The majority of the bacterial genome sequences deposited in the National Center for Biotechnology Information database contain prophage sequences. Analysis of the prophages suggested that after being integrated into bacterial genomes, they undergo a complex decay process consisting of inactivating point mutations, genome rearrangements, modular exchanges, invasion by further mobile DNA elements, and massive DNA deletion. We review the technical difficulties in defining such altered prophage sequences in bacterial genomes and discuss theoretical frameworks for the phage-bacterium interaction at the genomic level. The published genome sequences from three groups of eubacteria (low- and high-G+C gram-positive bacteria and γ-proteobacteria) were screened for prophage sequences. The prophages from Streptococcus pyogenes served as test case for theoretical predictions of the role of prophages in the evolution of pathogenic bacteria. The genomes from further human, animal, and plant pathogens, as well as commensal and free-living bacteria, were included in the analysis to see whether the same principles of prophage genomics apply for bacteria living in different ecological niches and coming from distinct phylogenetical affinities. The effect of selection pressure on the host bacterium is apparently an important force shaping the prophage genomes in low-G+C gram-positive bacteria and γ-proteobacteria

Nearly 100 years ago, Felix d'Herelle, the codiscoverer of bacteriophages, used bacteria to control insect pests and used phages against bacterial disease. His approaches reflected ecological insights before this branch of biology became an established scientific discipline. In fact, one might have predicted that phage research would become the springboard for biotechnology and ecology. However, d'Herelle was ahead of his time, and the zeitgeist in the 1930s pushed physicists into the question "What is life?" Phages as the simplest biological systems were the logical choice for this question, and phage research became the cradle of molecular biology. Now many researchers speak of a "new age of phage research." It is now realized that phages play an important role in ecology (e.g., phage impact on the cycling of organic matter in the biosphere at a global level) (27), that phages influence the evolution of bacterial genomes (most obviously in the development of bacterial pathogenicity) (7), and that phages might provide potential tools to face the antibiotic resistance crisis in medicine (59). With this new trend, we now see a clear shift from the reductionist approach, focusing on a handful of phages in carefully controlled laboratory conditions, towards the study of many different phages in the complexity of real-life situations.In contrast to the molecular biology-oriented phage research where the interaction of molecules took center stage, ecology focuses on the interactions between organisms and their physical environment. Much of ecology is therefore about the evolution of biological diversity in space and time. In contrast to many branches of biology, ecology attributes a great importance to quantitative relationships and numbers and aims at a mathematical formulation of its observations. It is thus appropriate to start this review with an overview of phage titers encountered in the biosphere. Next, we ask how a parasite targets its host if the latter is scarce or not in an appropriate physiological state. Finally, we report on research that tries to bridge phage ecology and genomics and cell biology approaches. It is concluded that the integration of phages into complex networks of interacting biological systems, and analysis by molecular techniques, could give phage research a model character in biology again.

Fifteen healthy adult volunteers received in their drinking water a lower Escherichia coli phage T4 dose (10 3 PFU/ml), a higher phage dose (10 5 PFU/ml), and placebo. Fecal coliphage was detected in a dose-dependent way in volunteers orally exposed to phage. All volunteers receiving the higher phage dose showed fecal phage 1 day after exposure; this prevalence was only 50% in subjects receiving the lower phage dose. No fecal phage was detectable a week after a 2-day course of oral phage application. Oral phage application did not cause a decrease in total fecal E. coli counts. In addition, no substantial phage T4 replication on the commensal E. coli population was observed. No adverse events related to phage application were reported. Serum transaminase levels remained in the normal range, and neither T4 phage nor T4-specific antibodies were observed in the serum of the subjects at the end of the study. This is, to our knowledge, the first safety test in the recent English literature which has measured the bioavailability of oral phage in humans and is thus a first step to the rational evaluation of phage therapy for diarrheal diseases.Antibiotic treatment of Escherichia coli diarrhea is frequently problematic, which raises interest in alternative approaches. Felix d'Hérelle, the codiscoverer of phages, advocated the idea of exploiting the lytic effect of phages on bacteria for therapeutic purposes. Phage therapy has a colorful history but became a common therapy for intestinal and skin infections only in the Soviet Union (17). Currently, we see a renewed interest in phage therapy (13). The present study describes the oral administration of phages to human volunteers and the subsequent clinical and microbiological analyses. This safety test is a follow-on from ecology studies of T4-like phages isolated from the stools of pediatric diarrhea patients (7), the analysis of their genomes (6), and their behavior in experimental animals (8). MATERIALS AND METHODS Subjects.Fifteen healthy adult volunteers between 23 and 54 years of age (six women and nine men) were recruited from the personnel at the Nestlé Research Center. Their heights ranged from 150 to 187 cm, and their weights ranged from 56 to 85 kg (body mass index range, 21.4 to 32.1 kg/m 2 ). All subjects were Caucasians. Exclusion criteria for enrollment were immunosuppression, gastric problems, raised serum transaminase levels, antibiotic treatment during the preceding 4 weeks, laxative use, pregnancy, and participation in other trials. The protocol was approved by the local ethical committee, and the participants provided written consent.Study design. The study was designed as a single-center, randomized, and placebo-controlled study. The trial was a double-blinded, three-period crossover comparison of two dosages of oral T4 phage conducted in June 2003 at our research center. Each subject received a higher phage dose (dose A, with 10 5 PFU/ml), a lower phage dose (dose B, with 10 3 PFU/ml), and placebo (dose C). The subjects were randomly assigned to one o...

The gut microbiota enhances the host’s metabolic capacity for processing nutrients and drugs and modulate the activities of multiple pathways in a variety of organ systems. We have probed the systemic metabolic adaptation to gut colonization for 20 days following exposure of axenic mice (n = 35) to a typical environmental microbial background using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy to analyze urine, plasma, liver, kidney, and colon (5 time points) metabolic profiles. Acquisition of the gut microbiota was associated with rapid increase in body weight (4%) over the first 5 days of colonization with parallel changes in multiple pathways in all compartments analyzed. The colonization process stimulated glycogenesis in the liver prior to triggering increases in hepatic triglyceride synthesis. These changes were associated with modifications of hepatic Cyp8b1 expression and the subsequent alteration of bile acid metabolites, including taurocholate and tauromuricholate, which are essential regulators of lipid absorption. Expression and activity of major drug-metabolizing enzymes (Cyp3a11 and Cyp2c29) were also significantly stimulated. Remarkably, statistical modeling of the interactions between hepatic metabolic profiles and microbial composition analyzed by 16S rRNA gene pyrosequencing revealed strong associations of the Coriobacteriaceae family with both the hepatic triglyceride, glucose, and glycogen levels and the metabolism of xenobiotics. These data demonstrate the importance of microbial activity in metabolic phenotype development, indicating that microbiota manipulation is a useful tool for beneficially modulating xenobiotic metabolism and pharmacokinetics in personalized health care.

The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3×10(9) and 3×10(7) plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.

Four T4-like coliphages with broad host ranges for diarrhea-associated Escherichia coli serotypes were isolated from stool specimens from pediatric diarrhea patients and from environmental water samples. All four phages showed a highly efficient gastrointestinal passage in adult mice when added to drinking water. Viable phages were recovered from the feces in a dose-dependent way. The minimal oral dose for consistent fecal recovery was as low as 10 3 PFU of phage per ml of drinking water. In conventional mice, the orally applied phage remained restricted to the gut lumen, and as expected for a noninvasive phage, no histopathological changes of the gut mucosa were detected in the phage-exposed animals. E. coli strains recently introduced into the intestines of conventional mice and traced as ampicillin-resistant colonies were efficiently lysed in vivo by phage added to the drinking water. Likewise, an in vitro phage-susceptible E. coli strain freshly inoculated into axenic mice was lysed in vivo by an orally applied phage, while an in vitro-resistant E. coli strain was not lysed. In contrast, the normal E. coli gut flora of conventional mice was only minimally affected by oral phage application despite the fact that in vitro the majority of the murine intestinal E. coli colonies were susceptible to the given phage cocktail. Apparently, the resident E. coli gut flora is physically or physiologically protected against phage infection.

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells.

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients.