Visualisation of Mycobacterium avium subsp. paratuberculosis in cultured cells, infected sheep and human tissue sections using fluorescent in situ hybridization (FISH).

We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity.

Klebsiella pneumoniae subsp. pneumoniae-bacteriophage combination from the caecal effluent of a healthy woman.

A sample of caecal effluent was obtained from a female patient who had undergone a routine colonoscopic examination. Bacteria were isolated anaerobically from the sample, and screened against the remaining filtered caecal effluent in an attempt to isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolated on a strain identified as Klebsiella pneumoniae subsp. pneumoniae (capsular type K2, rmpA (+)). This Siphoviridae phage presents a rosette-like tail tip and exhibits depolymerase activity, as demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1 was shown to infect and lyse capsular type K2 strains, though it did not exhibit depolymerase activity on such hosts. The genome of KLPN1 was determined to be 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which 23 represented genes associated with structure, host recognition, packaging, DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1 (GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank: KP658157) were found to be two new members of the genus "Kp36likevirus."

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

BACKGROUND: Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. METHODS: Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. RESULTS: E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. CONCLUSION: In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Effect of breadmaking process on in vitro gut microbiota parameters in irritable bowel syndrome.

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria-mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small-molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.

Quantification and characterization of mucosa-associated and intracellular Escherichia coli in inflammatory bowel disease.

BACKGROUND: Mucosa-associated Escherichia coli are abundant in inflammatory bowel disease (IBD), but whether these bacteria gain intracellular access within the mucosa is uncertain. If E. coli does gain intracellular access, the contribution of bacterial pathogenicity to this requires further elucidation. This study aimed to quantify and characterize mucosa-associated and intracellular E. coli in patients with IBD and in healthy control subjects (HC). METHODS: Mucosal biopsies from 30 patients with Crohn's disease (CD), 15 with ulcerative colitis (UC), and 14 HC were cultured with or without gentamicin protection to recover intracellular or mucosa-associated E. coli, respectively. Overall, 40 strains (CD: n = 24, UC: n = 9, and HC: n = 7) were characterized by phylogenetic typing, adhesion and invasion assays, detection of virulence factors, antimicrobial resistance genes, and proteomic analysis. RESULTS: Mucosa-associated E. coli were more abundant in CD and UC than in HC (2750 versus 1350 versus 230 median colony-forming units per biopsy; P = 0.01). Intracellular E. coli were more prevalent in CD (90%) than in UC (47%) or HC mucosal biopsies (0%) (P < 0.001). Of 24 CD strains, 2 were adherent and invasive, but there were no unifying pathogenicity determinants that could distinguish most CD strains from UC or HC strains, or intracellular isolates from mucosa-associated isolates. CONCLUSIONS: Intracellular E. coli are more common in CD than in UC and not identified in HC. Most intracellular E. coli did not have characterizing pathogenic features, suggesting a significant role for defects in mucosal immunity or barrier dysfunction in their ability to gain intracellular access.

High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease.

BACKGROUND: The gut microbiota is thought to play a key role in the development of the inflammatory bowel diseases Crohn's disease (CD) and ulcerative colitis (UC). Shifts in the composition of resident bacteria have been postulated to drive the chronic inflammation seen in both diseases (the "dysbiosis" hypothesis). We therefore specifically sought to compare the mucosa-associated microbiota from both inflamed and non-inflamed sites of the colon in CD and UC patients to that from non-IBD controls and to detect disease-specific profiles. RESULTS: Paired mucosal biopsies of inflamed and non-inflamed intestinal tissue from 6 CD (n = 12) and 6 UC (n = 12) patients were compared to biopsies from 5 healthy controls (n = 5) by in-depth sequencing of over 10,000 near full-length bacterial 16S rRNA genes. The results indicate that mucosal microbial diversity is reduced in IBD, particularly in CD, and that the species composition is disturbed. Firmicutes were reduced in IBD samples and there were concurrent increases in Bacteroidetes, and in CD only, Enterobacteriaceae. There were also significant differences in microbial community structure between inflamed and non-inflamed mucosal sites. However, these differences varied greatly between individuals, meaning there was no obvious bacterial signature that was positively associated with the inflamed gut. CONCLUSIONS: These results may support the hypothesis that the overall dysbiosis observed in inflammatory bowel disease patients relative to non-IBD controls might to some extent be a result of the disturbed gut environment rather than the direct cause of disease. Nonetheless, the observed shifts in microbiota composition may be important factors in disease maintenance and severity.

Independent and population-specific association of risk variants at the IRGM locus with Crohn's disease.

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohn's disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5' untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 x 10(-5) to 1.40 x 10(-9)), and that the CNV and the 5'-untranslated region variant -308(GTTT)(5) contribute independently to CD susceptibility (P = 2.6 x 10(-7) and P = 2 x 10(-5), respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10(-12)) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case-control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene-gene or gene-environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European-Asian split.

Molecular characterization of rectal mucosa-associated bacterial flora in inflammatory bowel disease.

BACKGROUND: Colorectal bacteria may play a role in the pathogenesis of inflammatory bowel disease (IBD). To test the hypothesis that, in affected patients, the numbers of potentially protective mucosal bacteria might be reduced and pathogenic species increased, we compared rectal mucosa-associated flora in patients with IBD and normal controls. METHODS: Snap-frozen rectal biopsies taken at routine diagnostic colonoscopy from 33 patients with ulcerative colitis, 6 patients with Crohn's disease, and 14 controls with normal colonoscopy were processed, and individual bacterial groups were counted using fluorescent in situ hybridization. RESULTS: Bacteria were mostly found apposed to the epithelial surface and within crypts. Epithelium-associated counts of bifidobacteria in active [median 15/mm of epithelial surface (range, 4-56), n = 14] and quiescent ulcerative colitis [26/mm (range, 11-140), n = 19] were lower than in controls [56/mm (range, 0-144), n = 14; P = 0.006 and P = 0.03, respectively]. Conversely, epithelium-associated Escherichia coli counts were higher in active [82/mm (range, 56-136)] than inactive ulcerative colitis [6/mm (range, 0-136), P = 0.0001] or controls [0/mm (range, 0-16), P < 0.0001]. Epithelium-associated clostridia counts were also higher in active [3/mm (range, 0-9)] than inactive colitis [0/mm (range, 0-9), P = 0.03] or controls [0/mm (range, 0-1); P = 0.0007]. Epithelium-associated E. coli counts were higher in Crohn's disease [42/mm (range, 3-90), n = 6] than controls (P = 0.0006). E. coli were also found as individual bacteria and in clusters in the lamina propria in ulcerative colitis and Crohn's disease but in none of the controls (P < 0.01). Numbers of Lactobacillus and Bacteroides showed no differences between patient groups. CONCLUSIONS: The reduction in mucosa-associated bifidobacteria and increase in E. coli and clostridia in patients with IBD supports the hypothesis that an imbalance between potentially beneficial and pathogenic bacteria may contribute to its pathogenesis.

An in vitro assessment of the effects of broad-spectrum antibiotics on the human gut microflora and concomitant isolation of a Lactobacillus plantarum with anti-Candida activities.

Chemostat culture was used to determine the effects of the antimicrobial agents tetracycline and nystatin on predominant components of the human gut microflora. Their addition to mixed culture systems caused a non-specific, and variable, decrease in microbial populations, although tetracycline allowed an increase in numbers of yeasts. Both had a profound inhibitory effect upon populations seen as important for gut health (bifidobacteria, lactobacilli). However, a tetracycline resistant Lactobacillus was enriched from the experiments. A combination of genotypic and phenotypic characterisation confirmed its identity as Lactobacillus plantarum. This strain exerted powerful inhibitory effects against Candida albicans. Because of its ability to resist the effects of tetracycline, this organism may be useful as a probiotic for the improved management of yeast related conditions such as thrush and irritable bowel syndrome.

In vitro studies on colonization resistance of the human gut microbiota to Candida albicans and the effects of tetracycline and Lactobacillus plantarum LPK.

An anaerobic three-vessel continuous-flow culture system, which models the three major anatomical regions of the human colon, was used to study the persistence of Candida albicans in the presence of a faecal microbiota. During steady state conditions, overgrowth of C. albicans was prevented by commensal bacteria indigenous to the system. However antibiotics, such as tetracycline have the ability to disrupt the bacterial populations within the gut. Thus, colonization resistance can be compromised and overgrowth of undesirable microorganisms like C. albicans can then occur. In this study, growth of C. albicans was not observed in the presence of an established faecal microbiota. However, following the addition of tetracycline to the growth medium, significant growth of C. albicans occurred. A probiotic Lactobacillus plantarum LPK culture was added to the system to investigate whether this organism had any effects upon the Candida populations. Although C. albicans was not completely eradicated in the presence of this bacterium, cell counts were markedly reduced, indicating a compromised physiological function. This study shows that the normal gut flora can exert 'natural' resistance to C. albicans, however this may be diminished during antibiotic intake. The use of probiotics can help fortify natural resistance.