and zonulin) and toxin/endocrine indicators (beta-glucuronidase) (Khaki-Khatibi et al., 2020; Malinen et al., 2005; Singh et al., 2019; Wang et al., 2015). The test can provide clues related to hypochlorhydria as elevated H. pylori is associated with hypochlorhydria (Wang et al., 2015).
The aforementioned markers can help detect the presence and underlying contributors to: gastrointestinal inflammation and inflammatory pathologies such as IBD, digestive problems such as constipation, diarrhea, gas, bloating, IBS, hypochlorhydria, maldigestion, malabsorption, autoimmune disease triggers, dysbiosis and more. To highlight the clinical application of one of the markers, calprotectin is a well studied and accepted marker for inflammation in the colon. Those with IBD (UC) are found to have elevated calprotectin (>50). The higher calprotectin is, the more likely the presence of a disease state (Benítez & García-Sánchez, 2015).
Products of protein breakdown (valerate, isobutyrate and isovalerate) are not included in GI Map whereas they are included in Genova Diagnostics GI Effects stool test. These markers indicate protein maldigestion which can contribute to dysbiosis (Bures et al., 2010; Pezzilli et al., 2013; Rios-Covian et al., 2020).
SCFAs are not included on GI Map, whereas they are included in GI Effects (Diagnostic Solutions, 2023; Genova Diagnostics, 2023). SCFA levels can be implied by the GI Map by knowing which bacterial species are producers. For example, F. prausnitzii, Eubacterium rectale, Roseburia spp., Butyrivibrio spp., and Clostridium spp. produce butyrate. Healthy butyrate levels are key to maintain healthy gut barrier integrity, reduce risk of cancer and inflammatory conditions (Diagnostic Solutions, 2023; Feng et al., 2018).
The SCFA acetate is produced by Coprococcus spp., Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., Eubacterium spp., Ruminococcus spp., Clostridium spp. and Anaerostipes spp. The SCFA propionate is derived from Coprococcus eutactus, Bacteroides spp., Akkermansia muciniphilia, Roseburia inulinivorans, Prevotella spp., Ruminococcus spp. and Veillonella spp. Like butyrate, acetate and propionate also have anti-inflammatory, anti-cancer properties and help regulate a healthy immune system and metabolic functions (Diagnostic Solutions, 2023; Feng et al., 2018; Lord, 2021).
The GI map primarily indicates the microbial health of the colon, as this is where the majority of the microbiota reside. Thus, there are limitations to the interpretations of stool testing. For example, fecal levels of bacteria are not sufficient to diagnose Small Intestine Bacterial Overgrowth (SIBO), and fecal levels of candida are not sufficient to diagnose candida or fungal overgrowth in the small intestine (Banaszak et al., 2023; Tang et al., 2020).
Diagnostic Solutions made the GI Map stool test available to the public in 2014. The GI Map is CLIA (Clinical Laboratory Improvement Amendments) approved, meaning it adheres to the set forth regulations and standards to ensure accurate, reliable and timely testing to aid in accurate assessment and treatment plans for disease management and prevention.
There is limited publicized research data specifically assessing the accuracy of GI Map, however, Diagnostic Solutions publishes accuracy testing data internally and it is approved during CLIA inspection. Accuracy of each of the test markers is ensured before adding to GI Map by testing the assay sensitivity, specificity and variation. They must be within an acceptable range for the marker to be added. For example, the variation (given by Coefficient of Variation, CV) tests to ensure consistency in results when the stool sample is tested multiple times, on different days and in different batches, and must be less than 10% (Diagnostic Solutions, 2023).
Although GI Map is a relatively newer test used clinically and more research will be useful to better understand the accuracy and limitations of the GI Map, several research studies have suggested high accuracy of qPCR stool testing in assessing various aspects of gastrointestinal health.
For example, a meta-analysis assessing the accuracy of the qPCR testing method in diagnosing Clostridium difficile infection including 19 studies (7392 samples) concluded qPCR was a highly accurate method. An average sensitivity and specificity of 90% and 96% respectively was found (Deshpande et al., 2011). In other words, qPCR testing can correctly identify the presence of C. difficile in 90% of infected individuals, and can correctly identify the absence of C. difficile in 96% of cases. While this would suggest a high degree of accuracy in such testing, there are still cases for which false positives and false negatives may occur.
Noteworthy is that this aforementioned meta-analysis was only looking at clostridium difficile and not specifically looking at GI Map. Other studies have shown that there can be considerable variability in results among different targeted species and also among different companies each using the qPCR method. For example, a study assessed the accuracy of four commercially available qPCR tests in detecting the parasites Cryptosporidium spp. and G. duodenalis. The R-Biopharm qPCR test resulted in an 87.5% sensitivity in detecting Cryptosporidium spp., whereas the Fast Track assay showed only a 53.1% sensitivity (Paulos et al., 2019). Thus, the company chosen for stool testing does matter.
The one PubMed diagnostic accuracy study specifically assessing GI Map concluded that GI Map has highly variable results with sensitivity of 80% and specificity of only 26%, thus likely to provide false positive results (Gingras & Maggiore, 2020). However, there were errors in the methodology and potential conflicts of interest. For example, the study was funded by Doctor’s Data, which is a direct competitor of Diagnostic Solutions. With regards to the methodology, a qualitative test (Biofire) cannot be used as a comparator to assess the accuracy of a quantitative test (GI Map). For example, a negative result from the Biofire test cannot rule out that the pathogen is present at lower levels, below the threshold to induce disease states. GI Map, unlike BioFire, does quantify the presence of the pathogen and would indicate its presence in such a scenario. This explains how the low specificity finding of 26% (many false positive results) is misleading, as GI Map identified organisms that were present at lower levels and therefore not identified by BioFire (Diagnostic Solutions, 2023).
To have a better idea of how the above findings of qPCR stool testing accuracy translates to clinical practice, studies have found significant alterations in the microbiota of individuals diagnosed with IBS versus healthy controls through use of qPCR stool testing. For example, a systematic review involving 360 individuals with IBS and 268 healthy controls found notable differences between groups for expression of Lactobacillus, Bifidobacterium, and F. prausnitzii (Liu et al., 2017). Therefore, despite potential inaccuracies in results, the accuracy of qPCR stool testing is high enough such that it appears to have significant clinical usefulness.
It is important to keep in mind that while working with clients that, although highly accurate, there is a degree of error within the GI Map and PCR stool testing. Thus, it is important to consider the results alongside the client’s complete health history and clinical picture.
Many potential confounders can affect results of the GI Map. Any user error and failure to comply with set instructions may result in erroneous results. The individual should discontinue probiotics, probiotic rich foods, antibiotics, antifungals, antiparasitics – which alter the gut microbiota levels – at least 2 weeks, but preferably 4 weeks prior to collecting their stool sample. If testing for H. pylori, PPIs and NSAIDs which alter stomach acid should be discontinued.
To develop ranges of the various markers on stool tests, a reference population was chosen and tested to establish. We assume the population from which the reference ranges are developed are the epitome of health. In reality, the current state of our world subjects humans to contaminants, toxins, drugs and poor quality food. We also assume the levels of the reference can be extrapolated to each individual client with their unique bio individuality (Lord, 2021).
Another consideration is that we may see metabolically and seemingly healthy individuals, free of clinical symptoms of disease that have elevations in a bacterial species that may be thought of as pathogenic when elevated. This highlights the complexity of the gut, and its interactions with our unique environment and genes (Lord, 2021).
Shifts in one’s diet can cause substantial (albeit temporary) shifts to the microbiome in a matter of days (Leeming et al., 2019). Thus, consuming a diet not representative of one’s standard diet leading up to stool sample collection could alter results and interpretations.
Furthermore, the gut microbiome changes with age (Xu et al., 2019). One perspective is that it would be expected to see differences in results from different age groups which may not necessarily indicate imbalance. Yet, another perspective is that such changes that occur with age may partly explain the negative manifestations associated with aging.
Finally, improper collection, storage and handling of the stool sample can result in errors. For example, the stool sample is not to be subject to extreme temperatures nor is an extended period of time expected to lapse between collection and testing (Diagnostic Solutions, 2023).
Roseburia is a genus of beneficial gut bacteria to which the species R. intestinalis, R. hominis, R. inulinivorans, R. faecis, and R. cecicola belong (Nie et al., 2021). Roseburia produces SCFAs – most notably butyrate – key to gastrointestinal health and healthy digestion. Low Roseburia is associated with a host of diseases including IBD (specifically UC), IBS, constipation, diarrhea, abdominal pain, metabolic syndrome, colorectal cancer, neurological disorders such as Parkinson’s Disease (PD), inflammatory conditions and immunological pathologies (Parthasarathy et al., 2016).
A study using qPCR stool analysis comparing UC patients with healthy controls found lower Roseburia hominis and F. prausnitzii (p< 0.0001) and lower SCFAs in UC patients. Furthermore, lower R. hominis levels were correlated with increased disease activity in UC patients. (Machiels et al., 2014).
A study involving 147 subjects comparing the microbiome of constipated individuals with healthy, non-constipated controls found low Roseburia, Bacteroides and Coprococcus 3 via PCR stool analysis (Mancabelli et al., 2017).
A recent meta-analysis including 1703 subjects compared the stool sample microbe composition between individuals with PD and healthy controls. The study found significant differences, most notably decreased Roseburia and increased Akkermansia (Toh et al., 2022).
Studies have found significantly lower abundance of Roseburia spp. and other butyrate producing bacteria in colorectal cancer patients compared to healthy controls (Rinninella et al., 2019). For example, a study of 102 subjects found such a correlation alongside a reduction in copy numbers of butyryl-coenzyme A CoA transferase genes (p<0.01) in colorectal cancer patients, further supporting the initial finding (Wang et al., 2012).
A 2019 systematic review comparing stool sample microbe composition between individuals with Major Depressive Disorder (MDD) and healthy controls found significant differences in microbe population abundance, however, both low levels and high levels of Roseburia, Bacteroides, Oscillibacter and Prevotella were found (Cheung et al., 2019).
Many of Roseburia’s beneficial characteristics may be associated with its ability to produce the health-promoting SCFA, butyrate, as a byproduct through feeding on various fiber sources (Morrison & Preston, 2016). Butyrate is primarily taken up by colocytes to undergo beta oxidation, and produce ATP used by colonocytes for their various functions, including cell proliferation, cell differentiation and transport of ions across membranes. Butyrate is also suggested to promote the growth of other beneficial gut microbes such as Bifidobacterium. To explain the latter, R. intestinalis is believed to play a role in encoding β-endoxylanases, which is required for Bifidobacterium spp. to use arabinoxylan as a substrate to feed on and grow (Rivière et al., 2016). Through these roles, butyrate maintains gut epithelial integrity, reduces intestinal permeability, reduces intestinal inflammation and promotes healthy digestion (Chen & Vitetta, 2020; Kircher et al., 2022).
The connection between low Roseburia and neurological disorders such as PD can (at least partly) be explained by butyrate’s beneficial roles in the brain; Butyrate is believed to also serve as an energy source for neurons in addition to colonocytes. Butyrate may have epigenetic effects by playing a regulatory role in methylation and histone acetylation, which in turn influence neuronal plasticity and function. Furthermore, butyrate is believed to have anti-inflammatory effects in the brain (Berni Canani et al., 2012; Bourassa et al., 2016).
In light of Roseburia's involvement of such critical roles in health, it may be considered among the Bellwether Targets, a group of commensal bacteria that are indicative of overall health of the microbiome as they should be at the proper levels to ensure health (Lord, 2021).
Dietary Management of Low Roseburia spp.
Consuming the substrates for which Roseburia spp. feeds on is arguably the best place to begin to help a client increase their levels. In general, consuming a diversity of plant based fibers is key. Specifically, Roseburia is found to grow through feeding on the following types of fibers: soluble fibers such as β-mannan (found in konjac flour, aloe vera, etc.), xylan (rice, quinoa, oats, buckwheat), inulin type fructans (leeks, chicory root, asparagus, garlic), and resistant starch (RS) (green bananas, cooked and cooled potatoes, plantains, legumes) (Flint et al., 2012; Rivière et al., 2016). Polyphenols (found in cocoa, herbal teas, fruits and vegetables, nuts and seeds, etc.) are also believed to increase Roseburia levels (Corrêa et al., 2019). Cinnamon and grape pomace extract, both rich in polyphenols, were shown to significantly increase Roseburia levels in mice after eight weeks of supplementation (Van Hul et al., 2018).
To apply the above knowledge, aim to consume about 4+ servings of different vegetables, 2+ servings of different fruits and 2+ servings of RS per day. Consume at least one food source per day from each of the different types of aforementioned soluble fibers (β-mannan, xylan and inulin type fructans). These strategies may help increase low Roseburia.
A randomized controlled trial study including 39 insulin resistant adults found that a high RS diet (44 to 66 g RS per day) for 2 weeks led to increased relative amounts of Roseburia spp., which was not observed for participants undergoing the low RS diet (4 to 5 g RS per day). This was accompanied by increased butyrate and propionate in the high RS group (Maier et al., 2017).
Given the interconnectedness and interplay among gut microbes, some research suggests that balanced levels of gut microbes as a whole can have a positive “snowball effect". For example, Roseburia is a “hydrogenogenic” (hydrogen-producing) major-abundance bacteria. Thus, it requires “hydrogenotrophic” (hydrogen-consuming) targets (such as M. smithii and D. piger) as hydrogenogenic bacteria are inhibited by excess hydrogen. To extrapolate this, excessive levels of other hydrogenogenic bacteria (Clostridium spp., Ruminococcus spp., Prevotella spp. etc.) may also inhibit Roseburia (Lord, 2021; Wolf et al., 2017). Therefore, taking measures to improve microbial balance as a whole by eating a Mediterranean-style whole foods diet rich in plant based foods may help to improve low Roseburia.
While preliminary research assessing the efficacy and safety of probiotics containing Roseburia strains seems promising, more long term studies will provide great insight into optimal dosage, potential long-term side effects and efficacy for specific health conditions such as IBD, IBS, colorectal cancer, UC, PD, etc. (Zhang et al., 2022).
Research suggests having adequate amounts of certain micronutrients is key to achieve healthy gut microbe levels. For example, zinc deficiency was shown to disrupt microbial balance and reduce SCFAs in animal studies. Folate deficiency impairs methylation which is suggested to have negative downstream consequences on the gut microbiota (Gagliardi et al., 2017).
Finally, avoiding a Western style diet high in processed food and avoiding overuse of certain medications like antibiotics when possible will further help to achieve and maintain healthy microbial and Roseburia levels. There may be some difficult cases for which a fecal transplant may be the best option to resolve the dysbiotic state (Gagliardi et al., 2017).
Given that diseases often begin in the gut, it stands to reason that stool testing has immense potential power to help individuals address root causes of symptoms ranging from digestive complaints (IBS, constipation, diarrhea, bloating, etc.) to neurological disturbances to inflammatory conditions to immune system dysregulation to endocrine disruption. Diagnostic Solutions GI Map is utilized by numerous healthcare practitioners to restore balance to their clients’ health. This CLIA approved test is also supported by several research studies validating the accuracy of the qPCR testing method used by GI Map.
Isolated commensal bacteria markers typically do not indicate health or disease alone, as the levels must be considered in conjunction with one’s overall microbial balance and diversity, environment, diet, lifestyle, genetics, health history and clinical picture. However, more importance can be placed on ensuring balanced levels of each of the “Bellwether Targets”, including Roseburia, as they each confer important positive health benefits to the point where they may be indicative of the overall state of health of one’s microbiome (Lord, 2021). Roseburia promotes gastrointestinal motility, neurological function, immune system function, and a healthy inflammatory response, all in large part due to its ability to produce butyrate. Note that Roseburia plays additional beneficial, unique roles such as its ability to encode β-endoxylanases needed for growth of other bacteria such as Bifidobacterium spp..
Clients with low Roseburia levels should be provided nutritional strategies to improve these levels. Roseburia grows through feeding upon resistant starch and soluble fiber substrates such as β-mannan, xylan and inulin type fructans. Thus, regular consumption of a variety of plant-based foods, specifically potatoes, green bananas, rice, oats, aloe, konjac flour, leeks, chicory root, asparagus and garlic can be great therapeutic strategies. Researchers describe a Roseburia probiotic as a “next generation probiotic.” Further research assessing the efficacy and long-term safety of such may offer great benefit to certain individuals (Zhang et al., 2022).
Measures to correct other gut microbe imbalances is also key to ensuring healthy Roseburia levels given the complex interactions among microbes. For example, Roseburia is a hydrogenogenic bacteria that is inhibited by excess hydrogen, thus relying upon balanced levels of other hydrogenogenic bacteria as well as hydrogenotrophic targets. Overall microbial balance is typically best achieved by eating a whole foods, Mediterranean-style diet rich in a diversity of plant-based foods with at least 5 servings of vegetables and fruits daily. It is also important to avoid overuse of medications, especially PPIs and antibiotics, and to minimize processed foods.
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