Bead-beating artefacts in the Bacteroidetes to Firmicutes ratio of the human stool metagenome.

We evaluated bead-beating cell-lysis in analysing the human stool metagenome, since this is a key step. We observed that two different bead-beating instruments from the same producer gave a three-fold difference in the Bacteroidetes to Firmicutes ratio. This illustrates that bead-beating can have a major impact on downstream metagenome analyses.

Temporal development of the infant gut microbiota in immunoglobulin E-sensitized and nonsensitized children determined by the GA-map infant array.

At birth, the human infant gut is sterile, but it becomes fully colonized within a few days. This initial colonization process has a major impact on immune development. Our knowledge about the correlations between aberrant colonization patterns and immunological diseases, however, is limited. The aim of the present work was to develop the GA-map (Genetic Analysis microbiota array platform) infant array and to use this array to compare the temporal development of the gut microbiota in IgE-sensitized and nonsensitized children during the first 2 years of life. The GA-map infant array is composed of highly specific 16S rRNA gene-targeted single nucleotide primer extension (SNuPE) probes, which were designed based on extensive infant 16S rRNA gene sequence libraries. For the clinical screening, we analyzed 216 fecal samples collected from a cohort of 47 infants (16 sensitized and 31 nonsensitized) from 1 day to 2 years of age. The results showed that at a high taxonomic level, Actinobacteria was significantly overrepresented at 4 months while Firmicutes was significantly overrepresented at 1 year for the sensitized children. At a lower taxonomic level, for the sensitized group, we found that Bifidobacterium longum was significantly overrepresented at the age of 1 year and Enterococcus at the age of 4 months. For most phyla, however, there were consistent differences in composition between age groups, irrespective of the sensitization state. The main age patterns were a rapid decrease in staphylococci from 10 days to 4 months and a peak of bifidobacteria and bacteroides at 4 months. In conclusion, our analyses showed consistent microbiota colonization and IgE sensitization patterns that can be important for understanding both normal and diseased immunological development in infants.

Comparative genomic analysis of pathogenic and probiotic Enterococcus faecalis isolates, and their transcriptional responses to growth in human urine.

Urinary tract infection (UTI) is the most common infection caused by enterococci, and Enterococcus faecalis accounts for the majority of enterococcal infections. Although a number of virulence related traits have been established, no comprehensive genomic or transcriptomic studies have been conducted to investigate how to distinguish pathogenic from non-pathogenic E. faecalis in their ability to cause UTI. In order to identify potential genetic traits or gene regulatory features that distinguish pathogenic from non-pathogenic E. faecalis with respect to UTI, we have performed comparative genomic analysis, and investigated growth capacity and transcriptome profiling in human urine in vitro. Six strains of different origins were cultivated and all grew readily in human urine. The three strains chosen for transcriptional analysis showed an overall similar response with respect to energy and nitrogen metabolism, stress mechanism, cell envelope modifications, and trace metal acquisition. Our results suggest that citrate and aspartate are significant for growth of E. faecalis in human urine, and manganese appear to be a limiting factor. The majority of virulence factors were either not differentially regulated or down-regulated. Notably, a significant up-regulation of genes involved in biofilm formation was observed. Strains from different origins have similar capacity to grow in human urine. The overall similar transcriptional responses between the two pathogenic and the probiotic strain suggest that the pathogenic potential of a certain E. faecalis strain may to a great extent be determined by presence of fitness and virulence factors, rather than the level of expression of such traits.

The Response of Enterococcus faecalis V583 to Chloramphenicol Treatment.

Many Enterococcus faecalis strains display tolerance or resistance to many antibiotics, but genes that contribute to the resistance cannot be specified. The multiresistant E. faecalis V583, for which the complete genome sequence is available, survives and grows in media containing relatively high levels of chloramphenicol. No specific genes coding for chloramphenicol resistance has been recognized in V583. We used microarrays to identify genes and mechanisms behind the tolerance to chloramphenicol in V583, by comparison of cells treated with subinhibitory concentrations of chloramphenicol and untreated V583 cells. During a time course experiment, more than 600 genes were significantly differentially transcribed. Since chloramphenicol affects protein synthesis in bacteria, many genes involved in protein synthesis, for example, genes for ribosomal proteins, were induced. Genes involved in amino acid biosynthesis, for example, genes for tRNA synthetases and energy metabolism were downregulated, mainly. Among the upregulated genes were EF1732 and EF1733, which code for potential chloramphenicol transporters. Efflux of drug out of the cells may be one mechanism used by V583 to overcome the effect of chloramphenicol.

The transcriptome of the nosocomial pathogen Enterococcus faecalis V583 reveals adaptive responses to growth in blood.

BACKGROUND: Enterococcus faecalis plays a dual role in human ecology, predominantly existing as a commensal in the alimentary canal, but also as an opportunistic pathogen that frequently causes nosocomial infections like bacteremia. A number of virulence factors that contribute to the pathogenic potential of E. faecalis have been established. However, the process in which E. faecalis gains access to the bloodstream and establishes a persistent infection is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: To enhance our understanding of how this commensal bacterium adapts during a bloodstream infection and to examine the interplay between genes we designed an in vitro experiment using genome-wide microarrays to investigate what effects the presence of and growth in blood have on the transcriptome of E. faecalis strain V583. We showed that growth in both 2xYT supplemented with 10% blood and in 100% blood had a great impact on the transcription of many genes in the V583 genome. We identified several immediate changes signifying cellular processes that might contribute to adaptation and growth in blood. These include modulation of membrane fatty acid composition, oxidative and lytic stress protection, acquisition of new available substrates, transport functions including heme/iron transporters and genes associated with virulence in E. faecalis. CONCLUSIONS/SIGNIFICANCE: The results presented here reveal that cultivation of E. faecalis in blood in vitro has a profound impact on its transcriptome, which includes a number of virulence traits. Observed regulation of genes and pathways revealed new insight into physiological features and metabolic capacities which enable E. faecalis to adapt and grow in blood. A number of the regulated genes might potentially be useful candidates for development of new therapeutic approaches for treatment of E. faecalis infections.

Transcriptional responses of Enterococcus faecalis V583 to bovine bile and sodium dodecyl sulfate.

Resistance to bile is a prerequisite property of the gastrointestinal bacterial flora. Bile acids are powerful detergents, and resistance to sodium dodecyl sulfate (SDS) has therefore often been considered relevant to studies of bile resistance. We have studied the effects of bovine bile (BB) and SDS on Enterococcus faecalis V583 by traditional growth studies and microarrays. Transcriptional responses were studied by time course experiments. In the presence of BB (V583-BB) or SDS (V583-SDS), 308 and 209 genes were identified as differentially expressed at one or more time points, respectively. In V583 treated with both BB and SDS (V583-BB-SDS), 254 genes showed differential expression. Detergents exert their toxic effects primarily on the microbial membrane. The enrichment of differentially transcribed genes that encode proteins with membrane-associated functions and/or locations indicates a major impact of all three treatments on the integrity and functionality of the cell membrane. Two gene clusters involved in fatty acid biosynthesis were repressed in V583-BB and V583-BB-SDS and partly induced in V583-SDS. Furthermore, two EmrB/QacA family drug resistance transporters and a vacuolar-type ATPase were induced in V583-BB and V583-BB-SDS. None of the putative bile salt hydrolase homologs in V583 showed differential expression during the bile treatments. The transcriptional profile of V583-BB-SDS was qualitatively more similar to the response in V583-BB than to that in V583-SDS, suggesting that the presence of bile suppresses the effects of SDS in V583-BB-SDS. The overall results presented here indicate that different mechanisms are involved in detergent resistance in E. faecalis.

Transcriptional response of Enterococcus faecalis V583 to erythromycin.

A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is presented. This is the first study describing a complete transcriptional profile of Enterococcus. E. faecalis is a common and nonvirulent bacterium in many natural environments, but also an important cause of nosocomial infections. We have used a genome-wide microarray based on the genome sequence of V583 to study gene expression in cells exposed to erythromycin. V583 is resistant to relatively high concentrations of erythromycin, but growth is retarded by the treatment. The effect of erythromycin treatment on V583 was studied by a time course experiment; samples were extracted at five time points over a period of 90 min. A drastic change in gene transcription was seen with the erythromycin-treated cells compared to the untreated cells. Altogether, 260 genes were down-regulated at one or more time points, while 340 genes were up-regulated. Genes encoding hypothetical proteins and genes encoding transport and binding proteins were the two most dominating groups of differentially expressed genes. The gene encoding ermB (EFA0007) was expressed, but not differentially, which indicated that other genes are important for the survival and growth maintenance of V583 treated with erythromycin. One of these genes is a putative MsrC-like protein, which was up-regulated at all time points studied. Other specific genes that were found to be up-regulated were genes encoding ABC transporters and two-component regulatory systems, and these may be genes that are important for the specific response of V583 to erythromycin.

Germinating fission yeast spores delay in G1 in response to UV irradiation.

BACKGROUND: Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified. RESULTS: We have studied the response of germinating Schizosaccharomyces pombe spores to UV irradiation in G1. When germinating spores are irradiated in early G1 phase, entry into S phase is delayed. We argue that the observed delay is caused by two separate mechanisms. The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation. Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast. We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation. The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint. If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation. CONCLUSIONS: We have studied the response of germinating S. pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses. Our results point to a mechanism delaying expression of proteins required for S phase entry.