Review Article

Small Intestinal Bacterial Overgrowth

Prevalence, Clinical Features, Current and Developing Diagnostic Tests, and Treatment

E. Grace; C. Shaw; K. Whelan; H. J. N. Andreyev

Disclosures

Aliment Pharmacol Ther. 2013;38(7):674-688. 

In This Article

Potential for New Diagnostic Tests for SIBO

The changes or dysbiosis of GI microbiota in SIBO are difficult to characterise in clinical practice. Although advances in genomic technology allow for phylogenetic analysis and typing in the research setting, such methods are laborious, expensive and not suitable for routine clinical application. More accessible means of gaining insight into the dysbiosis associated with SIBO include metabolic profiling of biofluids using metabonomics technology, the use of an electronic nose and/or field asymmetric ion mobility spectrometry (FAIMS) to detect volatile organic compounds (VOCs) from gases of luminal origin.

Metabonomics Using Proton Nuclear Magnetic Resonance Spectroscopy

Characterisation of the microbial content of the intestine is a concept that may prove useful in the identification of a new diagnostic method for SIBO. The GI microbiota have co-evolved with humans and metabonomics technology, when based on proton nuclear magnetic resonance (1H-NMR) spectroscopy, can exploit this co-evolution. It has the potential to identify biomarkers and prognostic factors and therefore might enhance the clinical diagnosis of SIBO. Each subject has their own 'metabolic fingerprint', which changes in response to disease, environmental or genetic perturbations. This concept can be applied to SIBO, by evaluating and comparing the metabolic fingerprints of healthy and diseased subjects.

Proton nuclear magnetic resonance spectroscopy is based on the application of a radiofrequency pulse to the nuclear ensemble placed in a magnetic field and observing the response after the duration of the pulse.[103,104] Parallel application of other analytical platforms, for example, gas chromatography mass spectrometry allows the comprehensive study of the metabolome (the quantitative complement of all the low molecular weight molecules present in a biological sample) (Figure 1).[105]

Figure 1.

Mass spectrometry-based metabonomics. GC/MS, gas chromatography mass spectrometry; LC/MS, liquid chromatography mass spectrometry. Reproduced from[106] with permission from The Royal Society of Chemistry.

It provides a robust, efficient, reproducible and relatively cheap approach for high-throughput metabolic screening of biofluids such as blood, urine, small intestinal fluid and faecal water. By combining 1H-NMR spectroscopy with multivariate analysis methodologies, there is growing evidence to suggest that the metabolic profile of biofluids shows clustering of specific components in diseased individuals.[107,108]

In a metabonomics study investigating the content of aspirates from the upper small bowel in patients with malabsorption syndrome, those with the syndrome had significantly higher median quantities of bile acids/cholesterol, acetate, lactate and formate than controls.[107] In those with malabsorption syndrome and SIBO, significantly greater quantities of acetate, lactate, formate and unconjugated bile acids were found compared with controls (P < 0.01 for all), implying that SIBO itself might elicit a specific, potentially diagnostic metabonomic signature.

In another study using faecal samples from patients with inflammatory bowel disease (IBD) (n = 10 Crohn's disease, CD and n = 10 ulcerative colitis, UC) and healthy controls (n = 13), a metabonomics approach was employed to aid with diagnosis.[109] The researchers reported that the faecal samples obtained from the patients with CD and UC manifested similar global differences in metabolic profiles compared with the healthy subjects. A depletion of short-chain fatty acids, including acetate and butyrate, was a prominent feature of CD patients when compared with healthy subjects. In addition, a high concentration of glycerol was found in the faeces of CD patients in comparison to UC patients. Higher concentrations of amino acids were also found in the faeces of patients with both CD and UC as compared with the healthy controls. This could be a consequence of a malabsorption caused by the inflammation.

Chronic inflammatory cells in the lamina propria have been reported in rats with SIBO secondary to an experimental blind loop.[110] Also, data in humans indicate that local lamina propria immunoglobulin A plasma cell and intraepithelial lymphocyte counts are increased in SIBO.[67,111,112] As the condition has been shown to result in microscopic mucosal inflammation, it is plausible to consider that overall differences in the metabolic profiles of SIBO patients and controls are likely to be found.[113] Also, as it is thought that a dysbiosis of the GI microbiota is involved in IBD, either in initiating it or in maintaining it, and as SIBO is also related to dysbiosis, it may be that following the successful application of metabonomics in IBD, it will also prove relevant in SIBO.[109,114]

Occasionally, a single marker molecule will provide an adequate measure of a disease. However, in reality, most human diseases are polygenic in origin and are conditionally linked to environmental influences.[105] Thus, it is more likely that multiple marker molecules will be needed. Studies have shown that inherent factors such as gender, age, circadian rhythms and external factors such as diet, physical activity, stress and drugs can modulate metabonomic profiles.[115,116] Sample collection, storage and preparation also need to be considered as sources of variation in the profiles.

Electronic Nose and Field Asymmetric ion Mobility Spectrometry

The concept of using volatile molecule detection as a means to diagnose SIBO is not a new one. Gas chromatography of jejunal fluid has previously been used to detect and identify volatile fatty acids (short-chain fatty acids) resulting from the fermentation of organic material by nonsporing anaerobic microorganisms. In one study, an increase in the concentrations of the fatty acids, acetate and propionate, was shown in the jejunal contents of patients with stagnant loop syndrome.[117]

In another series of patients thought to have SIBO, a complete microbiological analysis of jejunal aspirates was performed.[75] The results from this were then compared with other testing methods including gas chromatographic detection of the volatile fatty acids in the aspirates. The gas chromatography method was found to have a sensitivity of 56% and a specificity of 100%. Factors to explain the low sensitivity of this method include the preponderance of facultative Gram-negative over anaerobic bacteria in the study group and the required 12-h fast pre-intubation, which would have resulted in a lack of fermentable substrate available to the bacteria.

Gas chromatography, along with mass spectrometry is still considered the gold standard of sample analysis. These traditional approaches provide information on the individual chemical components within a sample, unlike the electronic nose, which analyses the sample as a whole to produce a 'chemical fingerprint'.

The electronic nose was first developed in the 1980s as a way of mimicking the biological olfactory system and used to detect VOCs from human samples, e.g. breath, sweat, blood, urine or faecal samples.[118] VOCs are an important component of the metabolome and include alcohols, aldehydes, furans, ketones, pyrroles, terpenes and others.[119] The electronic nose is not attempting to measure one specific compound, but is able to measure a collection of multiple marker molecules. Colonic fermentation creates several chemicals including VOCs, which may prove important for GI homeostasis. The electronic nose comprises an array of metal gas sensors, whose resistance is modulated in the presence of a target gas/vapour such as VOCs. Each sensor is different in some way (generally broadly tuned to a chemical group) and so the interaction between the sample and each sensor is unique.

The 'chemical fingerprint' produced by this method has been shown to be disparate in different disease groups due to a relative change in the proportions of the VOCs emitted in diseased individuals. The investigation of faecal VOCs may be a promising way of diagnosing SIBO because human faecal samples represent dietary end-products resulting from digestive and excretory processes and intestinal bacterial metabolism. In SIBO, the presence of anaerobic bacteria in the small bowel effectively leads to fermentation occurring in the small bowel in addition to the colon, i.e. an altered intestinal bacterial metabolism. This altered metabolism has the potential to be identified using the electronic nose method.

The electronic nose has not yet been piloted in patients with suspected SIBO. It has, however, proven successful in a range of GI, metabolic and infectious diseases.[120] A pilot study identified a distinct pattern of VOCs in the faeces of patients with UC, Clostridium difficile and Campylobacter jejuni, which strongly suggests that specific changes occur in the pattern of VOCs in GI disease.[121] In another study by this group, the analysis of VOCs from the faeces of Bangladeshi patients affected by cholera showed that fewer VOCs were detected in cholera samples in contrast to healthy controls.[122]

The electronic nose has recently been combined with a newer technology, FAIMS. This is a technique that is able to detect VOCs that emanate from biological material in real-time. It functions by introducing ionised samples (composed of ions of varying shapes, charges and sizes) between two metal plates. An asynchronous high-voltage waveform is applied between the plates and produces conditions whereby some ions drift and hit the plates, while others remain between the plates. Using different voltages, a complex mixture of gases can be separated by differences in mobility across the plates (Figure 2).

Figure 2.

Illustration of the FAIMS effect (parallel plate example), showing ion drift. V, volts; t, time. Source: Thermo Fisher Scientific, 2013.

A pilot study used both an electronic nose and FAIMS technology together to investigate if they could identify differences in faecal gas emissions between patients who developed high toxicity and low toxicity during pelvic radiotherapy.[123] The faecal samples from 23 patients were analysed (11 in the low-toxicity group and 12 in the high-toxicity group). Principle component analysis was applied to the electronic nose data and Fisher discriminant analysis to the FAIMS data. This showed that, perhaps unsurprisingly, it was possible to separate patients after treatment by their toxicity levels. However, distinct differences in the two groups were also identified in their pre-treatment samples, suggesting that severity of side effects can be predicted.

A combination of the electronic nose and FAIMS technologies were used to test their potential usefulness in differentiating between IBD subjects and controls using urine samples.[124] Secondly, the ability of the techniques to distinguish between active IBD compared with those in clinical remission was assessed. Of the 62 adults included, there were three groups: 24 patients with UC, 24 patients with CD and 14 controls. The first two groups were divided further into those with a relapse or in remission.

When the electronic nose samples were analysed using discriminant function analysis, there was a clear separation of groups. Classifications purely according to disease groups or control led to an accuracy of 88%. This distinction was confirmed by repeating the analysis with the FAIMS technology, which gave accuracy in excess of 75% (P < 0.001) as compared to random classification. The results also demonstrated that there is a fundamental difference in the VOCs emitted from the urine samples between groups in remission and those with relapse.

As with the pelvic radiotherapy and IBD patients, it is plausible to think that distinct differences in VOC patterns would also be observed between SIBO patients and healthy controls. The possibility of using an electronic nose for the detection of SIBO is particularly attractive due to its non-invasive nature, its portability and the potential to use this technology in the out-patient setting. It can be operated at room temperature and pressure. It is cheap, less time-consuming and complex than gas chromatography mass spectrometry. Many of the advantages of the electronic nose also apply to FAIMS and combining the two instruments may prove a powerful technique for the future development of a diagnostic tool for SIBO.

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