Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease

BACKGROUND: Clinical and experimental observations in animal models indicate that intestinal commensal bacteria are involved in the initiation and amplification of inflammatory bowel disease (IBD). No paediatric reports are available on intestinal endogenous microflora in IBD. AIMS: To investigate and characterise the predominant composition of the mucosa-associated intestinal microflora in colonoscopic biopsy specimens of paediatric patients with newly diagnosed IBD. METHODS: Mucosa-associated bacteria were quantified and isolated from biopsy specimens of the ileum, caecum and rectum obtained at colonoscopy in 12 patients with Crohn's disease, 7 with ulcerative colitis, 6 with indeterminate colitis, 10 with lymphonodular hyperplasia of the distal ileum and in 7 controls. Isolation and characterisation were carried out by conventional culture techniques for aerobic and facultative-anaerobic microorganisms, and molecular analysis (16S rRNA-based amplification and real-time polymerase chain reaction assays) for the detection of anaerobic bacterial groups or species. RESULTS: A higher number of mucosa-associated aerobic and facultative-anaerobic bacteria were found in biopsy specimens of children with IBD than in controls. An overall decrease in some bacterial species or groups belonging to the normal anaerobic intestinal flora was suggested by molecular approaches; in particular, occurrence of Bacteroides vulgatus was low in Crohn's disease, ulcerative colitis and indeterminate colitis specimens. CONCLUSION: This is the first paediatric report investigating the intestinal mucosa-associated microflora in patients of the IBD spectrum. These results, although limited by the sample size, allow a better understanding of changes in mucosa-associated bacterial flora in these patients, showing either a predominance of some potentially harmful bacterial groups or a decrease in beneficial bacterial species. These data underline the central role of mucosa-adherent bacteria in IBD.     

The intestinal mucus layer from patients with inflammatory bowel disease harbors high numbers of bacteria compared with controls.

BACKGROUND & AIMS: Whether the bacterial flora contributes to the pathogenesis of inflammatory bowel disease (IBD) by increased penetration in mucus, increased adherence to epithelial cells, or invasion of the epithelium is unknown. We therefore studied the spatial distribution of bacteria in the mucosa of rectal biopsy specimens from patients with IBD and from controls. METHODS: Rectal biopsy specimens from 19 patients with IBD and from 14 controls were studied by using nonradioactive ribosomal RNA in situ hybridization. Total mucosal surface length examined for each patient was measured, and the number of bacteria visualized was estimated semiquantitatively. RESULTS: No bacteria were observed in biopsy specimens from 10 controls (71%) and 6 IBD patients (32%) (P = 0.04; odds ratio, 5.42; 95% confidence interval, 1.23-23.9). IBD rectal specimens contained significantly more bacteria than control samples (P = 0.004). Bacteria were localized within the mucus layer but did not adhere to the epithelial cells and were not present within the lamina propria. There was no correlation between the numbers of bacteria present and either the degree of inflammation or the use of anti-inflammatory agents or sulfasalazine compounds. CONCLUSIONS: The intestinal mucus in IBD patients is less protective against the endogenous microflora than in controls, resulting in increased association of luminal bacteria with the mucus layer.     

Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope

It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD). However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear. We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection with Helicobacter hepaticus. We here show that H. hepaticus Ag (SHelAg)-specific CD4+ Th1 clones transfer disease to H. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a control Schistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice. The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certain Helicobacter species as they responded when stimulated in vitro with H. hepaticus and Helicobacter typhlonius Ag, but not when cultured with Ag preparations from Helicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice. Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) of H. hepaticus presented by I-Ab. Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.     

The effects of phthalylsulphathiazole on the bacteria of the colonic mucosa and intestinal contents as revealed by the examination of surgical samples.

The isolation of a bacterial flora specifically associated with the colonic mucosa of patients undergoing large-bowel surgery is described. This flora differed from that of faeces in both the numbers and the types of bacteria isolated. The most striking difference was the reduction in the number of anaerobic bacteria isolated from the colonic mucosa. The ratio of anaerobic to aerobic was approximately 1:1 for mucosa compared with 100:1 for faeces.     

Studies on the intestinal flora : Part II Bacterial flora of the small intestine in patients with gastrointestinal disorders

The type and distribution of bacteria in the jejunal juice of patients with a variety of gastrointestinal conditions that might affect the small intestinal flora were examined.Bacterial colonization of the jejunum defined, in this context, as the occurrence of a bile salttolerant flora consisting of both aerobic and anaerobic bacteria qualitatively resembling that of faeces, was observed only in patients with some form of blind loop. Prominent among the bacteria isolated from these colonized juices were non-sporing anaerobic bacteria, most usually Bacteroides, able to hydrolyse bile salts.No simple correlation between the patient's fat excretion and bacterial colonization of the jejunum could be demonstrated.     

肝硬化患者肠道菌群的研究

目的:研究肝硬化患者肠道菌群的变化、并分析血内毒素水平与肠道细菌的关系。方法:对37例肝硬化患者和18例健康者粪便中8种常见的厌氧菌及需氧菌进行定量研究,以偶氮基质显色法测外周血内毒素。结果:(1)肝硬化患者双歧杆菌、拟杆菌、真杆菌量明显低于正常组,而大肠杆菌、产气荚膜梭菌量高于正常组(P<0.05);(2)肝硬化患者肠菌失调程度与肝功能Child-Pugh分级有关;(3)肝硬化患者血内毒素水平与大肠杆菌量存在相关性。结论:肝硬化患者存在肠道菌群失调,具有代表性的厌氧菌减少,需氧菌增多。需氧革兰阴性杆菌大量繁殖可能是引起肝硬化肠源性内毒素血症的一个重要因素。     

Aerobic and anaerobic microbiology of paronychia

Pus specimens from 28 patients with paronychia of the finger yielded bacterial growth by techniques for cultivation of aerobic and anaerobic bacteria. Anaerobic and aerobic organisms only were isolated in pure culture in five (18%) and eight patients (29%), respectively; mixed aerobic and anaerobic flora were present in 15 patients (54%). Seventy-two isolates were recovered, or 2.6 isolates per specimen. The predominant anaerobic organisms were Gram-positive anaerobic cocci, Bacteroides species, and Fusobacterium species. The predominant aerobic organisms were Staphylococcus aureus, gamma-hemolytic streptococci, Eikenella corrodens, group A beta-hemolytic streptococci, alpha-hemolytic streptococci, and Klebsiella pneumoniae. Candida albicans was recovered in four cases. This study demonstrates the mixed aerobic and anaerobic bacteriology of paronychia.     

Bacteriology and beta-lactamase activity in acute exacerbation of chronic bronchitis.

OBJECTIVES: To assess the bacteriology of beta-lactamase (BL) enzyme activity in sputum of 40 patients with acute exacerbation of chronic bronchitis (AECB). METHODS: The microbiology, BL production by the different isolates, and BL contents in the sputum were determined. RESULTS: Eighty-four isolates were recovered (2.1 isolates per specimen), 44 aerobic and facultative (1.1 isolates per specimen), and 40 anaerobic (1.0 isolate per specimen). Aerobic bacteria were recovered in only 9 (22.5%) specimens, anaerobic bacteria in 9 (22.5%), and mixed aerobic and anaerobic bacteria were found in 22 (55%). The predominant aerobic isolates were Streptococcus pneumoniae (15 isolates), Haemophilus influenzae (11), Moraxella catarrhalis and Klebsiella pneumoniae (4 each). The predominant anaerobes were Peptostreptococcus sp. (19), Prevotella sp. (11), and Fusobacterium sp.(6). Mixed flora were present in 25 (62.5%) specimens, and the number of isolates varied from 2 to 5 per specimen. Thirty-nine beta-lactamase-producing bacteria (BLPB) were isolated in 33 (82.5%) of the 40 cases. The predominant aerobic BLPB were H. influenzae, M. catarrhalis, K. pneumoniae, Staphylococcus aureus, and Escherichia coli. The predominant anaerobic BLPB were Prevotella sp. and Fusobacterium sp. Beta-lactamase activity was detected in 26 (79%) of 33 of specimens in which BLPB were isolated, and in none of the seven specimens that did not harbor BLPB. CONCLUSIONS: The rapid detection of BL activity in sputum specimens may have implications for the antimicrobial management with AECB.     

非酒精性脂肪性肝炎患者肠道菌群的变化及意义

目的探讨非酒精性脂肪性肝炎肠道菌群的变化及意义。方法选择肠道菌群中具有代表性的细菌共5种进行培养和计数。选择健康正常成人、单纯性非酒精性脂肪肝和非酒精性脂肪性肝炎(NASH)患者各30例,计数各组肠道菌群中5种细菌的数量,比较各组细菌数量的变化。结果 NASH组患者存在不同程度的肠道菌群失调,主要表现为厌氧菌减少(P<0.05),而需氧菌显著增多(P<0.05),而兼性厌氧菌酵母样真菌没有显著变化(P>0.05)。结论 NASH患者存在肠道菌群失调、革兰阴性杆菌过度生长的现象,提示肠道微生态失衡可能参与了NASH的发生发展。     

肝硬化患者肠道菌群的研究

目的研究肝硬化患者肠道菌群的变化，并分析血浆内毒素水平与肠道细菌的关系。方法对３７例肝硬化患者和１８例健康者粪便中８种常见的厌氧菌及需氧菌进行定量研究，以偶氮基质显色法测外周血内毒素。结果（１）肝硬化患者双歧杆菌、拟杆菌、真杆菌量明显低于正常组，而大肠杆菌、产气荚膜杆菌量高于正常组（Ｐ＜０．０５）：（２）肠菌失调程度与肝功能Ｃｈｉｌｄ－Ｐｕｇｈ分级有关；（３）内毒素水平与大肠杆菌量存在相关性。结论肝硬化患者存在肠道菌群失调，具有代表性的厌氧菌减少，需氧菌增多。需氧革兰氏阴性杆菌大量繁殖可能是引起肝硬化肠源性内毒素血症的一个重要因素。     

Influence of decontamination on induction of arthritis in Lewis rats by cell wall fragments of Eubacterium aerofaciens. Arthropathic properties of indigenous anaerobic bacteria.

Although the cause (or causes) of rheumatoid arthritis is unknown, many workers have suggested that microorganisms play a part. The intestinal flora in particular has been related to the development of joint inflammation. It has been shown previously that cell wall fragments of several anaerobic Gram positive intestinal bacteria of human origin are arthritogenic after a single intraperitoneal injection in Lewis rats. The part played by indigenous microflora in this model has now been studied by decontaminating Lewis rats before the injection of Eubacterium aerofaciens cell wall fragments. The pattern and severity of arthritis appeared to be comparable in decontaminated and control rats. The second goal of this work was to isolate arthritogenic bacteria from the autochthonous intestinal flora of rats. Only a limited number of bacteria showing a resemblance to arthritogenic strains from human intestinal flora (i.e. E aerofaciens and Bifidobacterium adolescentis) could be isolated. These strains did not induce chronic arthritis after intraperitoneal injection. This may explain why spontaneous arthritis did not develop in Lewis rats.     

Translocation of indigenous bacteria from the gastrointestinal tract of mice after oral ricinoleic acid treatment

A single dose of ricinoleic acid, the active component of castor oil, administered intragastrically to specific pathogen-free mice produced significant alterations in the proximal small intestinal mucosa. Two hours after drug administration, the duodenal villi were markedly shortened with massive exfoliation of columnar and goblet cells. This disruption of the mucosal barrier resulted in continuity between the intestinal lumen and the lamina propria of the villi. Because of the loss of the mucosal barrier, bacteria of the indigenous gastrointestinal flora translocated from the gastrointestinal lumen to the mesenteric lymph nodes, spleen, and liver. The peak incidence of bacterial translocation occurred 4 days after the ricinoleic acid treatment. Strictly anaerobic bacteria, which normally colonize the gastrointestinal tract at greater levels than aerobic or facultatively anaerobic bacteria, were translocated at a greater incidence to the mesenteric lymph nodes than were the other indigenous bacteria. The mucosa began regenerating within 4 h after the ricinoleic acid treatment and viable translocated bacteria were no longer cultured from the mesenteric lymph nodes by 7 days after treatment.     

Chemokine receptor CXCR3 expression in inflammatory bowel disease.

CD4+ T lymphocytes in the lamina propria (LP) of the gut play a central role in the immune response in inflammatory bowel disease (IBD). CXCR3 is a chemokine receptor expressed on activated T lymphocytes, and a key component for the recruitment of T helper (Th1) effector cells to the site of inflammation. To determine if CXCR3 is involved in localization of T cells to the gut in IBD patients, we investigated the expression of CXCR3 on CD4+ T lymphocytes in the LP and in the submucosa of resection specimens from 51 IBD patients and 15 control patients. Positive cells were microscopically scored using a semiquantitative analysis on a five-point scale. We found that CD4+ T cells, CXCR3+ cells, and CD4+CXCR3+ T cells in the LP were slightly increased in both IBD groups compared with control non-IBD specimens. In addition, CD4+ and CXCR3+ cells in the submucosa were significant increased in the CD group compared with the control group. CD4+ and CXCR3+ expression was not statistically different between CD and UC. Flow cytometry was used to analyze the percentage of CXCR3+ cells within the CD4+ T-cell population isolated from biopsy specimens and peripheral blood from IBD patients and control patients. There was no difference in the percentage of CD4+CXCR3+ cells between the different groups in the gut as well as in the circulation. These results suggest that CD4+CXCR3+ T cells migrate to the normal and inflamed intestinal mucosa, indicating a role in maintaining normal gut homeostasis. The selective expression of CXCR3+ cells in the submucosa of CD patients might also indicate that these cells play a role in inflammation.     

T-cell responses to viral, bacterial and protozoan antigens in rheumatoid inflammation. Selective migration of T cells to synovial tissue

OBJECTIVE: To identify any preferential or selective migration of T-cell specificities to inflamed tissues of rheumatoid arthritis (RA) patients. METHODS: Lymphocytes from peripheral blood (PB) and synovial tissue (ST) were isolated from RA patients and stimulated with a panel of crude antigen preparations from 18 bacterial, protozoan and viral sources. Proliferative responses of the T lymphocytes to each antigen and group of antigens were compared in PB and ST. Antigen-specific T-cell clones were developed and their migratory capacities towards synovial chemokines were compared. RESULTS: ST-derived T cells showed a small but significantly higher stimulation index (SI) to the group of intestinal bacteria compared with PB T cells. Conversely, responses of ST-derived T cells to Acanthamoeba polyphaga (AP) were both profoundly and significantly lower compared with PB-derived T cells. The viral antigens as a whole gave comparable reactivities in blood and ST. The migratory capacity of AP-specific T-cell clones towards chemokines produced by ST was profoundly poorer compared with Campylobacter jejuni- and herpes simplex virus-specific T-cell clones. CONCLUSIONS: The results indicate a selective migration of T cells of given specificities to the inflamed rheumatoid synovium.     

Recovery of potential pathogens and interfering bacteria in the nasopharynx of smokers and nonsmokers

BACKGROUND: Active smoking and passive exposure to cigarette smoke are associated with colonization by some potentially pathogenic species of bacteria and an increased risk of respiratory tract infection in both adults and children. In an attempt to explain these observations, this study compared the frequency of isolation of potential pathogens, and aerobic and anaerobic bacteria that possess interfering capabilities (ie, interfering with the in vitro growth of potential pathogens) in the nasopharynx of smokers to their recovery in nonsmokers. METHODS: Nasopharyngeal specimens for cultures were taken from 20 smokers and 20 nonsmokers. Potential pathogens, and aerobic and anaerobic bacteria with interfering capabilities against these organisms were identified. RESULTS: Fourteen potential pathogens (0.7 per patient) were isolated from nasopharyngeal cultures obtained from 11 of the 20 smokers, and 4 (0.2 per patient) were recovered from 3 of the 20 nonsmokers (p < 0.01). In vitro bacterial interference between two aerobic (alpha-hemolytic and nonhemolytic streptococci) and two anaerobic species (Prevotella and Peptostreptococcus species), and four potential pathogens (Streptococcus pneumoniae, Haemophilus influenzae [non-type b], Moraxella catarrhalis, and Streptococcus pyogenes) was observed. Bacterial interference was noted in 61 instances against the four potential pathogens by 22 normal flora isolates that were recovered from the group of smokers, and in 155 instances by 50 isolates from the group of nonsmokers (p < 0.01). CONCLUSIONS: These findings illustrate for the first time that the nasopharyngeal flora of smokers contains fewer aerobic and anaerobic organisms with interfering capabilities and more potential pathogens compared with those of nonsmokers.     

Mucosal and Invading Bacteria in Patients with Inflammatory Bowel Disease Compared with Controls

Background: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. Methods: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. Results: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls ( P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria , the Enterobacteriaceae , the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. Conclusion: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.     

Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls

BACKGROUND: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. METHODS: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. RESULTS: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls (P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria, the Enterobacteriaceae, the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. CONCLUSION: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.     

Impact of different betalactam antibiotics on the normal human flora,and colonization of the oral cavity,throat and colon

Summary Phenoxymethylpenicillin was given orally in doses of 800 mg twice daily for seven days to six patients, and bacampicillin was given in doses of 400 mg three times per day for seven days to another six patients. Saliva, throat and faecal specimens were taken for cultivation of aerobic and anaerobic bacteria. Only small changes in the normal saliva and throat flora were observed, and no changes in the faecal flora were noticed during the observation period. Cefoxitin was administered parenterally in doses of 2 g at 6 h intervals for 12 h to six other patients. Pronounced changes in the colon flora occurred. Of the aerobic bacteria, enterobacteria decreased and cefoxitin-resistant enterococci increased in number; of the anaerobic bacteria, gramnegative rods decreased in number. At the end of the administration period, all cefoxitin-resistant strains decreased, and suppressed enterobacteria and bacteroides increased in number.

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Einfluß verschiedener -Laktam Antibiotika auf die normale Flora und auf die Kolonisation der Mundhöhle, des Rachens und des Colon beim Menschen

Zusammenfassung Sechs Personen erhielten sieben Tage lang Phenoxymethylpenicillin oral in Dosen von 800 mg zweimal täglich. Sechs weitere Personen erhielten sieben Tage lang Bacampicillin in Tablettenform in Dosen von 400 mg dreimal täglich. Zur Kultivierung aerober und anaerober Bakterien wurden Speichel, Rachenabstriche und Stuhlproben entnommen. Im Beobachtungszeitraum waren nur geringe Veränderungen der normalen Speichel- und Rachenflora und keine Veränderungen der faekalen Flora zu beobachten. Sechs Patienten erhielten über einen Zeitraum von zwölf Stunden 2 g Cefoxitin in sechsstündlichen Abständen parenteral appliziert. Daraufhin traten ausgeprägte Veränderungen in der Zusammensetzung der Colonflora auf. Unter den aeroben Bakterien nahmen die Enterobakterien ab, cefoxitinresistente Enterokokken nahmen zu; unter den anaeroben Bakterien war die Zahl der gramnegativen Stäbchen reduziert. In der Phase nach der Antibiotikaapplikation nahm die Zahl aller cefoxitinresistenten Stämme ab, und die supprimierten Enterobakterien und Bacteroidesstämme vermehrten sich wieder.