Microbial community analysis reveals high level phylogenetic alterations in the overall gastrointestinal microbiota of diarrhoea-predominant irritable bowel syndrome sufferers.

BACKGROUND: A growing amount of scientific evidence suggests that microbes are involved in the aetiology of irritable bowel syndrome (IBS), and the gastrointestinal (GI) microbiota of individuals suffering from diarrhoea-predominant IBS (IBS-D) is distinguishable from other IBS-subtypes. In our study, the GI microbiota of IBS-D patients was evaluated and compared with healthy controls (HC) by using a high-resolution sequencing method. The method allowed microbial community analysis on all levels of microbial genomic guanine plus cytosine (G+C) content, including high G+C bacteria. METHODS: The collective faecal microbiota composition of ten IBS-D patients was analysed by examining sequences obtained using percent G+C (%G+C) -based profiling and fractioning combined with 16S rRNA gene clone library sequencing of 3267 clones. The IBS-D library was compared with an analogous healthy-control library of 23 subjects. Real-time PCR analysis was used to identify phylotypes belonging to the class Gammaproteobacteria and the order Coriobacteriales. RESULTS: Significant differences were found between clone libraries of IBS-D patients and controls. The microbial communities of IBS-D patients were enriched in Proteobacteria and Firmicutes, but reduced in the number of Actinobacteria and Bacteroidetes compared to control. In particular, 16S rDNA sequences belonging to the family Lachnospiraceae within the phylum Firmicutes were in greater abundance in the IBS-D clone library. CONCLUSIONS: In the microbiota of IBS-D sufferers, notable differences were detected among the prominent bacterial phyla (Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria) localized within the GI tract.

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype.

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (

Characterization of genes involved in fructose utilization by Lactobacillus fermentum.

The genes encoding phosphoglucose isomerase (fruI) and fructokinase (fruK) of Lactobacillus fermentum NRRL-B-1932 were sequenced. They constituted an operon, which is involved in fructose metabolism of this strain by channeling intracellular fructose into the phosphoketolase pathway. A third open reading frame, unkR, upstream of the operon was identified as homologous to genes of LacI/GalR family repressors. The UnkR repressor's role in transcriptional control of the fruIK operon could, however, not be established by electrophoretic mobility shift assay (EMSA) analysis. Sequence analysis revealed two putative catabolite responsive elements (cre) in the promoter region of fruIK suggesting that the fruIK operon is under negative regulatory control by carbon catabolite repression. Expression and enzyme activity data were compatible with the assumption that the fruIK operon is repressed by glucose. No sugar specific phosphoenolpyruvate sugar transferase system activity for the transport of fructose, glucose, sucrose or mannose could be detected in L. fermentum NRRL-B-1932 cells, which suggest that fructose is taken up by a permease system.

Improved mannitol production by a random mutant of Leuconostoc pseudomesenteroides.

A mutant of Leuconostoc pseudomesenteroides ATCC12291 that was unable to grow on fructose was constructed by chemical mutagenesis. The fructose uptake of this mutant, designated as BPT143, was unaltered and allowed fructose still to be converted into mannitol when glucose was present in the growth medium. The mutant grew and consumed fructose faster than the parent strain when grown in a medium containing both glucose and fructose. The specific activity of fructokinase, the enzyme involved in phosphorylation of fructose to fructose-6-phosphate, was decreased to about 10% of that of the parent strain, and resulted in a reduced leakage of fructose into the phosphoketolase (PK) pathway. The yield of mannitol from fructose was improved from 74 to 86 mol%. The increased fructose consumption rate and higher mannitol yield of the mutant also resulted in improvement of volumetric mannitol productivity. In addition, isolation and characterization of the wild type L. pseudomesenteroides fructokinase gene (fruK) was performed. DNA sequence analysis of the fruK gene region of BPT143 revealed only one silent mutation which does not explain the highly reduced fructokinase activity of the mutant. The genetic characterization of fruK was completed by analyzing the expression, size and 5' end of fruK transcripts. Expression data with BPT143, revealing absence of fruK transcripts, was in accordance with the reduced fructokinase activity of the mutant.

Probiotic and milk technological properties of Lactobacillus brevis.

Two Lactobacillus brevis strains ATCC 8287 and ATCC 14869(T), were evaluated for their applicability as putative probiotics in dairy products. The strains expressed good in vitro adherence to human Caco-2 and Intestine 407 cells and tolerated well low pH, bile acids and pancreatic fluid under in vitro conditions. In antimicrobial activity assays, strain ATCC 8287 showed inhibitory properties toward selected potential harmful microorganisms, particularly against Bacillus cereus. Both L. brevis strains were resistant to vancomycin, which is typical for the genus Lactobacillus. The L. brevis strains were not able to acidify milk to yoghurt but were suitable as supplement strains in yoghurts. This was shown by producing a set of yoghurt products and analysing their rheological and sensory properties during a cold storage period of 28 days. Survival of the strains through human intestine was examined in 1-week feeding trials. Despite its human origin, L. brevis ATCC 14869(T) could not survive through the gastrointestinal (GI) tract, whereas L. brevis ATCC 8287 was detected in the faecal samples taken during and immediately after ingestion of the strain. In conclusion, L. brevis ATCC 8287 is a promising candidate as a probiotic supplement in dairy products.

Metabolic engineering of Lactobacillus fermentum for production of mannitol and pure L-lactic acid or pyruvate.

For production of mannitol in combination with pure L-lactic acid or pyruvate, the D- and L-lactate dehydrogenase genes (ldhD and ldhL) of a mannitol-producing Lactobacillus fermentum strain were cloned and stepwise inactivated. For inactivation of both ldh genes by a gene replacement technique, deletion constructs removing a 0.4-kb fragment from the promoter and the 5' end region of the ldh genes were used. The first inactivation mutant, designated L. fermentum GRL1030, carried the deletion in ldhD (DeltaldhD). A double mutant, DeltaldhD-DeltaldhL, was constructed by the inactivation of the ldhL gene of strain GRL1030, resulting in strain L. fermentum GRL1032. The correctness of the both mutants was confirmed at the DNA level by polymerase chain reaction, as shown by the absence of ldh transcripts by northern blotting and as a lack of the corresponding enzyme activity. In bioreactor cultivations, the single mutant GRL1030 produced mannitol and L-lactic acid as expected. Mannitol and lactic acid yields and productivities were practically unaffected by deletion of the ldhD gene. The double mutant GRL1032 produced mannitol and pyruvate as expected. However, although the yield of mannitol from fructose remained high, its volumetric productivity was reduced. The double mutation negatively affected the glucose consumption rate, resulting in reduced cellular growth. In addition to pyruvate, the double mutant produced 2,3-butanediol. More surprisingly, some lactic acid was still produced.