Abundance of Enterobacteriaceae in the colon mucosa in diverticular disease

ARTICLE HIGHLIGHTS

Research background

Diverticular disease (DD) is a common gastrointestinal disease of unknown etiology. The symptoms of DD are similar with symptoms of irritable bowel syndrome (IBS). The gut microbiota is discussed as important for the etiology and pathophysiology in a wide range of diseases. Bacterial diversity is higher in lean compared to obese individuals, and in healthy states compared to unhealthy states, and some bacterial groups, e.g., Enterobacteriaceae, are associated with over-weight and inflammation. The family Enterobacteriaceae is commonly found in the gut ecosystem, where Escherichia coli is the most abundant species of the family. Only a few studies have been performed regarding microbial composition in DD. Recently, lower amounts of Enterobacteriaceae were found in the colon mucosa of DD patients compared with healthy controls, whereas higher amounts of Akkermansia and no difference in the Escherichia coli subgroup were found in feces in another DD cohort. Thus, it is hypothesized that gut microbiota is involved in the etiology and pathophysiology of DD, but the few studies performed so far have shown inconclusive results.

Research motivation

Today, there is no efficient treatment option for DD, neither to prevent disease development nor to reduce the symptoms when the disease has been established, which renders a lot of suffering to the patients. To find out the etiology is crucial to be able to prevent and efficiently treat the disease. New knowledge within this disease field may point out the direction for future research.

Research objectives

The primary aim of the present study was to compare the level of the large Gram-negative bacterial family Enterobacteriaceae and gut bacterial diversity in colon mucosa between consecutive patients diagnosed with DD and patients with normal endoscopic findings. Secondary aims were to evaluate the influence of demography, socioeconomic status, lifestyle habits, inflammatory parameters and gastrointestinal symptoms on the gut microbiota. These objectives were possible to realize by the present study design. Further studies according to the same study design, but with larger patient cohorts, are important to perform to confirm the results.

Research methods

All consecutive patients referred to elective colonoscopy at the Department of Endoscopy, Skåne University Hospital, Malmö, were invited to participate in the study. If the patients agreed to participate, they had to complete a study questionnaire about demography, socioeconomic status, lifestyle habits, family history and medical history, the Visual Analog Scale for Irritable Bowel Syndrome (VAS-IBS), and a nutrition questionnaire to analyze dietary habits. The colonoscopy was performed according to clinical routines. Four different mucosa biopsies were obtained from the mid part of the colon descendens. Samples were stored at -80 °C until the gut microbiota was analyzed by quantitative polymerase chain reaction (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Blood samples were collected according to clinical routines and analyzed at the Department of Clinical Chemistry. A study protocol was completed by the physician about clinical findings and histopathological diagnoses. The patients were divided into two groups depending on the colonoscopy finding: patients with DD, and patients without any diverticula who served as controls. Three of the four mucosa samples, mean weight 15 ± 0.6 mg, were used for DNA extraction. DNA was isolated and purified by EZ1 Advanced XL (EZ1 DNA Tissue Kit and Bacteria Card; Qiagen, Hilden, Germany). The amount of Enterobacteriaceae was estimated using a qPCR assay according to Karlsson et al. Primers used for the qPCR assay have been used and published previously. The detection limit was 10² genes/reaction. For standard curves, 10-fold dilution series of the target DNA were made in EB buffer (Qiagen). Number of bacteria was expressed as log₁₀ 16S rRNA genes/g feces. T-RFLP was applied to assess the microbial diversity, as previously described. Thresholds for internal standard and terminal restriction fragments (T-RFs) were set to 5 and 15 fluorescence units, respectively. Microbial diversity was estimated by calculation of richness (number of T-RFs) and Shannon-Wiener and Simpson’s diversity indices as described by Karlsson et al, with the exception that T-RFs within 40-580 base pairs were included in the T-RFLP profile analysis and calculation. The diversity indices take into account both richness and evenness when considering the relative abundance of bacterial groups. Both indices are commonly used to assess microbial diversity. Samples below the limit of detection (in qPCR) were replaced by the limit of detection for statistical analysis.

Research results

Finally, 51 patients were included in the present study, 16 with DD and 35 controls without organic changes visible at the colonoscopy or at the histopathological examination (n = 12), except non-malignant polyps (n = 23). The reasons for referral to colonoscopy were presence of gastrointestinal symptoms which rendered a colonoscopy to exclude inflammatory bowel disease, malignancy or DD (n = 17), follow-up after previous resection of polyps (n = 17), rectal bleeding (n = 11), screening for cancer due to heredity (n = 4), or perforation to the urinary tract (n = 2). Only one subject in the DD group had a history of verified acute diverticulitis. There was an equal sex distribution in the groups. Subjects without DD were slightly older than controls [68 (62-76) years vs 62 (40-74) years, P = 0.072]. Altogether, 22 patients (43.1%) fulfilled the Rome IV criteria for IBS. The prevalence of functional dyspepsia, IBS, gastric ulcer, lactose intolerance and reflux was equally distributed between groups. Each symptom item estimated by the VAS-IBS questionnaire was present in about half of all patients examined. Only 4 patients in each group did not have any form of gastrointestinal symptoms. There was a wide variety in symptom intensity within each group also. None of the items in VAS-IBS correlated with age. Those who had homemade lunch suffered from more gastrointestinal symptoms compared with those who did not eat lunch, had lunch at a restaurant or had precooked meals, although bloating and flatulence was the only item that reached statistical significance [52 (25-93) vs 88 (70-100), P = 0.024]. The difference could not be related to any differences in socioeconomic factors or smoking or alcohol habits or in age span [66 (50-76) vs 65 (59-72), P = 0.851]. Patients with DD had significantly higher levels of Enterobacteriaceae than patients without diverticula (P = 0.043). Although patients with DD more often had lower education and less physical activity, the different subgroups of these parameters did not affect the amount of Enterobacteriaceae, diversity indices of Shannon-Wiener or Simpson, or the number of T-RFs (P = 0.413, P = 0.803, P = 0.770, and P = 0.588, respectively, vs P = 0.684, P = 0.616, P = 0.745, and P = 0.316, respectively). There were no differences in any parameters between controls with and without polyps. There was an inverse correlation between the amount of Enterobacteriaceae and Simpson’s index (rs = -0.361, P = 0.033) and a tendency to correlation between Enterobacteriaceae and Shannon-Wiener index (rs = -0.299, P = 0.081). The Shannon-Wiener and Simpson’s indices correlated with each other (rs = 0.947, P < 0.001) and number of T-RFs (rs = 0.917, P < 0.001 and rs = 0.772, P < 0.001, respectively). Several of the patients had humoral inflammatory parameters above or beneath the reference values, i.e. plasma-C-reactive protein (CRP): < 3 mg/L; blood-leucocytes: 3.5-8.8 × 10⁹/L; blood-thrombocytes 125-340 × 10⁹/L; and plasma-albumin: 36-48 g/L. The level of inflammatory biomarkers did not differ between patients with or without DD. Neither did presence nor absence of IBS affect the plasma levels of CRP (P = 0.194) and albumin (P = 0.902), or blood levels of leukocytes (P = 0.912) and thrombocytes (P = 0.509). There was no correlation between any of the inflammatory biomarkers and the level of Enterobacteriaceae or bacterial diversity. Neither the amount of Enterobacteriaceae nor the diversity indices correlated with age, BMI, or any items of the VAS-IBS. When calculating differences between patients with and without any of the gastrointestinal symptoms, there were no differences in amount of Enterobacteriaceae or diversity indices (data not shown). Presence of IBS did not affect the amount of Enterobacteriaceae (P = 0.867), Shannon-Wiener index (P = 0.533), Simpson’s index (P = 0.478), or number of T-RFs (P = 0.828). There were no differences in the amount of Enterobacteriaceae or the diversity indices between those who had a regular vs irregular breakfast intake of coffee/tea, dairy products, or cereals. The gut microbiota parameters examined were not influenced by intake of homemade lunch or dinner, smoking and alcohol habits, intake of probiotics and antibiotics, or movement patterns. The problems that remain to be solved are whether the difference in gut microbiota composition are primary events in the disease development or secondary to the DD. The causality to DD must still be defined.

Research conclusions

The new finding of the present study is the abundance of Enterobacteriaceae in colon mucosa in DD, and that this abundance was not related to age, BMI, socioeconomic parameters, gastrointestinal symptoms or lifestyle habits. Microbial diversity was not affected by DD or any other parameters measured. The new theory that this study proposes is that the composition of gut microbiota is involved in DD. The summarization of this study is that gut microbiota may be affected in patients with DD. This study is the first study where a clinical cohort of patients is consecutively enrolled during colonoscopy to analyze gut microbiota in colon mucosa, where the only difference between the groups compared is the presence or absence of colon diverticula. Previous studies have enrolled participants in screening programs or analyzed microbiota composition in feces. The authors also studied socioeconomic features and lifestyle habits in the cohort, to be able to adjust for confounders. The new hypotheses proposed are that gut microbiota is involved in DD and that demography, socioeconomic parameters and dietary habits may be of less importance for the microbiota than the presence or absence of colon diverticula. The new methods proposed are the enrolment of consecutive clinical patients in scientific trials, analyses of gut microbiota in mucosa instead of feces, analysis of microbial diversity to get a general reflection of the gut microbiota, analysis of the amount of Enterobacteriaceae or other bacteria by qPCR, and estimation of gastrointestinal symptoms by the VAS-IBS questionnaire. The new phenomenon found were that presence or absence of colon diverticula are more important for gut microbiota than demography, socioeconomic parameters, gastrointestinal symptoms, or lifestyle habits. Another new phenomenon was that patients with homemade lunch had more gastrointestinal symptoms than patients who did not eat lunch or had lunch at a restaurant. The authors confirmed the hypothesis that the amount of Enterobacteriaceae was affected by DD, but failed to confirm the hypothesis that overall bacterial diversity was influenced by colon diverticula. The authors also failed to confirm the hypotheses that demography, socioeconomic parameters, gastrointestinal symptoms and lifestyle habits were associated with gut microbiota composition. The major implication for clinical practice in the future is to consider dysbiosis in patients with DD. Tests to determine gut microbiota are available for clinical use, and should be considered in the management of these patients.

Research perspectives

The experience the authors have learnt from this study is that presence or absence of DD is more important for the gut microbiota composition than demography, socioeconomic parameters, gastrointestinal symptoms, and lifestyle habits. The authors have also learnt from this study that homemade food is not always the best for patients in the management of gastrointestinal symptoms. The authors must further study the importance of gut microbiota in DD. The authors should continue to include patients with DD in experiments to analyze gut microbiota composition to get larger cohorts, and to perform clinical trials to evaluate the effect of probiotics in symptom management of DD. The best method is to analyze gut microbiota in colon mucosa instead of feces. The VAS-IBS is also a useful tool to estimate gastrointestinal symptoms.