Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

The gastrointestinal microbiome plays a significant role in modulating numerous host processes, including metabolism. Prior studies show that when mice receive fecal transplants from obese donors on high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes, demonstrating the prominent role that gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, the impact of gut viruses on these phenotypes is understudied. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow over a 4-week study. By characterizing the gut bacterial biota via 16S rRNA amplicon sequencing and measuring mouse weights over time, we demonstrate that transplanted viruses affect the gut bacterial community, as well as weight gain/loss. Notably, mice fed chow but gavaged with HFD-derived viromes gained more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained less weight than their counterparts receiving HFD-derived viromes. Results were replicated in two independent experiments and phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Due to methodological limitations, we were unable to identify the specific bacterial strains responsible for respective phenotypic changes. This study confirms that virome-mediated perturbations can alter the fecal microbiome in vivo and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

Background: The gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mice’s gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss. Results: We gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes. Conclusions: This study confirms that virome-mediated perturbations can alter the fecal microbiome in an in vivo model and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Gram-Negative Bacterial Lysins.

Antibiotics have had a profound impact on human society by enabling the eradication of otherwise deadly infections. Unfortunately, antibiotic use and overuse has led to the rapid spread of acquired antibiotic resistance, creating a major threat to public health. Novel therapeutic agents called bacteriophage endolysins (lysins) provide a solution to the worldwide epidemic of antibiotic resistance. Lysins are a class of enzymes produced by bacteriophages during the lytic cycle, which are capable of cleaving bonds in the bacterial cell wall, resulting in the death of the bacteria within seconds after contact. Through evolutionary selection of the phage progeny to be released and spread, these lysins target different critical components in the cell wall, making resistance to these molecules orders of magnitude less likely than conventional antibiotics. Such properties make lysins uniquely suitable for the treatment of multidrug resistant bacterial pathogens. Lysins, either naturally occurring or engineered, have the potential of being developed into fast-acting, narrow-spectrum, biofilm-disrupting antimicrobials that act synergistically with standard of care antibiotics. This review focuses on newly discovered classes of Gram-negative lysins with emphasis on prototypical enzymes that have been evaluated for efficacy against the major antibiotic resistant organisms causing nosocomial infections.

The Virome of Cerebrospinal Fluid: Viruses Where We Once Thought There Were None.

Traditionally, medicine has held that some human body sites are sterile and that the introduction of microbes to these sites results in infections. This paradigm shifted significantly with the discovery of the human microbiome and acceptance of these commensal microbes living across the body. However, the central nervous system (CNS) is still believed by many to be sterile in healthy people. Using culture-independent methods, we examined the virome of cerebrospinal fluid (CSF) from a cohort of mostly healthy human subjects. We identified a community of DNA viruses, most of which were identified as bacteriophages. Compared to other human specimen types, CSF viromes were not ecologically distinct. There was a high alpha diversity cluster that included feces, saliva, and urine, and a low alpha diversity cluster that included CSF, body fluids, plasma, and breast milk. The high diversity cluster included specimens known to have many bacteria, while other specimens traditionally assumed to be sterile formed the low diversity cluster. There was an abundance of viruses shared among CSF, breast milk, plasma, and body fluids, while each generally shared less with urine, feces, and saliva. These shared viruses ranged across different virus families, indicating that similarities between these viromes represent more than just a single shared virus family. By identifying a virome in the CSF of mostly healthy individuals, it is now less likely that any human body site is devoid of microbes, which further highlights the need to decipher the role that viral communities may play in human health.

Transmission of viruses via our microbiomes.

BACKGROUND: Bacteria inhabiting the human body have important roles in a number of physiological processes and are known to be shared amongst genetically-related individuals. Far less is known about viruses inhabiting the human body, but their ecology suggests they may be shared between close contacts. RESULTS: Here, we report the ecology of viruses in the guts and mouths of a cohort and demonstrate that substantial numbers of gut and oral viruses were shared amongst genetically unrelated, cohabitating individuals. Most of these viruses were bacteriophages, and each individual had distinct oral and gut viral ecology from their housemates despite the fact that some of their bacteriophages were shared. The distribution of bacteriophages over time within households indicated that they were frequently transmitted between the microbiomes of household contacts. CONCLUSIONS: Because bacteriophages may shape human oral and gut bacterial ecology, their transmission to household contacts suggests they could have substantial roles in shaping the microbiota within a household.

Immunogenicity and protective efficacy of recombinant Clostridium difficile flagellar protein FliC.

Clostridium difficile is a Gram-positive bacillus and is the leading cause of toxin-mediated nosocomial diarrhea following antibiotic use. C. difficile flagella play a role in colonization, adherence, biofilm formation, and toxin production, which might contribute to the overall virulence of certain strains. Human and animal studies indicate that anti-flagella immune responses may play a role in protection against colonization by C. difficile and subsequent disease outcome. Here we report that recombinant C. difficile flagellin (FliC) is immunogenic and protective in a murine model of C. difficile infection (CDI) against a clinical C. difficile strain, UK1. Passive protection experiments using anti-FliC polyclonal serum in mice suggest this protection to be antibody-mediated. FliC immunization also was able to afford partial protection against CDI and death in hamsters following challenge with C. difficile 630Δerm. Additionally, immunization against FliC does not have an adverse effect on the normal gut flora of vaccinated hamsters as evidenced by comparing the fecal microbiome of vaccinated and control hamsters. Therefore, the use of FliC as a vaccine candidate against CDI warrants further testing.

Immunogenicity and protective efficacy of Clostridium difficile spore proteins.

Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.

The prospect for vaccines to prevent Clostridium difficile infection.

Clostridium difficile is a spore-forming anaerobic gram-positive organism that is the leading cause of antibiotic-associated nosocomial infectious diarrhea in the Western world. This article describes the evolving epidemiology of C difficile infection (CDI) in the twenty-first century, evaluates the importance of vaccines against the disease, and defines the roles of both innate and adaptive host immune responses in CDI. The effects of passive immunotherapy and active vaccination against CDI in both humans and animals are also discussed.

Toll-like receptor 5-dependent immunogenicity and protective efficacy of a recombinant fusion protein vaccine containing the nontoxic domains of Clostridium difficile toxins A and B and Salmonella enterica serovar typhimurium flagellin in a mouse model of Clostridium difficile disease.

Clostridium difficile is a spore-forming bacillus that produces toxin-mediated enteric disease. C. difficile expresses two major virulence factors, toxin A (TcdA) and toxin B (TcdB). Human and animal studies demonstrate a clear association between humoral immunity to these toxins and protection against C. difficile infection (CDI). The receptor binding-domains (RBDs) of TcdA and TcdB are known to be immunogenic. Here, we tested the immunoadjuvant properties of Salmonella enterica serovar Typhimurium flagellin (FliC) subunit D1 as an innate immune agonist expressed as a recombinant fusion vaccine targeting the RBDs of TcdA and TcdB in mice. Intraperitoneally immunized mice developed prominent anti-TcdA and anti-TcdB immunoglobulin G in serum. The protective efficacy of the recombinant vaccines, with or without an adjuvant, was tested in a mouse model of CDI that closely represents the human disease. Following intraperitoneal immunization equivalent to two doses of toxoid A and toxoid B vaccine adjuvanted with alum and oral challenge with C. difficile VPI 10463, C57BL/6 mice were able to mount a protective immune response that prevented diarrhea and death compared to mice immunzed with alum alone. These results are significantly different from those for control mice (P < 0.001). These results provide evidence that a recombinant protein-based vaccine targeting the RBDs of the C. difficile toxins adjuvanted with S. Typhimurium flagellin can induce rapid, high-level protection in a mouse model of CDI when challenged with the homologous strain from which the vaccine antigens were derived and warrant further preclinical testing against clinically relevant C. difficile strains in the mouse and hamster models of CDI.

Clostridium difficile infection in the twenty-first century.

Clostridium difficile is a spore-forming gram-positive bacillus, and the leading cause of antibiotic-associated nosocomial diarrhea and colitis in the industrialized world. With the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027), the epidemiology of C. difficile infection has rapidly changed in the last decade. C. difficile infection, once thought to be an easy to treat bacterial infection, has evolved into an epidemic that is associated with a high rate of mortality, causing disease in patients thought to be low-risk. In this review, we discuss the changing face of C .difficile infection and the novel treatment and prevention strategies needed to halt this ever growing epidemic.

Transcutaneous immunization with Clostridium difficile toxoid A induces systemic and mucosal immune responses and toxin A-neutralizing antibodies in mice.

Clostridium difficile is the leading cause of nosocomial infectious diarrhea. C. difficile produces two toxins (A and B), and systemic and mucosal anti-toxin A antibodies prevent or limit C. difficile-associated diarrhea. To evaluate whether transcutaneous immunization with formalin-treated C. difficile toxin A (CDA) induces systemic and mucosal anti-CDA immune responses, we transcutaneously immunized three cohorts of mice with CDA with or without immunoadjuvantative cholera toxin (CT) on days 0, 14, 28, and 42. Mice transcutaneously immunized with CDA and CT developed prominent anti-CDA and anti-CT immunoglobulin G (IgG) and IgA responses in serum and anti-CDA and anti-CT IgA responses in stool. Sera from immunized mice were able to neutralize C. difficile toxin A activity in an in vitro cell culture assay. CDA itself demonstrated adjuvant activity and enhanced both serum and stool anti-CT IgA responses. Our results suggest that transcutaneous immunization with CDA toxoid may be a feasible immunization strategy against C. difficile, an important cause of morbidity and mortality against which current preventative strategies are failing.

Serological assays for identification of human gastric colonization by Helicobacter pylori strains expressing VacA m1 or m2.

The Helicobacter pylori vacA gene encodes a secreted protein (VacA) that alters the function of gastric epithelial cells and T lymphocytes. H. pylori strains containing particular vacA alleles are associated with differential risk of disease. Because the VacA midregion may exist as one of two major types, m1 or m2, serologic responses may potentially be used to differentiate between patients colonized with vacA m1- or vacA m2-positive H. pylori strains. In this study, we examined the utility of specific antigens from the m regions of VacA as allele-specific diagnostic antigens. We report that serological responses to P44M1, an H. pylori m1-specific antigen, are observed predominantly in patients colonized with m1-positive strains, whereas responses to VacA m2 antigens, P48M2 and P55M2, are observed in patients colonized with either m1- or m2-positive strains. In an Asian-American population, serologic responses to VacA m region-specific antigens were not able to predict the risk of development of gastric cancer.

Evidence of host-virus co-evolution in tetranucleotide usage patterns of bacteriophages and eukaryotic viruses.

BACKGROUND: Virus taxonomy is based on morphologic characteristics, as there are no widely used non-phenotypic measures for comparison among virus families. We examined whether there is phylogenetic signal in virus nucleotide usage patterns that can be used to determine ancestral relationships. The well-studied model of tail morphology in bacteriophage classification was used for comparison with nucleotide usage patterns. Tetranucleotide usage deviation (TUD) patterns were chosen since they have previously been shown to contain phylogenetic signal similar to that of 16S rRNA. RESULTS: We found that bacteriophages have unique TUD patterns, representing genomic signatures that are relatively conserved among those with similar host range. Analysis of TUD-based phylogeny indicates that host influences are important in bacteriophage evolution, and phylogenies containing both phages and their hosts support their co-evolution. TUD-based phylogeny of eukaryotic viruses indicates that they cluster largely based on nucleic acid type and genome size. Similarities between eukaryotic virus phylogenies based on TUD and gene content substantiate the TUD methodology. CONCLUSION: Differences between phenotypic and TUD analysis may provide clues to virus ancestry not previously inferred. As such, TUD analysis provides a complementary approach to morphology-based systems in analysis of virus evolution.

East Asian genotypes of Helicobacter pylori strains in Amerindians provide evidence for its ancient human carriage.

Phylogenies of indigenous microbes have been used as surrogates for the origins of the hosts that carry them. Conversely, polymorphisms may be used to date the spread of a microbial species when information about their host populations is available. Therefore, we examined polymorphisms in Helicobacter pylori, which persistently colonize the human stomach, to test the hypothesis that they have been ancient inhabitants of humans. Three H. pylori loci that previously have been shown to have phylogeographic affinity have been analyzed for two populations with different ethnic origins from Venezuela. In a group of Amerindian subjects from Amazonia, East Asian H. pylori genotypes were present for each of the loci examined but were absent in a mestizo population from Caracas. These findings provide evidence that H. pylori has been present in humans at least since ancestors of Amerindians migrated from Asia more than 11,000 years ago.