Phageome Analysis of Bifidobacteria-Rich Samples.

Bifidobacteria are important early colonizers of the human intestinal tract. The relative abundance of bifidobacterial species may be modulated, in part, by bacteriophage activity. Metagenomic studies of these populations is a crucial step in understanding this important interaction. This chapter outlines the technical instructions required to analyze the virome of a bifidobacteria-rich sample, for example, an infant fecal sample.

Honey bees harbor a diverse gut virome engaging in nested strain-level interactions with the microbiota.

The honey bee gut microbiota influences bee health and has become an important model to study the ecology and evolution of microbiota-host interactions. Yet, little is known about the phage community associated with the bee gut, despite its potential to modulate bacterial diversity or to govern important symbiotic functions. Here we analyzed two metagenomes derived from virus-like particles, analyzed the prevalence of the identified phages across 73 bacterial metagenomes from individual bees, and tested the host range of isolated phages. Our results show that the honey bee gut virome is composed of at least 118 distinct clusters corresponding to both temperate and lytic phages and representing novel genera with a large repertoire of unknown gene functions. We find that the phage community is prevalent in honey bees across space and time and targets the core members of the bee gut microbiota. The large number and high genetic diversity of the viral clusters seems to mirror the high extent of strain-level diversity in the bee gut microbiota. We isolated eight lytic phages that target the core microbiota member Bifidobacterium asteroides, but that exhibited different host ranges at the strain level, resulting in a nested interaction network of coexisting phages and bacterial strains. Collectively, our results show that the honey bee gut virome consists of a complex and diverse phage community that likely plays an important role in regulating strain-level diversity in the bee gut and that holds promise as an experimental model to study bacteria-phage dynamics in natural microbial communities.

Genome analysis of the temperate bacteriophage PMBT6 residing in the genome of Bifidobacterium thermophilum MBT94004.

The Siphoviridae phage PMBT6 was identified by transmission electron microscopy in the supernatant of Bifidobacterium thermophilum MBT94004 bioreactor fermentation culture, where it occurred at a moderately high titer. Genome analysis of the bacterial DNA confirmed the presence of this prophage within the genome of the lysogenic host. Under laboratory conditions, the prophage could not be induced by mitomycin C, ultraviolet C irradiation or hydrogen peroxide, suggesting that the prophage was released by spontaneous induction under (yet unknown) bioreactor conditions. Genome sequencing of the virion resulted in a linear, double-stranded DNA molecule of 36,561 bp with a mol% G + C content of 61.7 and 61 predicted open reading frames with low similarity to other Bifidobacterium spp. genomes, confirming that PMBT6 represents a novel temperate phage for this genus.

Alterations in the diversity and composition of mice gut microbiota by lytic or temperate gut phage treatment.

Phages, the most abundant species in the mammalian gut, have numerous advantages as biocontrol agent over antibiotics. In this study, mice were orally treated with the lytic gut phage PA13076 (group B), the temperate phage BP96115 (group C), no phage (group A), or streptomycin (group D) over 31 days. At the end of the experiment, fecal microbiota diversity and composition was determined and compared using high-throughput sequencing of the V3-V4 hyper-variable region of the 16S rRNA gene and virus-like particles (VLPs) were quantified in feces. There was high diversity and richness of microbiota in the lytic and temperate gut phage-treated mice, with the lytic gut phage causing an increased alpha diversity based on the Chao1 index (p < 0.01). However, the streptomycin treatment reduced the microbiota diversity and richness (p = 0.0299). Both phage and streptomycin treatments reduced the abundance of Bacteroidetes at the phylum level (p < 0.01) and increased the abundance of the phylum Firmicutes. Interestingly, two beneficial genera, Lactobacillus and Bifidobacterium, were enhanced by treatment with the lytic and temperate gut phage. The abundance of the genus Escherichia/Shigella was higher in mice after temperate phage administration than in the control group (p < 0.01), but lower than in the streptomycin group. Moreover, streptomycin treatment increased the abundance of the genera Klebsiella and Escherichia/Shigella (p < 0.01). In terms of the gut virome, fecal VLPs did not change significantly after phage treatment. This study showed that lytic and temperate gut phage treatment modulated the composition and diversity of gut microbiota and the lytic gut phage promoted a beneficial gut ecosystem, while the temperate phage may promote conditions enabling diseases to occur.

Characterization and induction of prophages in human gut-associated Bifidobacterium hosts.

In the current report, we describe the identification of three genetically distinct groups of prophages integrated into three different chromosomal sites of human gut-associated Bifidobacterium breve and Bifidobacterium longum strains. These bifidobacterial prophages are distantly related to temperate actinobacteriophages of several hosts. Some prophages, integrated within the dnaJ2 gene, are competent for induction, excision, replication, assembly and lysis, suggesting that they are fully functional and can generate infectious particles, even though permissive hosts have not yet been identified. Interestingly, several of these phages harbor a putative phase variation shufflon (the Rin system) that generates variation of the tail-associated receptor binding protein (RBP). Unlike the analogous coliphage-associated shufflon Min, or simpler Cin and Gin inversion systems, Rin is predicted to use a tyrosine recombinase to promote inversion, the first reported phage-encoded tyrosine-family DNA invertase. The identification of bifidobacterial prophages with RBP diversification systems that are competent for assembly and lysis, yet fail to propagate lytically under laboratory conditions, suggests dynamic evolution of bifidobacteria and their phages in the human gut.

Impact of gut-associated bifidobacteria and their phages on health: two sides of the same coin?

Bifidobacteria are among the first microbial colonisers of the human infant gut post-partum. Their early appearance and dominance in the human infant gut and the reported health-promoting or probiotic status of several bifidobacterial strains has culminated in intensive research efforts that focus on their activities as part of the gut microbiota and the concomitant implications for human health. In this mini-review, we evaluate current knowledge on the genomics of this diverse bacterial genus, and on the genetic and functional adaptations that have underpinned the success of bifidobacteria in colonising the infant gut. The growing interest in functional genomics of bifidobacteria has also created interest in the interactions of bifidobacteria and their (bacterio)phages. While virulent phages of bifidobacteria have yet to be isolated, the incidence of integrated (pro)phages in bifidobacterial genomes are widely reported and this mini-review considers the role of these so-called bifidoprophages in modulating bifidobacterial populations in the human gastrointestinal tract and the implications for existing and future development of probiotic therapies.

Maternal inheritance of bifidobacterial communities and bifidophages in infants through vertical transmission.

BACKGROUND: The correct establishment of the human gut microbiota represents a crucial development that commences at birth. Different hypotheses propose that the infant gut microbiota is derived from, among other sources, the mother's fecal/vaginal microbiota and human milk. RESULTS: The composition of bifidobacterial communities of 25 mother-infant pairs was investigated based on an internal transcribed spacer (ITS) approach, combined with cultivation-mediated and genomic analyses. We identified bifidobacterial strains/communities that are shared between mothers and their corresponding newborns. Notably, genomic analyses together with growth profiling assays revealed that bifidobacterial strains that had been isolated from human milk are genetically adapted to utilize human milk glycans. In addition, we identified particular bacteriophages specific of bifidobacterial species that are common in the viromes of mother and corresponding child. CONCLUSIONS: This study highlights the transmission of bifidobacterial communities from the mother to her child and implies human milk as a potential vehicle to facilitate this acquisition. Furthermore, these data represent the first example of maternal inheritance of bifidobacterial phages, also known as bifidophages in infants following a vertical transmission route.

Prophages of the genus Bifidobacterium as modulating agents of the infant gut microbiota.

Phage predation is one of the key forces that shape genetic diversity in bacterial genomes. Phages are also believed to act as modulators of the microbiota composition and, consequently, as agents that drive bacterial speciation in complex bacterial communities. Very little is known about the occurrence and genetic variability of (pro)phages within the Bifidobacterium genus, a dominant bacterial group of the human infant microbiota. Here, we performed cataloguing of the predicted prophage sequences from the genomes of all currently recognized bifidobacterial type strains. We analysed their genetic diversity and deduced their evolutionary development, thereby highlighting an intriguing origin. Furthermore, we assessed infant gut microbiomes for the presence of (pro)phage sequences and found compelling evidence that these viral elements influence the composition of bifidobacterial communities in the infant gut microbiota.

Analyses of bifidobacterial prophage-like sequences.

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.

Comparative analyses of prophage-like elements present in bifidobacterial genomes.

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.

[Bifidobacterial plasmids and their application to genetic engineering].

Representatives of Bifidobacterium genus are considered to play many important roles in intestinal homeostasis. On the other hand, their molecular biology and genetics have been poorly studied. In order to broaden our understanding of their health-promoting mechanisms, it is extremely important to possess tools to manipulate them genetically. Another challenging task is to take advantage of genetic engineering technology for designing new probiotic bifidobacteria with unique therapeutic properties. An important step in such work is to isolate and characterize small bifidobacterial plasmids, which can be applied to the construction of cloning vectors. This article presents a review of several pioneering studied devoted to bifidobacterial plasmids and genetic engineering with bifidobacteria. Trends in and prospects of molecular genetics of bifidobacteria are discussed as well.

A novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria.

As shown in many intervention studies, probiotic bacteria can have a beneficial effect on rotavirus and HIV-induced diarrhoea. In spite of that fact, antiviral effects of probiotic bacteria have not been systematically studied yet. Non-tumorigenic porcine intestinal epithelial cells (IPEC-J2) and alveolar macrophages (3D4/2) were treated in different experimental designs with probiotic and other lactic bacteria and their metabolic products. Vesicular stomatitis virus (VSV) was used in the study as a model virus. Cell survival and viral inhibition were determined by antiviral assay and confirmed by immunofluorescence. Pre-incubation of cell monolayers with probiotic bacteria reduced viral infectivity up to 60%. All bacteria used prevented VSV binding to the cell monolayers by direct binding of VSV to their surface. Probiotic and other lactic bacteria prevented viral infection also by establishment of the antiviral state in pre-treated cell monolayers. Probiotic and other lactic bacteria secreted antiviral substances during their growth, as the infectivity of the virus was diminished by 68% when bacterial supernatants were tested. It was shown for the first time that probiotic and other lactic bacteria exhibit an antiviral activity in a cell culture model. Possible mechanisms of antiviral activity include: 1) hindering the adsorption and cell internalisation of the VSV due to the direct trapping of the virus by the bacteria, 2) "cross-talk" with the cells in establishing the antiviral protection and 3) production of metabolites with a direct antiviral effect.

Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis.

So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus Bifidobacterium. In this report we analyzed three prophage-like elements that are present in the genomes of Bifidobacterium breve UCC 2003, Bifidobacterium longum NCC 2705, and Bifidobacterium longum DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNA(Met) gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the attB sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a B. longum DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among B. breve strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.

Protection against influenza virus infection of mice fed Bifidobacterium breve YIT4064.

Mice fed Bifidobacterium breve YIT4064 and immunized orally with influenza virus were more strongly protected against influenza virus infection of the lower respiratory tract than ones immunized with influenza virus only. The number of mice with enhanced anti-influenza virus immunoglobulin G (IgG) in serum upon oral administration of B. breve YIT4064 and oral immunization with influenza virus was significantly greater than that upon oral immunization with influenza virus only. These findings demonstrated that the oral administration of B. breve YIT4064 increased anti-influenza virus IgG antibodies in serum and protected against influenza virus infection. The oral administration of B. breve YIT4064 may enhance antigen-specific IgG against various pathogenic antigens taken orally and induce protection against various virus infections.

Reduction of virus shedding by B. bifidum in experimentally induced MRV infection. Statistical application for ELISA.

The protective effect of a human strain of Bifidobacterium bifidum (B. bifidum) against murine group A rotavirus (MRV) was examined in the intestines of BALB/c infected mice. In experiments designed to determine whether B. bifidum mediated MRV shedding during diarrheal disease, pregnant dams (and their expected litters) were randomly assigned to the following groups: (1) mice infected with MRV alone; (2) B. bifidum-treated + MRV-infected mice; (3) B. bifidum-treated controls; and (4) saline control animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of group A rotavirus was used to measure virus protein. The sensitivity of the MRV antigen detector ELISA was determined by serially diluting the rotavirus antigen in test samples. Antigen was detected in dilution ranges of 1:256-1:4096 during the acute phase and 1:16-1:512 in the recovery phase of MRV clinical disease, in the samples tested. Treatment with B. bifidum significantly reduced shedding of MRV antigen (P < 0.009) on days 2-10 postinoculation. The reduction in shedding of virus protein corresponded well with delayed onset of acute diarrhea (P < 0.02). Closer examination of tissue cross sections under electron microscopy revealed that the B. bifidum-ingested strain adhered to the epithelium of the small intestine. These results suggest that priming the intestine with B. bifidum is effective against experimental MRV challenge and confirmed the potential usefulness of this detector ELISA for studying the kinetics of group A rotavirus infection in animals and humans.