Bifidobacteria on the spot: a genomics approach on population dynamaics and interactions in the intestinal tract

This thesis combines comprehensive microarray-based studies contributing to a better understanding of the role of bifidobacteria in relation to the human host. It reviews recently described modes of interaction between bifidobacteria and human gastrointestinal cells and highlights the unique characteristics of the genus Bifidobacterium that are indicative for its role in our gut. A microarray platform has been developed that enables genomic comparison of Bifidobacterium species originating from our gastrointestinal tract (GIT). Based on the obtained high-resolution data, species-unique genomic sequences could be identified. A large fraction of these predicted genes encode proteins belonging to the bifidobacterial glycobiome. An unique ability of the microarray platform is to zoom in on the strain level. Direct mapping of genomic hybridization patterns was applied on different B. breve isolates. This revealed a relatively high genomic variation, testifying for the existence of various subspecies within the species B. breve. Clustering of the same hybridization patterns resulted in clear grouping of isolates originating from the same infant, indicating specific niche adaption. Additionally, DNA extracts from Bifidobacterium populations from different infant fecal samples were analyzed. This enabled the analysis of the bifidobacterial population dynamics in breast- and formula-fed infants. The applied microarray platform showed the potential to monitor temporal development and effects of dietary regimens. The observed differences in the composition of bifidobacterial populations could be linked to dietary effects. Additionally, mapping of hybridization patterns enabled monitoring shifts in genomic content within one bifidobacterial species in time. Sequence analysis of DNA fragments showing discriminating hybridization characteristics, resulted in the selection of genes that are either conserved or strain-specific within the species B. breve. Next to studying genomic variation, transcript profiling experiments in both bifidobacterial cells and human intestinal epithelial cell lines were performed. Analysis of bifidobacterial transcriptional responses provided clear proof of transcriptional activity in bifidobacterial cells isolated from infant feces. To the best of our knowledge, this is the first demonstration of in situ activity of bifidobacteria in the human GIT. Furthermore, our results indicate a link between transcription patterns and the infants’ diet, as bifidobacteria in fecal samples from breast-fed infants showed differential transcriptional responses in comparison to those in fecal samples from formula-fed infants. Additionally, transcript sequence analysis revealed expression of genes that are homologous to genes known to be involved in folate production, testifying for the production of this important vitamin in early life. Finally, transcriptome analysis on human intestinal epithelial cells (HIECs) showed species-specific suppression by B. breve M-16V of genes upregulated by TNF-α. Other B. breve strains showed an extreme mild or no effect on TNF-α stimulation. Although we did not observe complete suppression of the TNF effect, we could show that apoptotic and immune regulatory pathways were affected by incubation with cells of B. breve M-16V. In conclusion, the work presented in the thesis, which formed part of a larger IOP Genomics project, contributed to an advanced insight in the interaction between bifidobacteria and the human host. Furthermore, it resulted in the development of genome-based molecular platforms suited for analyzing genomic diversity between and within species, as well as population dynamics in complex microbial communities. We anticipate that the molecular approaches pioneered in this thesis will be instrumental in the further elucidation of the host-microbe interactions in the GIT of human an other animals. <br/

Interactomics in the human intestine: Lactobacilli and Bifidobacteria make a difference.

Scientific evidence that supports a correlation between our intestinal microbiota and health status has caused significant interest in microbe-host interaction studies. It has generated a paradigm shift from analyzing pathogens to that involving commensal and probiotic bacteria. This review summarizes the interaction mechanisms described for Lactobacilli and Bifidobacteria based on recent omics-based developments. This information is expected to provide new avenues for further unravelling the set of interactions that includes the interactome of microbial and host cells

Analysis of infant isolates of Bifidobacterium breve by comparative genome hybridization indicates the existence of new subspecies with marked infant specificity

A total of 20 Bifidobacterium strains were isolated from fecal samples of 4 breast- and bottle-fed infants and all were characterized as Bifidobacterium breve based on 16S rRNA gene sequence and metabolic analysis. These isolates were further characterized and compared to the type strains of B. breve and 7 other Bifidobacterium spp. by comparative genome hybridization. For this purpose, we constructed and used a DNA-based microarray containing over 2000 randomly cloned DNA fragments from B. breve type strain LMG13208. This molecular analysis revealed a high degree of genomic variation between the isolated strains and allowed the vast majority to be grouped into 4 clusters. One cluster contained a single isolate that was virtually indistinguishable from the B. breve type strain. The 3 other clusters included 19 B. breve strains that differed considerably from all type strains. Remarkably, each of the 4 clusters included strains that were isolated from a single infant, indicating that a niche adaptation may contribute to variation within the B. breve species. Based on genomic hybridization data, the new B. breve isolates were estimated to contain approximately 60-90% of the genes of the B. breve type strain, attesting to the existence of various subspecies within the species B. breve. Further bioinformatic analysis identified several hundred diagnostic clones specific to the genomic clustering of the B. breve isolates. Molecular analysis of representatives of these revealed that annotated genes from the conserved B. breve core encoded mainly housekeeping functions, while the strain-specific genes were predicted to code for functions related to life style, such as carbohydrate metabolism and transport. This is compatible with genetic adaptation of the strains to their niche, a combination of infants and die

A Bifidobacterium mixed-species microarray for high resolution discrimination between intestinal bifidobacteria

A genomic DNA-based microarray was constructed containing over 6000 randomly cloned genomic fragments of approximately 1-2 kb from six mammalian intestinal Bifidobacterium spp. including B. adolescentis, B. animalis, B. bifidum, B. catenulatum, B. longum and B. pseudolongum. This Bifidobacterium Mixed-Species (BMS) microarray was used to differentiate between type strains and isolates belonging to a set of nine Bifidobacterium spp. Hierarchical clustering of genomic hybridization data confirmed the grouping of the Bifidobacterium spp. according to the 16S rRNA-based phylogenetic clusters. In addition, these genomic hybridization experiments revealed high homology between the type-strain B. animalis subsp. lactis LMG18314 and B. animalis subsp. animalis LMG10508 (79%) as well as between the type strains B. longum biotype longum LMG13197 and B. longum biotype infantis LMG8811 (72%) - nevertheless, discrimination between these species was possible due to the high resolution output of the BMS-array. In addition, it was shown that the BMS-array could be used for assigning unknown Bifidobacterium isolates to a species group. Finally, a set of 54 diagnostic clones for Bifidobacterium identification was selected and sequenced to advance the understanding of the species-related differences. Remarkably, a large fraction (31%) of these was predicted to encode proteins that belong to the bifidobacterial glycobiome and another 11% had functional homology with genes involved in the protection against foreign DNA. Overall, the BMS-microarray is a high-resolution diagnostic tool that is able to facilitate the detection of strain- and species-specific characteristics of bifidobacteria. © 2008 Elsevier B.V. All rights reserved

Analysis of infant isolates of Bifidobacterium breve by comparative genome hybridization indicates the existence of new subspecies with marked infant specificity

A total of 20 Bifidobacterium strains were isolated from fecal samples of 4 breast- and bottle-fed infants and all were characterized as Bifidobacterium breve based on 16S rRNA gene sequence and metabolic analysis. These isolates were further characterized and compared to the type strains of B. breve and 7 other Bifidobacterium spp. by comparative genome hybridization. For this purpose, we constructed and used a DNA-based microarray containing over 2000 randomly cloned DNA fragments from B. breve type strain LMG13208. This molecular analysis revealed a high degree of genomic variation between the isolated strains and allowed the vast majority to be grouped into 4 clusters. One cluster contained a single isolate that was virtually indistinguishable from the B. breve type strain. The 3 other clusters included 19 B. breve strains that differed considerably from all type strains. Remarkably, each of the 4 clusters included strains that were isolated from a single infant, indicating that a niche adaptation may contribute to variation within the B. breve species. Based on genomic hybridization data, the new B. breve isolates were estimated to contain approximately 60-90% of the genes of the B. breve type strain, attesting to the existence of various subspecies within the species B. breve. Further bioinformatic analysis identified several hundred diagnostic clones specific to the genomic clustering of the B. breve isolates. Molecular analysis of representatives of these revealed that annotated genes from the conserved B. breve core encoded mainly housekeeping functions, while the strain-specific genes were predicted to code for functions related to life style, such as carbohydrate metabolism and transport. This is compatible with genetic adaptation of the strains to their niche, a combination of infants and die

Interactomics in the human intestine: Lactobacilli and Bifidobacteria make a difference

Scientific evidence that supports a correlation between our intestinal microbiota and health status has caused significant interest in microbe-host interaction studies. It has generated a paradigm shift from analyzing pathogens to that involving commensal and probiotic bacteria. This review summarizes the interaction mechanisms described for Lactobacilli and Bifidobacteria based on recent omics-based developments. This information is expected to provide new avenues for further unravelling the set of interactions that includes the interactome of microbial and host cell

A Bifidobacterium mixed-species microarray for high resolution discrimination between intestinal bifidobacteria

A genomic DNA-based microarray was constructed containing over 6000 randomly cloned genomic fragments of approximately 1-2 kb from six mammalian intestinal Bifidobacterium spp. including B. adolescentis, B. animalis, B. bifidum, B. catenulatum, B. longum and B. pseudolongum. This Bifidobacterium Mixed-Species (BMS) microarray was used to differentiate between type strains and isolates belonging to a set of nine Bifidobacterium spp. Hierarchical clustering of genomic hybridization data confirmed the grouping of the Bifidobacterium spp. according to the 16S rRNA-based phylogenetic clusters. In addition, these genomic hybridization experiments revealed high homology between the type-strain B. animalis subsp. lactis LMG18314 and B. animalis subsp. animalis LMG10508 (79%) as well as between the type strains B. longum biotype longum LMG13197 and B. longum biotype infantis LMG8811 (72%) - nevertheless, discrimination between these species was possible due to the high resolution output of the BMS-array. In addition, it was shown that the BMS-array could be used for assigning unknown Bifidobacterium isolates to a species group. Finally, a set of 54 diagnostic clones for Bifidobacterium identification was selected and sequenced to advance the understanding of the species-related differences. Remarkably, a large fraction (31%) of these was predicted to encode proteins that belong to the bifidobacterial glycobiome and another 11% had functional homology with genes involved in the protection against foreign DNA. Overall, the BMS-microarray is a high-resolution diagnostic tool that is able to facilitate the detection of strain- and species-specific characteristics of bifidobacteri

Bifidobacterium population analysis in the infant gut by direct mapping of genomic hybridization patterns: potential for monitoring temporal development and effects of dietary regimens

A bifidobacterial mixed-species microarray platform was used in a proof-of-principle study to address the composition and development of bifidobacteria in DNA extracted from faecal samples. These were collected in a time-course of 2 years since birth and derived from human infants that were breastfed, standard formula-fed or received a prebiotic formula during their weaning period. A set of over 50 samples was analysed, testifying for the throughput of the designed platform for multiple genome hybridizations. The generated data revealed that faecal samples of breastfed infants contained a high abundance of genomic DNA homologous to Bifidobacterium breve. In contrast, faecal samples from standard formula-fed infants lacked detectable amounts of this B. breve DNA but contained genes with high similarity to B. longum. Remarkably, infants that received breastmilk and later a prebiotic formula consisting of a standard formula milk containing a mixture of specific galacto- and fructo-oligosaccharides, continued to harbour a B. breve-dominant faecal population. One infant that received standard formula in combination with the additional B. lactis Bb12 culture, contained significant amounts of faecal DNA belonging to Bb12 but only during the period of ingestion. The microarray platform showed sufficient sensitivity to analyse the B. breve group at the strain level. Overall, the B. breve populations observed in the faecal samples of the studied infants showed a stable composition over time and were unique per infant. In conclusion, our results show the applicability of comparative genome hybridization to study bifidobacterial populations in infant faecal samples without the use of any amplification ste

Bifidobacterium population analysis in the infant gut by direct mapping of genomic hybridization patterns: potential for monitoring temporal development and effects of dietary regimens

A bifidobacterial mixed-species microarray platform was used in a proof-of-principle study to address the composition and development of bifidobacteria in DNA extracted from faecal samples. These were collected in a time-course of 2 years since birth and derived from human infants that were breastfed, standard formula-fed or received a prebiotic formula during their weaning period. A set of over 50 samples was analysed, testifying for the throughput of the designed platform for multiple genome hybridizations. The generated data revealed that faecal samples of breastfed infants contained a high abundance of genomic DNA homologous to Bifidobacterium breve. In contrast, faecal samples from standard formula-fed infants lacked detectable amounts of this B. breve DNA but contained genes with high similarity to B. longum. Remarkably, infants that received breastmilk and later a prebiotic formula consisting of a standard formula milk containing a mixture of specific galacto- and fructo-oligosaccharides, continued to harbour a B. breve-dominant faecal population. One infant that received standard formula in combination with the additional B. lactis Bb12 culture, contained significant amounts of faecal DNA belonging to Bb12 but only during the period of ingestion. The microarray platform showed sufficient sensitivity to analyse the B. breve group at the strain level. Overall, the B. breve populations observed in the faecal samples of the studied infants showed a stable composition over time and were unique per infant. In conclusion, our results show the applicability of comparative genome hybridization to study bifidobacterial populations in infant faecal samples without the use of any amplification ste

Bifidobacterium breve - HT-29 cell line interaction: modulation of TNF-a induced gene expression

To provide insight in the molecular basis for intestinal host-microbe interactions, we determined the genome-wide transcriptional response of human intestinal epithelial cells following exposure to cells of Bifidobacterium breve. To select an appropriate test system reflecting inflammatory conditions, the responsiveness to TNF-a was compared in T84, Caco-2 and HT-29 cells. The highest TNF-a response was observed in HT-29 cells and this cell line was selected for exposure to the B. breve strains M-16V, NR246 and UCC2003. After one hour of bacterial pre-incubation followed by two hours of additional TNF-a stimulation, B. breve M-16V (86%), but to a much lesser extent strains NR246 (50%) or UCC2003 (32%), showed a strain-specific reduction of the HT-29 transcriptional response to the inflammatory treatment. The most important functional groups of genes that were transcriptionally suppressed by the presence of B. breve M-16V, were found to be involved in immune regulation and apoptotic processes. About 54% of the TNF-a induced genes were solely suppressed by the presence of B. breve M-16V. These included apoptosis-related cysteine protease caspase 7 (CASP7), interferon regulatory factor 3 (IRF3), amyloid beta (A4) precursor proteinbinding family A member 1 (APBA1), NADPH oxidase (NOX5), and leukemia inhibitory factor receptor (LIFR). The extracellular IL-8 concentration was determined by an immunological assay but did not change significantly, indicating that B. breve M-16V only partially modulates the TNF-a pathway. In conclusion, this study shows that B. breve strains modulate gene expression in HT-29 cells under inflammatory conditions in a strain-specific wa