One of the great things about keeping up-to-date on the literature and operating outside of the traditional medical paradigm is that we can implement cutting-edge findings from the scientific literature years and sometimes even decades before they will ever make their way into conventional clinical practice.
It’s one of the things I love most about writing this blog – that I can break down the latest insights in gut health, nutrition, and the microbiome, facilitate meaningful discussion, and empower you to make more knowledgeable decisions about your health.
But science isn’t perfect. Being at the forefront of the latest research also means that sometimes we have to back-track, be willing to accept when we are wrong, and acknowledge when our previous scientific understanding was misguided. I’m grateful for your trust in me to review the literature objectively and to be transparent in my journey and commitment to scientific truth — even if it means occasionally putting my own foot in my mouth!
Thanks to newly published evidence, I’ve recently had paradigm shifts regarding the merits of taking probiotics after antibiotics and the use of culture-based methodologies for comprehensive stool testing. A few months ago, I also came across a new study that called into question the validity of breath testing for small intestinal bacterial overgrowth (SIBO).1
This article will review the most recent evidence surrounding SIBO, including whether our “gold standard” diagnostic methodologies are outdated, if breath testing is clinically useful, and if SIBO is really what we think it is. If you’re not familiar with SIBO, you may want to read my previous article first. Just know that some of the research in the initial article is outdated and that my most up-to-date recommendations can be found here!
Is quantitative culture, the “gold standard” for diagnosing SIBO, outdated?
For years, the “gold standard” for diagnosing SIBO has been small-bowel aspiration and quantitative culture. This involves undergoing an upper endoscopy, where a scope is inserted via the mouth down to the small intestine, and having a sample taken through a sterile catheter using a small amount of suction. The aspirate is typically taken from the jejunum, the middle section of the small intestine, and is then promptly cultured and quantified in a lab.
While new sampling techniques have been able to sidestep contamination issues, quantitative culture poses another major issue that is not often discussed – chiefly, that most species that live in the gut cannot be effectively cultured. I’ve written about this before in the context of stool testing. The number of colony-forming units (CFU) detected in a sample will greatly depend on whether the microbes in a given individual’s gut can be effectively grown in culture – and how rapidly they grow in the particular growth media chosen.
Recent studies have confirmed that culture-based methods severely underestimate the number of bacteria in the small intestine – by about a hundred-fold!1 With next-generation sequencing widely available, why are we still using quantitative culture as the “gold standard” in research settings? Until we adopt sequence-based methodologies for answering questions about SIBO, we’re likely to continue spreading confusion about what it is and isn’t.
(Note: This may be why there has been such disagreement over the proper diagnostic threshold for SIBO using quantitative culture. While many studies use >105 CFU/mL as indicative of SIBO, a recent consensus document suggests using >103 CFU/mL as the cut-off.2)
Due to the expense and invasive nature of small intestinal aspirates, breath testing has carried favor for diagnosing SIBO in clinical practice. But does breath testing accurately reflect the degree of bacterial overgrowth?
A group of researchers at Texas Tech University wanted to find out. They performed glucose breath testing, quantitative culture, and next-generation sequencing (quantitative PCR) of jejunal aspirates in parallel for 18 participants who reported experiencing symptoms commonly associated with SIBO, such as bloating, abdominal discomfort, gas, or irregular bowel habits.1
Breath testing was performed using a test dose of 90 grams of glucose, and both hydrogen and methane were measured in the breath every 20 minutes for three hours. Based on a quantitative culture threshold of 105 CFU/mL, the authors diagnosed 10 of the 18 symptomatic patients with SIBO.
However, a recent consensus document suggests that cultured bacterial concentrations greater than 103 CFU/mL should be considered positive for SIBO.2 Using this more liberal cut-off, all 18 symptomatic patients were positive for SIBO.
Nevertheless, regardless of which diagnostic threshold was used for quantitative culture, breath test results did not accurately predict SIBO:
- Of the 10 patients with >105 CFU/mL by jejunal culture, only four had a positive breath test and six had a negative breath test.
- Of the 18 patients with >103 CFU/mL by jejunal culture, only seven had a positive breath test result and eleven had a negative breath test.
Moreover, bacterial load was similar for breath test positive and breath test negative patients, and there was no correlation between breath test gas measurements and bacterial load by quantitative culture. In fact:
“When aspirated bacteria were measured by colony forming units, there was actually a weak negative correlation, which meant that higher hydrogen and methane production was associated with lower numbers of jejunal bacteria.”
Furthermore, there was no significant correlation between breath test gas measurements and bacterial load in jejunal aspirates by quantitative PCR.
In the discussion of the paper, the researchers further mused whether SIBO-level bacterial loads could even produce a positive breath test. Through a series of calculations, they estimated that bacterial overgrowth at the level considered to be positive for SIBO diagnosis could not produce enough hydrogen in the breath to result in a positive breath test:
“We need to consider the possibility that positive breath test signals of all except the most extreme postsurgical SIBO patients are the result of colonic, and not jejunal fermentation.”
Indeed, several other studies suggest that a positive breath test may be a result of colonic fermentation due to rapid transit time or carbohydrate malabsorption. I’ll discuss these in the next section. As for whether SIBO-level bacterial loads can’t produce enough gas to ever produce a positive breath test, I think the jury is still out on this and will require more careful analysis of gas production in the lumen to determine if this hypothesis is true.
In interpreting breath tests, it is commonly assumed that gases produced in the first 90 minutes are indicative of small intestinal fermentation, whereas gases produced after 90 minutes are indicative of colonic fermentation. But is this true for everyone?
In a 2011 study, Yu et al. combined gastric emptying scintigraphy with lactulose breath tests in 25 patients with irritable bowel syndrome (IBS).3 By administering a radioisotope along with 10 grams of lactulose, the researchers were able to track the lactulose as it traveled through the GI tract and match this up against measurements of exhaled breath.
Here’s what they found:
- Oro-cecal transit time ranged from as little as 10 minutes to as long as 220 minutes between individuals.
- In 22 patients (88 percent), the radioisotope reached the end of the small intestine before the concentration of hydrogen ever reached abnormal levels
- Only 3 patients had abnormal hydrogen levels while the radioisotope was still in the small intestine.
In other words, they found that the rise in hydrogen on the breath test, regardless of how early it occurred, almost always reflected the arrival of the lactulose substrate in the colon. This would indicate rapid small intestinal transit, rather than bacterial overgrowth.
Other studies that have concurrently assessed transit time and exhaled gases in breath have corroborated these results:
- In 2014, Zhao et al. concluded that lactulose breath testing alone “is not a valid method for SIBO diagnosis in IBS due to the high level of variation in oro-cecal transit time.”4
- In 2016, Lin et al. found that 13 percent of patients with no prior GI surgery had false-positive glucose breath test results due to shorter oro-cecal transit time and colonic fermentation.5 Of patients with prior upper GI surgery, 48 percent had false positive results.
Lactulose has also been shown to have laxative properties and may itself decrease oro-cecal transit time.6
Positive breath tests may also indicate carbohydrate malabsorption, rather than bacterial overgrowth. Inflammation in the small intestine can damage the gut epithelium and reduce the number of functional glucose transporters.7 This can result in malabsorption, increasing the amount of glucose that remains in the lumen and is available for fermentation by microbes in the small intestine or colon.
Long-term dietary patterns can also influence glucose absorption. Glucose is normally absorbed in the small intestine through sodium-dependent glucose transporters. When dietary sugar intake is high, a second type of glucose transporter is also inserted into the apical epithelial membrane, increasing the capacity for glucose absorption from the gut lumen.8,9 Obese individuals have been shown to have higher levels of these glucose transporters.10
In contrast, regular fasting or habitual consumption of a low-carbohydrate diet cuts the capacity of the intestinal epithelium to absorb glucose nearly in half.11
This is an important consideration. Let’s say you give 75 grams of glucose to two individuals as part of a breath test. One individual has been consuming a very low-carb, ketogenic diet for years and has a healthy gut, while the other has SIBO but is obese, and eats a diet high in processed sugar. Given the differences in habitual dietary intake, the individual on the ketogenic diet may not absorb the entire glucose bolus, potentially leading to an early rise in breath hydrogen, while the obese individual will absorb the glucose rapidly, leading to little gas production despite having bacterial overgrowth.
While this example is hypothetical, it highlights another potentially significant issue with breath testing:
“The capacity for glucose absorption likely varies between individuals, and could, in part, determine who produces a positive glucose breath test.”
Future studies should seek to determine the extent to which this individual variability in carbohydrate absorption can influence breath test results.
Looking deeper into the literature, we can also find several recent studies that call into question the reproducibility of breath test results. This is a huge issue, as reproducibility is one of the key factors in determining whether a diagnostic test is clinically useful.
In 2017, Yao et al. performed lactulose breath testing on a group of 21 patients with functional bowel disorders and repeated the test two weeks later under the exact same conditions.12
- All 21 patients had a rise in breath hydrogen on the initial test, and all but one of these patients also produced hydrogen upon re-testing.
- However, there was no correlation in time of the first rise in hydrogen or the peak amount of hydrogen between the two tests.
- Six patients produced significant quantities of methane on the initial test, but only three patients produced significant methane upon re-testing.
Okay, so maybe there are issues with diagnosing SIBO, and the peak in hydrogen is subject to transit time and the rate of carbohydrate absorption. But aren’t breath tests still useful to identify possible overgrowths of methane or hydrogen-sulfide producing microbes? Let’s take a look at the evidence for each gas in turn.
Possibly. However, several studies have shown that breath methane excretion is a poor reflection of gut methane production.
Di Stefano et al. measured methane excreted in breath and from the rectum in a group of 103 IBS patients and 28 healthy volunteers after consuming lactulose.17 They found that 48 of the IBS patients had detectable rectal methane excretion, but only 27 of them excreted methane in the breath.
A recent review article suggests one potential explanation for this discrepancy:
“The journey a given gas takes from its production in the intestinal lumen, across the gut wall, into the circulation and out into the breath is clearly a perilous one with all but a fraction of that originally produced surviving to appear in the breath.”
Indeed, transport of intestinal gases across the intestinal wall has been shown to be limited by mucosal diffusion and intestinal blood flow, so it’s possible that individuals with constipation simply have more time for methane to diffuse into the blood due to slow transit time.
Thus, methane could identify individuals with constipation, but this could also be done with a simple and much less time-consuming clinical questionnaire.
Maybe. In 2014, Pimentel et al. did find that neomycin plus rifaximin was nearly twice as effective as rifaximin alone for constipation-predominant IBS patients with methane on the initial breath test.19
However, few studies have directly compared the response of IBS patients with or without positive breath methane results to neomycin. In one double-blind, placebo-controlled trial published in 2006, researchers looked at the clinical response to neomycin alone in 111 participants with IBS who underwent pretreatment breath testing. Overall, those with methane production on the breath test had 67.6 percent improvement in symptoms from neomycin, versus 32.7 percent improvement in those with hydrogen production alone.
However, it’s important to note that only 12 of the 111 study participants had methane on their pretreatment breath test. Only 10 of these had complete data, five of which received neomycin and five of which received placebo – an awfully small sample size to make population-level generalizations for clinical practice. Those with methane on the pretreatment breath test also had more severe constipation at baseline, so they may have had more room for improvement.
Furthermore, while neomycin may be most effective in those with methane on the lactulose breath test (based on a very small number of subjects), neomycin is still much more effective than placebo in all individuals with constipation-predominant IBS.20 And we still don’t know whether the lactulose breath test can adequately distinguish those who might benefit most from rifaximin plus neomycin. Based on the current data we have, it’s possible that this combination (rifaximin + neomycin) is useful in IBS-C, regardless of breath methane excretion.
The herbal formulation Atrantil has also shown to be helpful in IBS-C independent of breath methane excretion. One study that did not account for breath methane results found that 21 of 24 patients with IBS-C given Atrantil for two weeks had a significant response to the treatment.21
Hydrogen sulfide is another gas that has garnered major attention as the “missing gas” in diagnosing SIBO, and has been particularly associated with diarrhea-predominant IBS and visceral hypersensitivity.22 In this section, I’ll answer a few important questions surrounding this mysterious gas:
In 2016, Banik et al. assessed breath excretion of both hydrogen and hydrogen sulfide after ingestion of 50 grams of glucose and performed jejunal aspiration and quantitative culture in 151 patients with diarrhea-predominant IBS.23
They found that many glucose breath test results that would be considered negative for hydrogen were positive for hydrogen sulfide, and that hydrogen sulfide production was able to predict which individuals were positive for SIBO.
However, SIBO diagnosis was based on the “gold standard” of quantitative culture. Hydrogen-sulfide producers like Citrobacter, Escherichia coli, Pseudomonas, and Klebsiella tend to grow really well in laboratory culture media. Thus, if hydrogen-sulfide producers were present in the small intestine, even at very low abundance, it would not be surprising to see a high number of colony-forming units in quantitative culture.
Until we see this study repeated using quantitative PCR analysis of jejunal aspirates, I’m not entirely convinced that elevated breath hydrogen-sulfide is indicative of bacterial overgrowth of the small intestine.
Rather than hydrogen sulfide overgrowth, a flat-line result may indicate inefficient gas exchange from the colon to the lungs, possibly due to inflammation, rapid transit, or hyper-absorption of the substrate in the case of glucose.
Rather than making speculative diagnoses based on breath testing, I have increasingly relied on PCR-based stool tests to screen for potential overgrowths of methane and hydrogen-sulfide producing taxa. I should note that this has major limitations as well, as the ideal scenario would involve assessing the composition of microbes in the small intestine and colonic mucosa. However, fecal samples may provide an adequate proxy.
One study found that higher fecal abundance of Methanobrevibacter smithii¸ the predominant methanogen in patients with IBS-C, was strongly correlated with reduced stool frequency.24 Another small study found that M. smithii needed to reach 1.2 percent of total stool bacteria to produce a positive methane result on the lactulose breath test,25 suggesting that stool profiling might be more sensitive for detection of methanogen overgrowth than breath testing.
Hydrogen sulfide-producers may also be more abundant in the stool of IBS patients with this type of overgrowth. A 2011 study of 37 IBS patients and 20 healthy subjects found that the hydrogen sulfide producer Pseudomonas aeruginosa was significantly more abundant in both the small intestine and stool of IBS patients than in healthy controls.26
Thus, if you’re already doing a qPCR test like GI-MAP or a 16S rRNA gene sequencing test like uBiome as part of a functional gut-work up, you can look for the overabundance of certain taxa in the raw data:
- GI-MAP and uBiome can both identify the major known hydrogen-sulfide producing taxa, including Pseudomonas, Desulfovibrio, Citrobacter, Salmonella, and Aeromonas
- uBiome can also identify known methanogens, including Methanobrevibacter smithii and Methanosphaera
These tests are useful for identifying other gut imbalances as well. If a client has already done breath testing, I will certainly consider the results, but will do so in light of all of the research discussed here and their stool test results.
With all of this information, it really begs the question: is SIBO really what we think it is? SIBO has now been associated with over a hundred different conditions, largely based on breath testing. Could an increase in bacterial numbers in the small intestine alone cause this much individual variability in symptoms, clinical response, and breath test results? Or is SIBO as diverse a condition as IBS?
“It is unlikely that a mere increase in bacterial numbers, as measured by the culture of jejunal aspirates, may alone explain the protean clinical manifestations of SIBO. It is much more likely that these reflect the nature of the contaminating species and their unique biology.”
Fortunately, new technologies are on the horizon that may offer a better understanding of what microbes are present in the various regions of the GI tract, and which gases and other metabolites they are producing.
In April of 2018, a group of Australian researchers published a pilot study of a new telemetric capsule that is swallowed, travels through the GI tract, and can provide real-time measurement of the major gases in the GI tract. The concentration of oxygen allows the capsule to be accurately localized to the stomach, small intestine, or colon, while the concentrations of hydrogen and methane reflect microbial fermentation. Incredibly, the device detected concentrations of hydrogen that were 3000 times higher than those currently detected by breath tests!27
Dr. Kyle Berean, lead author on the pilot study, is now the Chief Technology officer at Atmo Biosciences, a company that is trying to bring these gas-sensing capsules to consumers. The Atmo Gas Capsule will be able to provide real-time measurements of hydrogen, methane, oxygen, and carbon dioxide as the capsule travels through your GI tract and send the data directly to your phone! The group is also working to incorporate measurement of hydrogen sulfide and short-chain fatty acids.
The capsules have already gone through phase 1 clinical trials, and the company hopes to launch the device to consumers in 2021 or 2022.
Direct measurement of the small intestinal microbiome and metabolome will also play a major role in the future of SIBO and IBS research and treatment:
Future studies that utilize mucosal biopsies or small intestinal aspirates combined with next-generation sequencing and metabolomics will be key to understanding which microbes or metabolites are implicated in states of health and disease, including in IBS and SIBO. Until then, empiric treatment may be worthwhile.
1) We desperately need a new “gold standard” for SIBO. Quantitative culture significantly underestimates the true bacterial load in the small intestine and should be a thing of the past. The only reliable means to diagnose bacterial overgrowth is a small intestinal aspirate coupled with PCR to quantify bacterial load.
2) Breath testing is unreliable for diagnosing SIBO. Breath tests do not adequately predict bacterial load in the small intestine using quantitative culture or PCR-based methods, and results are influenced by gut transit time, carbohydrate absorption, and habitual dietary intake.
3) Breath testing might be useful for detecting other small intestinal pathologies, but we don’t really know enough to be able to properly interpret the results. Breath testing may also guide treatment, but large clinical trials are lacking.
4) Stool testing may be a more sensitive way to screen for potential overgrowths of methanogens or hydrogen sulfide producers. A functional gut-work up that includes GI-MAP and a 16S test like uBiome probably eliminates the need for breath testing in most cases.
5) SIBO is NOT the explanation for all bloating, abdominal pain, or altered bowel habits. Small intestinal dysbiosis or other gut pathologies are likely the true cause behind many cases of suspected SIBO.
6) The next generation of SIBO and IBS research will include direct (intraluminal) gas sampling and microbiomics. Understanding the kinetics of gas production throughout the GI tract with easily swallowed capsules and identifying which microbes are contributing in the small and large intestine will be key to rewriting our understanding of SIBO as a condition. There is an enormous amount to be learned in this regard, including what a “normal” small intestinal microbiota looks like.
7) Until then, empiric treatment may be the best way to go. Given the many issues with breath testing, a treat-and-see approach may be worthwhile in individuals that are having consistent SIBO or IBS-type symptoms. Herbal antimicrobials are a particularly good option, as they can also treat colonic dysbiosis and have few adverse effects. While rifaximin does not appear to adversely impact the gut microbiome, clinical response rates are quite low and repeated courses are often needed. Targeted probiotics, dietary approaches, and gut-directed stress management techniques should also be considered as part of a comprehensive treatment approach.
That’s all for now! What do you make of this research? Do you distrust breath testing? What new studies would you like to see? Share your thoughts in the comments below.
- Sundin, O. H. et al. Does a glucose‐based hydrogen and methane breath test detect bacterial overgrowth in the jejunum? Neurogastroenterology & Motility 30, (2018).
- Rezaie, A. et al. Hydrogen and Methane-Based Breath Testing in Gastrointestinal Disorders: The North American Consensus. Am. J. Gastroenterol. 112, 775–784 (2017).
- Yu, D., Cheeseman, F. & Vanner, S. Combined oro-caecal scintigraphy and lactulose hydrogen breath testing demonstrate that breath testing detects oro-caecal transit, not small intestinal bacterial overgrowth in patients with IBS. Gut 60, 334–340 (2011).
- Zhao, J. et al. A study of the methodological and clinical validity of the combined lactulose hydrogen breath test with scintigraphic oro-cecal transit test for diagnosing small intestinal bacterial overgrowth in IBS patients. Neurogastroenterology & Motility 26, 794–802 (2014).
- Lin, E. C. & Massey, B. T. Scintigraphy Demonstrates High Rate of False-positive Results From Glucose Breath Tests for Small Bowel Bacterial Overgrowth. Clinical Gastroenterology and Hepatology 14, 203–208 (2016).
- Fritz, E. et al. Effects of lactulose and polyethylene glycol on colonic transit. Alimentary Pharmacology & Therapeutics 21, 259–268 (2005).
- Sundaram, U., Wisel, S., Rajendren, V. M. & West, A. B. Mechanism of inhibition of Na+-glucose cotransport in the chronically inflamed rabbit ileum. American Journal of Physiology-Gastrointestinal and Liver Physiology 273, G913–G919 (1997).
- Gouyon, F. et al. Simple-sugar meals target GLUT2 at enterocyte apical membranes to improve sugar absorption: a study in GLUT2-null mice. J. Physiol. (Lond.) 552, 823–832 (2003).
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- Ait-Omar, A. et al. GLUT2 Accumulation in Enterocyte Apical and Intracellular Membranes. Diabetes 60, 2598–2607 (2011).
- Cheeseman, C. I. & Harley, B. Adaptation of glucose transport across rat enterocyte basolateral membrane in response to altered dietary carbohydrate intake. The Journal of Physiology 437, 563–575 (1991).
- Yao, C. K. et al. Poor reproducibility of breath hydrogen testing: Implications for its application in functional bowel disorders. United European Gastroenterol J 5, 284–292 (2017).
- Chatterjee, S., Park, S., Low, K., Kong, Y. & Pimentel, M. The Degree of Breath Methane Production in IBS Correlates With the Severity of Constipation. The American Journal of Gastroenterology 102, 837–841 (2007).
- Hwang, L. et al. Evaluating Breath Methane as a Diagnostic Test for Constipation-Predominant IBS. Dig Dis Sci 55, 398–403 (2010).
- Kunkel, D. et al. Methane on Breath Testing Is Associated with Constipation: A Systematic Review and Meta-analysis. Dig Dis Sci 56, 1612–1618 (2011).
- Pimentel, M. et al. Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G1089-1095 (2006).
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- Pimentel, M. et al. Antibiotic treatment of constipation-predominant irritable bowel syndrome. Dig. Dis. Sci. 59, 1278–1285 (2014).
- Pimentel, M., Chow, E. J. & Lin, H. C. Normalization of lactulose breath testing correlates with symptom improvement in irritable bowel syndrome. a double-blind, randomized, placebo-controlled study. Am. J. Gastroenterol. 98, 412–419 (2003).
- Brown, K., Scott-Hoy, B. & Jennings, L. W. Response of irritable bowel syndrome with constipation patients administered a combined quebracho/conker tree/M. balsamea Willd extract. World J Gastrointest Pharmacol Ther 7, 463–468 (2016).
- Xu, G.-Y. et al. The endogenous hydrogen sulfide producing enzyme cystathionine-β synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome. Molecular Pain 5, 44 (2009).
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- Ghoshal, U., Shukla, R., Srivastava, D. & Ghoshal, U. C. Irritable Bowel Syndrome, Particularly the Constipation-Predominant Form, Involves an Increase in Methanobrevibacter smithii, Which Is Associated with Higher Methane Production. Gut Liver 10, 932–938 (2016).
- Kim, G. et al. Methanobrevibacter smithii Is the Predominant Methanogen in Patients with Constipation-Predominant IBS and Methane on Breath. Dig Dis Sci 57, 3213–3218 (2012).
- Kerckhoffs, A. P. M. et al. Molecular analysis of faecal and duodenal samples reveals significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel syndrome. Journal of Medical Microbiology 60, 236–245 (2011).
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