Fungi and inflammatory bowel diseases: Alterations of composition and diversity.

OBJECTIVE: Altered bacterial diversity of the intestinal mucosa-associated microbiota may reflect the net influence of lifestyle factors associated with the development of chronic inflammatory bowel diseases (IBD). While a reduced bacterial diversity has been reported in IBD, little is known about the fungal microbiota. The aim of this study was to carry out a systematic analysis of intestinal fungal microbiota in IBD. MATERIAL AND METHODS: The mucosa-associated fungal microbiota of 104 colonic biopsy tissues from 47 controls and 57 IBD patients was investigated using metagenomic 18S rDNA-based denaturing gradient gel electrophoresis (DGGE), clone libraries, sequencing, and in situ hybridization techniques. RESULTS: Fungi-specific 18S rDNA signatures could be detected in all 104 patients, accounting for only a small proportion of the intestinal microbiota (0.02% of the mucosal and 0.03% of the fecal microbiota). An overall fungal biodiversity of 43 different operational taxonomic units (OTUs) was found in the clone libraries. The qualitative composition of fungal microbiota was different between patients with IBD and controls. The DGGE profiles showed a higher mean fungal diversity in patients with Crohn's disease (CD) in comparison with controls (10.8+/-3.1 versus 6.2+/-2.4 for CD, p

Life-threatening chronic enteritis due to colonization of the small bowel with Stenotrophomonas maltophilia.

Chronic diarrheal illness and malabsorption are challenging diagnostic and clinical problems. The identification of the causative pathogens that are involved in gastrointestinal infections is often difficult. It took 85 years after the first description of a case of intestinal lipodystrophy by Georg Whipple in 1907 until the causative bacterium was characterized by using molecular genetics techniques. We here report the complicated clinical course of a young patient with chronic diarrhea accompanied by severe, life-threatening malabsorption with extensive weight loss. Histology and glucose hydrogen breath test were suggestive of a bacterial overgrowth syndrome in the small bowel, but standard culture-based techniques and serology failed to identify the causative bacteria. Thus, bacterial ribosomal DNA (16S ribosomal DNA) was extracted from duodenal biopsy samples and analyzed by community fingerprinting and species-specific polymerase chain reaction. Stenotrophomonas maltophilia was identified as the cause of chronic infectious enteritis. Only specific long-term antibiotic treatment with co-trimoxazole had a durable clinical effect and led to normalization of 16S ribosomal DNA profiles. This case shows the role of rare and uncommon bacteria in refractory and chronic human gastrointestinal infections. Genomic techniques, including 16S-based single-strand conformation polymorphism analysis, will play an increasing role in the diagnosis of chronic infections with facultatively pathogenic bacteria or in the clinical analysis of complex bacterial communities such as the intestinal bacterial microflora. Future enhancements in detection techniques will show that chronic bacterial infections are more frequent as a cause of gastrointestinal malfunction than commonly thought.

In vitro alterations of intestinal bacterial microbiota in fecal samples during storage.

The human gastrointestinal tract harbors an extremely diverse and complex microbial ecosystem. Most of the existent data about the enteric microflora have been generated using stool samples, but the collection and storage of fecal samples are often problematic. The influence of the storage of stool samples on the bacterial diversity and the degradation of bacterial DNA was analysed in this study. Stool samples from 5 healthy volunteers were exposed to different storage temperatures and durations. The bacterial diversity and the amount of intact bacterial DNA were analysed by single-stranded conformation polymorphism analysis (SSCP) and real-time polymerase chain reaction (PCR), both using a 16S rDNA approach. Additionally, biopsy specimens were taken from 3 of the 5 individuals to compare fecal and mucosal flora. The bacterial diversity of the fecal flora and the total number of bacteria were significantly reduced after 8 and 24 hours at both room temperature and 4 degrees C. The mucosa-associated bacterial microflora showed substantial differences compared with the fecal flora. The observed alterations of fecal flora during storage point to the difficulty of the molecular analysis of the bacterial diversity and the enumeration of bacterial cells in fecal samples.

ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme.

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.

Novel type of ADP-forming acetyl coenzyme A synthetase in hyperthermophilic archaea: heterologous expression and characterization of isoenzymes from the sulfate reducer Archaeoglobus fulgidus and the methanogen Methanococcus jannaschii.

Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) right harpoon over left harpoon acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers (alpha(2), beta(2)). The coding genes for the alpha and beta subunits are located at different sites in the P. furiosus chromosome. Based on significant sequence similarity of the P. furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the alpha and beta subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus and ORF MJ0590 of M. jannaschii were cloned and functionally overexpressed in Escherichia coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P. furiosus alpha and beta subunits in an alphabeta (AF1211 and MJ0590) or betaalpha (AF1938) orientation. The data indicate that A. fulgidus and M. jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused.