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This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by serRK , which is believed to control the expression of the ser 2003 locus in Bifidobacterium breve UCC2003. The ser 2003 locus consists of two genes, Bbr_1319 ( sagA ) and Bbr_1320 ( serU ), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region of ser 2003 , and the probable recognition sequence of SerR was determined by a combinatorial approach of in vitro site-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of the serRK 2CRS in the response of B. breve to protease-mediated induction was confirmed by generating a B. breve serR insertion mutant, which was shown to exhibit altered ser 2003 transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of a B. breve serU mutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

Bifidobacteria have attracted significant scientific attention due to their perceived role as health-promoting microorganisms, although the genetics of the bacterial group is still underexplored. In this study, we investigated the transcriptome of Bifidobacterium bifidum PRL2010 during in vitro growth by microarray technology. When B. bifidum PRL2010 was grown in liquid broth, 425 of the 1,644 PRL2010 genes represented on the array were expressed in at least one of the three investigated growth phases, i.e., the lag, exponential, and stationary phases. These transcriptional analyses identified a core in vitro transcriptome encompassing 150 genes that are expressed in all phases. A proportion of these genes were further investigated as potential reference genes by quantitative real-time reverse transcription-PCR (qRT-PCR) assays. Their expression stability was evaluated under different growth conditions, which included cultivation on different carbon sources, exposure to environmental stresses (thermal, acidic, and osmotic), and growth phases. Our analyses validated six reference genes suitable for normalizing mRNA expression levels in qRT-PCR experiments applied to bifidobacteria.

We have investigated the occurrence of bifidobacteria in human milk samples, and we provide evidence regarding the predominance of members of the Bifidobacterium breve species in this environment. Moreover, evaluation of the growth capabilities and transcriptomic analyses of one representative isolate of this species, i.e., B. breve 4L, on different milk types were performed.

Whole-genome sequencing efforts have revolutionized the study of bifidobacterial genetics and physiology. Unfortunately, the sequence of a single genome does not provide information on bifidobacterial genetic diversity and on how genetic variability supports improved adaptation of these bacteria to the environment of the human gastrointestinal tract (GIT). Analysis of nine genomes from bifidobacterial species showed that such genomes display an open pan-genome structure. Mathematical extrapolation of the data indicates that the genome reservoir available to the bifidobacterial pan-genome consists of more than 5000 genes, many of which are uncharacterized, but which are probably important to provide adaptive abilities pertinent to the human GIT. We also define a core bifidobacterial gene set which will undoubtedly provide a new baseline from which one can examine the evolution of bifidobacteria. Phylogenetic investigation performed on a total of 506 orthologues that are common to nine complete bifidobacterial genomes allowed the construction of aBifidobacteriumsupertree which is largely concordant with the phylogenetic tree obtained using 16S rRNA genes. Moreover, this supertree provided a more robust phylogenetic resolution than the 16S rRNA gene-based analysis. This comparative study of the genusBifidobacteriumthus presents a foundation for future functional analyses of this important group of GIT bacteria.

The genomes of 22 putative prophages (bifidoprophages), previously identified in bifidobacterial genomes, were analyzed to detect the presence and organization of functional modules. Bifidoprophages were shown to display a classical modular genomic organization in which the DNA lysogeny module and the DNA packaging regions are the most highly conserved. Furthermore, single phage gene as well as multiple phage gene-based phylogenetic analyses clearly revealed the chimeric make-up of the genomes of bifidoprophages.

Although the health-promoting roles of bifidobacteria are widely accepted, the diversity of bifidobacteria among the human intestinal microbiota is still poorly understood. We performed a census of bifidobacterial populations from human intestinal mucosal and fecal samples by plating them on selective medium, coupled with molecular analysis of selected rRNA gene sequences (16S rRNA gene and internally transcribed spacer [ITS] 16S-23S spacer sequences) of isolated colonies. A total of 900 isolates were collected, of which 704 were shown to belong to bifidobacteria. Analyses showed that the culturable bifidobacterial population from intestinal and fecal samples include six main phylogenetic taxa, i.e., Bifidobacterium longum , Bifidobacterium pseudocatenulatum , Bifidobacterium adolescentis , Bifidobacterium pseudolongum , Bifidobacterium breve , and Bifidobacterium bifidum , and two species mostly detected in fecal samples, i.e., Bifidobacterium dentium and Bifidobacterium animalis subp. lactis . Analysis of bifidobacterial distribution based on age of the subject revealed that certain identified bifidobacterial species were exclusively present in the adult human gut microbiota whereas others were found to be widely distributed. We encountered significant intersubject variability and composition differences between fecal and mucosa-adherent bifidobacterial communities. In contrast, a modest diversification of bifidobacterial populations was noticed between different intestinal regions within the same individual (intrasubject variability). Notably, a small number of bifidobacterial isolates were shown to display a wide ecological distribution, thus suggesting that they possess a broad colonization capacity.

This chapter discusses biology of bifidobacteria for probiotic and replacement therapy. Bifidobacteria were originally isolated from a breast-fed infant. Since then several species have been identified in the gastrointestinal tract (GIT) of mammals and insects. The availability of whole-genome sequences from bifidobacteria is extremely informative for the understanding of the processes underlying speciation and evolution in this genus as well as the adaptation to its specific habitat (e.g., the human intestine). Since bifidobacteria encounter a vast array of substrates in the GIT, information regarding their metabolic properties is crucial to formulate an optimal growth medium. Prebiotics are food substances which resist digestion in the proximal GIT and reach intact the distal region of the gut, where, besides their direct physiological effect, they also affect the GIT ecosystem by specifically stimulating the growth and activity of the probiotic components of the GIT microbiota (e.g., bifidobacteria), thereby eliciting a beneficial effect on the host’s health. Prebiotics are typically oligosaccharides that consist of a mixture of hexose oligomers with a variable extent of polymerization. The transmission of novel genes through horizontal gene transfer (HGT) is generally supported by vectors such as bacteriophages or plasmids which may become integrated into the bacterial chromosome. Generally, plasmids are rarely found in bifidobacteria. So far the majority of bifidobacterial plasmids have been identified within different B. longum biotype longum strains. The chapter ends with a discussion on taxonomy of bifidobacteria.

Turroni, Francesca et al., Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity., This work was supported in part by Fondazione Cariplo Grant 2010-0678 (to S.G. and V.T.); Irish Research Council for Science, Engineering, and Technology Embark postdoctoral Fellowship (to F.T.); and by PhD Fellowship Spinner 2013, Regione Emilia Romagna (to S.D.) and a Heath Research Board grant (to M.O.M.). D.v.S., M.O.M., and F.T. are members of The Alimentary Pharmabiotic Centre; D.v.S. is also a member of the Alimentary Glycoscience Research Cluster, both funded by Science Foundation Ireland through the Irish Government’s National Development Plan (Grant numbers 07/CE/B1368 and 08/SRC/B1393, respectively). M.O.M. is the recipient of a Health Research Board postdoctoral Fellowship Grant PDTM/20011/9.

Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group.

Serafini, Fausta et al., Bifidobacteria are extensively exploited by the food industry as health-promoting microorganisms. However, very little is known about the molecular mechanisms responsible for these beneficial activities, or the molecular players that sustain their ability to colonize and persist within the human gut. Here, we have investigated the enteric adaptation features of the gut commensal Bifidobacterium bifidum PRL2010, originally isolated from infant feces. This strain was able to survive under gastrointestinal challenges, while it was shown to adhere to human epithelial intestinal cell monolayers (Caco 2 and HT-29), thereby inhibiting adhesion of pathogenic bacteria such as Escherichia coli and Cronobacter sakazakii. © 2013 Elsevier Ltd., This work was also financially supported by Fondazione Cariparma to MV, by FEMS Advanced Fellowship 2011 and an IRCSET Embark postdoctoral fellowship to FT. DvS is a member of The Alimentary Pharmabiotic Centre, which is a Centre for Science and Technology (CSET) funded by the Science Foundation Ireland (SFI), through the Irish Government's National Development Plan (Grant no. 02/CE/B124 and 07/CE/B1368). The grant I-LINK2010-0122, funded by CSIC, is also acknowledged.

Turroni Francesca, Francesca et al., [Background] The human gastrointestinal tract (GIT) represents one of the most densely populated microbial ecosystems studied to date. Although this microbial consortium has been recognized to have a crucial impact on human health, its precise composition is still subject to intense investigation. Among the GIT microbiota, bifidobacteria represent an important commensal group, being among the first microbial colonizers of the gut. However, the prevalence and diversity of members of the genus Bifidobacterium in the infant intestinal microbiota has not yet been fully characterized, while some inconsistencies exist in literature regarding the abundance of this genus., [Methods/Principal Findings] In the current report, we assessed the complexity of the infant intestinal bifidobacterial population by analysis of pyrosequencing data of PCR amplicons derived from two hypervariable regions of the 16 S rRNA gene. Eleven faecal samples were collected from healthy infants of different geographical origins (Italy, Spain or Ireland), feeding type (breast milk or formula) and mode of delivery (vaginal or caesarean delivery), while in four cases, faecal samples of corresponding mothers were also analyzed., [Conclusions] In contrast to several previously published culture-independent studies, our analysis revealed a predominance of bifidobacteria in the infant gut as well as a profile of co-occurrence of bifidobacterial species in the infant’s intestine., This work was financially supported by the Italian Award for Outstanding Young Researcher scheme “Incentivazione alla mobilità di studiosi stranieri e italiani residenti all’estero” and by the Cariparma Bank Foundation to MV, by Spinner 2013, Regione Emilia Romagna and EF. This work was also financially supported by the FIRB-MIUR NG-LAB Research Program, Project RBLA03ER38_004 to GDB, by a FEMS Advanced Fellowship 2011 and an IRCSET Embark postdoctoral fellowship to FT. DvS and PO are members of The Alimentary Pharmabiotic Centre, which is a Centre for Science and Technology (CSET) funded by the Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant no. 02/CE/B124 and 07/CE/B1368).

The Bifidobacterium bifidum PRL2010 genome encodes a relatively small set of predicted carbohydrate transporters. Growth experiments and transcriptome analyses of B. bifidum PRL2010 revealed that carbohydrate utilization in this microorganism appears to be restricted to a relatively low number of carbohydrates.

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis , Bifidobacterium catenulatum , and Bifidobacterium pseudocatenulatum , by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays.

Botaccini, Francesca et al., Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notion that respiration is a common metabolic feature of this ancient bifidobacterial species, which has been lost in currently known mammal-derived Bifidobacterium species. In fact, phylogenomic based analyses suggested an ancient origin of B. asteroides and indicates it as an ancestor of the genus Bifidobacterium. Furthermore, the B. asteroides PRL2011 genome encodes various enzymes for coping with toxic products that arise as a result of oxygen-mediated respiration. © 2012 Bottacini et al., This work was financially supported by the Cariparma Bank Foundation to MV. This work was also financially supported by a Federation European Microbiology society (FEMS) Advanced Fellowship 2011 and an Irish Research Council for Science, Engineering & Technology (IRCSET) Embark postdoctoral fellowship to FT. DV is a member of The Alimentary Pharmabiotic Centre and the Alimentary Glycoscience Research Cluster, both funded by Science Foundation Ireland (SFI), through the Irish Government's National Development Plan (grant nos. 07/CE/B1368 and 08/SRC/B1393, respectively).

Mupirocin is an antibiotic commonly used in selective media for the isolation of bifidobacteria. However, little is known about the genetic traits responsible for bifidobacterial resistance to mupirocin. Our investigation demonstrates that all of the bifidobacteria tested exhibit a phenotype of generally high resistance to this antibiotic. The genotypic reason for bifidobacterial mupirocin resistance was further characterized by sequencing of the isoleucyl-tRNA synthetase gene ( ileS ) coupled with three-dimensional modeling of the encoded protein and cloning of the ileS gene of Bifidobacterium bifidum PRL2010 in a mupirocin-sensitive Escherichia coli strain. These analyses revealed key amino acid residues of the IleS protein that apparently are crucial for conferring a mupirocin resistance phenotype to bifidobacteria.

Turroni, Francesca et al., The human intestine is densely populated by a microbial consortium whose metabolic activities are influenced by, among others, bifidobacteria. However, the genetic basis of adaptation of bifidobacteria to the human gut is poorly understood. Analysis of the 2,214,650-bp genome of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a nutrient-acquisition strategy that targets host-derived glycans, such as those present in mucin. Proteome and transcriptome profiling revealed a set of chromosomal loci responsible for mucin metabolism that appear to be under common transcriptional control and with predicted functions that allow degradation of various O-linked glycans in mucin. Conservation of the latter gene clusters in various B. bifidum strains supports the notion that host-derived glycan catabolism is an important colonization factor for B. bifidum with concomitant impact on intestinal microbiota ecology., This work was supported by National Institutes of Health–National Institute of Child Health and Human Development Grant 1R01HD061923 (to D.M.). D.v.S., A.Z., and F.B. are members of the Alimentary Pharmabiotic Centre, a Center for Science and Technology funded by Science Foundation Ireland, through the Irish Government’s National Development Plan Grant 07/CE/B1368.

Members of the serpin ( ser ine p rotease in hibitor) superfamily have been identified in higher multicellular eukaryotes, as well as in bacteria, although examination of available genome sequences has indicated that homologs of the bacterial serpin-encoding gene ( ser ) are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least 5, and perhaps up to 9, of the 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria produce serpins that form a separate clade. We characterized the ser 210B locus of Bifidobacterium breve 210B, which encompasses a number of genes whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, microarray, reverse transcription-PCR (RT-PCR), and quantitative real-time PCR (qRT-PCR) analyses revealed that a 3.5-kb polycistronic mRNA encompassing the ser 210B operon with a single transcriptional start site is strongly induced following treatment of B. breve 210B cultures with some proteases. Interestingly, transcription of other bifidobacterial ser homologs appears to be triggered by different proteases.

So far, only scarce and rather diffuse information is available on bacteriophages infecting members of the genus Bifidobacterium . In the current study, we investigated the genetic organization, phylogenetic relationships, and, in some cases, transcription profiles and inducibility of 19 prophage-like elements present on the individual chromosomes of nine bifidobacterial strains, which represent six different species.

One of the most complex microbial ecosystems is represented by the microbiota of the human gastrointestinal tract (GIT). Although this microbial consortium has been recognized to have a crucial effect on human health, its precise composition is still not fully established. Among the GIT bacteria, bifidobacteria represent an important commensal group whose presence is often associated with health-promoting effects. In this work, we assessed the complexity of the human intestinal bifidobacterial population by analysing the diversity of several 16S rRNA gene-based libraries. These analyses showed the presence of novel bifidobacterial phylotypes, which had not been found earlier and may thus represent novel taxa within the genus Bifidobacterium.

The human gut is the home of an estimated 10(18) bacterial cells, many of which are uncharacterized or unculturable. Novel culture-independent approaches have revealed that the majority of the human gut microbiota consists of members of the phyla Bacteroidetes and Firmicutes. Nevertheless the role of bifidobacteria in gut ecology illustrates the importance of Actinomycetes and other Actinobacteria that may be underestimated. The human gut microbiota represents an extremely complex microbial community the collective genome of which, the microbiome, encodes functions that are believed to have a significant impact on human physiology. The microbiome is assumed to significantly enhance the metabolism of amino and glycan acids, the turnover of xenobiotics, methanogenesis and the biosynthesis of vitamins. Co-colonisation of the gut commensals Bifidobacterium longum and Bacteroides thetaiotaomicron in a murine model system revealed that the presence of bifidobacteria induced an expansion in the diversity of polysaccharides targeted for degradation by Bacteroides and also induced host genes involved in innate immunity. In addition, comparative analysis of individual human gut microbiomes has revealed various strategies that the microbiota use to adapt to the intestinal environment while also pointing to the existence of a distinct infant and adult-type microbiota.

In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages.

Bifidobacterium animalis subsp. lactis BLC1 is a probiotic bacterium that is widely exploited by food industries as the active ingredient of various functional foods. Here we report the complete genome sequence of B. animalis subsp. lactis BLC1, which is expected to provide insights into the biology of this health-promoting microorganism and improve our understanding of its phylogenetic relatedness with other members of the B. animalis subsp. lactis taxon.

Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.

Background Cell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria. Results and discussion In this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fim A or fim P) together with one or two minor pilin subunit-encoding genes (designated as fim B and/or fim Q), and a gene encoding a sortase enzyme (str A). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fim A/P and fim B/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.