Phages are the most abundant entity in the biosphere and outnumber bacteria by a factor of 10. Phage DNA may also constitute 20% of bacterial genomes; however, its role is ill defined. Here, we explore the impact of cryptic prophages on cell physiology by precisely deleting all nine prophage elements (166 kbp) using Escherichia coli. We find that cryptic prophages contribute significantly to resistance to sub-lethal concentrations of quinolone and β-lactam antibiotics primarily through proteins that inhibit cell division (for example, KilR of rac and DicB of Qin). Moreover, the prophages are beneficial for withstanding osmotic, oxidative and acid stresses, for increasing growth, and for influencing biofilm formation. Prophage CPS-53 proteins YfdK, YfdO and YfdS enhanced resistance to oxidative stress, prophages e14, CPS-53 and CP4-57 increased resistance to acid, and e14 and rac proteins increased early biofilm formation. Therefore, cryptic prophages provide multiple benefits to the host for surviving adverse environmental conditions.

Members of the genus Bifidobacterium can be found as components of the gastrointestinal microbiota, and are believed to play an important role in maintaining and promoting human health by eliciting a number of beneficial properties. Bifidobacteria can utilize a diverse range of dietary carbohydrates that escape degradation in the upper parts of the intestine, many of which are plantderived oligo-and polysaccharides. The gene content of a bifidobacterial genome reflects this apparent metabolic adaptation to a complex carbohydrate-rich gastrointestinal tract environment as it encodes a large number of predicted carbohydrate-modifying enzymes. Different bifidobacterial strains may possess different carbohydrate utilizing abilities, as established by a number of studies reviewed here. Carbohydrate-degrading activities described for bifidobacteria and their relevance to the deliberate enhancement of number and/or activity of bifidobacteria in the gut are also discussed in this review.

BackgroundRecurrent abdominal pain is a common and costly health‐care problem attributed, in part, to visceral hypersensitivity. Increasing evidence suggests that gut bacteria contribute to abdominal pain perception by modulating the microbiome‐gut‐brain axis. However, specific microbial signals remain poorly defined. γ‐aminobutyric acid (GABA) is a principal inhibitory neurotransmitter and a key regulator of abdominal and central pain perception from peripheral afferent neurons. Although gut bacteria are reported to produce GABA, it is not known whether the microbial‐derived neurotransmitter modulates abdominal pain.MethodsTo investigate the potential analgesic effects of microbial GABA, we performed daily oral administration of a specific Bifidobacterium strain (B. dentium ATCC 27678) in a rat fecal retention model of visceral hypersensitivity, and subsequently evaluated pain responses.Key ResultsWe demonstrate that commensal Bifidobacterium dentium produces GABA via enzymatic decarboxylation of glutamate by GadB. Daily oral administration of this specific Bifidobacterium (but not a gadB deficient) strain modulated sensory neuron activity in a rat fecal retention model of visceral hypersensitivity.Conclusions & InferencesThe functional significance of microbial‐derived GABA was demonstrated by gadB‐dependent desensitization of colonic afferents in a murine model of visceral hypersensitivity. Visceral pain modulation represents another potential health benefit attributed to bifidobacteria and other GABA‐producing species of the intestinal microbiome. Targeting GABAergic signals along this microbiome‐gut‐brain axis represents a new approach for the treatment of abdominal pain.

Cellodextrins, the incomplete hydrolysis products from insoluble cellulose, are accessible as a carbon source to certain members of the human gut microbiota, such as Bifidobacterium breve UCC2003. Transcription of the cldEFGC gene cluster of B. breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating this cluster in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this commensal. Moreover, our results suggest that transcription of the cld cluster is controlled by a LacI-type regulator encoded by cldR, located immediately upstream of cldE. Gel mobility shift assays using purified CldR His (produced by the incorporation of a His 12 -encoding sequence into the 3 end of the cldC gene) indicate that the cldEFGC promoter is subject to negative control by CldR His , which binds to two inverted repeats. Analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose, and cellopentaose, with cellotriose apparently representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 is, to the best of our knowledge, the first described bifidobacterial ␤-glucosidase exhibiting hydrolytic activity toward various cellodextrins.One of the dominant bacterial groups in the human and mammalian intestinal microbiota is represented by members of the genus Bifidobacterium (47, 48), which are high-GC-content Gram-positive, non-spore-forming, "bifid"-shaped, anaerobic bacteria. Specific bifidobacterial strains have been implicated in promoting host health through one or more beneficial activities, which include prevention of diarrhea, reduction of cholesterol levels, symptom alleviation of inflammatory bowel disease or irritable bowel syndrome, immunomodulation, anticarcinogenicity, easing of lactose intolerance, improving mineral adsorption, and production of vitamins (12,46). The mechanism(s) of probiotic action is largely unknown and thus merits further investigation of bifidobacteria, which includes research into its genomic content, genetics, biochemistry, and metabolism (52).Bifidobacterial growth and/or metabolism in the human gastrointestinal tract can be selectively stimulated by various dietary compounds, particularly by so-called prebiotic carbohydrates (22, 35). A prebiotic has been defined as "a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well being and health" (35). According to this definition, the potential prebiotic component must fulfill the following criteria: nondigestible by the host, fermentation by the intestinal microbiota, and selective stimulation of growth and activity of beneficial intestinal bacteria....

SummaryGrowth of Bifidobacterium breve UCC2003 on ribose leads to the transcriptional induction of the rbsACBDK gene cluster. Generation and phenotypic analysis of an rbsA insertion mutant established that the rbs gene cluster is essential for ribose utilization, and that its transcription is likely regulated by a LacI‐type regulator encoded by rbsR, located immediately upstream of rbsA. Gel mobility shift assays using purified RbsRHis indicate that the promoter upstream of rbsABCDK is negatively controlled by RbsRHis binding to an 18 bp inverted repeat and that RbsRHis binding activity is modulated by d‐ribose. The rbsK gene of the rbs operon of B. breve UCC2003 was shown to specify a ribokinase (EC 2.7.1.15), which specifically directs its phosphorylating activity towards d‐ribose, converting this pentose sugar to ribose‐5‐phosphate.

Two ␣-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the ␣-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His 12 sequence was incorporated (Agl1 His and Agl2 His , respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers-palatinose, trehalulose, turanose, and maltulose-while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1 His exhibits higher thermo and pH optima than Agl2 His . The two purified ␣-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.

Four novel heat-stable bacteriocin-like substances were found to be produced by Geobacillus stearothermophilus strains isolated from oil-wells in Lithuania. Geobacillus stearothermophilus 32A, 17, 30 and 31 strains were identified as producers of bacteriocins with bactericidal activity against closely related Geobacillus species and several pathogenic strains: Bacillus cereus DSM 12001 and Staphylococcus haemolyticus P903. The secretion of the analysed bacteriocins started during early logarithmic growth and dropped sharply after the culture entered the stationary phase of growth. The antimicrobial activity of the bacteriocins against sensitive indicator cells disappeared after treatment with proteolytic enzymes, indicating their proteinaceous nature. Bacteriocins were stable throughout the pH range between 4 and 10, and no loss in activity was noted following temperature exposures up to 100ºC. Direct detection of antibacterial activity on SDS-PAGE suggests that the inhibitory peptides have a molecular weight of 6 -7.5 kDa. Such bacteriocins with broad activity spectra, including antipathogenic action, are attractive to the biotechnology industry as they could be used as antimicrobial agents in medicine, agriculture and food products.

c Clostridium difficile is a leading cause of antibiotic-associated diarrhea and the etiologic agent responsible for C. difficile infection. Toxin A (TcdA) and toxin B (TcdB) are nearly indispensable virulence factors for Clostridium difficile pathogenesis. Given the toxin-centric mechanism by which C. difficile pathogenesis occurs, the selective sequestration with neutralization of TcdA and TcdB by nonantibiotic agents represents a novel mode of action to prevent or treat C. difficile-associated disease. In this preclinical study, we used quantitative enzyme immunoassays to determine the extent by which a novel drug, calcium aluminosilicate uniform particle size nonswelling M-1 (CAS UPSN M-1), is capable of sequestering TcdA and TcdB in vitro. The following major findings were derived from the present study. First, we show that CAS UPSN M-1 efficiently sequestered both TcdA and TcdB to undetectable levels. Second, we show that CAS UPSN M-1's affinity for TcdA is greater than its affinity for TcdB. Last, we show that CAS UPSN M-1 exhibited limited binding affinity for nontarget proteins. Taken together, these results suggest that ingestion of calcium aluminosilicate might protect gastrointestinal tissues from antibiotic-or chemotherapy-induced C. difficile infection by neutralizing the cytotoxic and proinflammatory effects of luminal TcdA and TcdB.C lostridium difficile is a leading cause of antibiotic-associated diarrhea (AAD) and is the etiologic agent responsible for C. difficile-associated infection (CDI). CDI typically starts as a mild diarrhea but rapidly degenerates into a variety of potentially lifethreatening conditions, including sepsis syndrome and pseudomembranous colitis (1). In the United States, approximately 330,000 cases of CDI are estimated to occur each year (2); however, the incidence of C. difficile infection continues to increase (2, 3). The increasing CDI rates highlight the fact that the current infection control procedures and treatment options are insufficient.In the health care setting, C. difficile endospores are transmitted to patients via the fecal-oral route (4). Following exposure, the host's gastrointestinal microbiota typically either quells a nascent C. difficile infection or suppresses it to subclinical levels (5). As a result of the latter, approximately 20% of hospitalized adults become asymptomatic C. difficile carriers, and the carriage rates approach 50% for patients in long-term care (6-9). The likelihood of development of CDI increases in patients with dysbiotic gastrointestinal microbiota, since C. difficile can thrive in the dysbiotic niche (5, 10). This dysbiosis is often the result of nonspecific chemotherapies that are used to treat conditions unrelated to C. difficile infection (e.g., antibacterial agents or antineoplastic drugs).The antibiotics metronidazole and vancomycin are currently used to treat CDI (11). Unfortunately, given the conflicting roles of antibiotics in the establishment and resolution of CDI, C. difficile AAD recurs in up to 1 in 5 patients (12)...