Bacteriophages, gut bacteria, and microbial pathways interplay in cardiometabolic health.

Cardiometabolic diseases are leading causes of mortality in Western countries. Well-established risk factors include host genetics, lifestyle, diet, and the gut microbiome. Moreover, gut bacterial communities and their activities can be altered by bacteriophages (also known simply as phages), bacteria-infecting viruses, making these biological entities key regulators of human cardiometabolic health. The manipulation of bacterial populations by phages enables the possibility of using phages in the treatment of cardiometabolic diseases through phage therapy and fecal viral transplants. First, however, a deeper understanding of the role of the phageome in cardiometabolic diseases is required. In this review, we first introduce the phageome as a component of the gut microbiome and discuss fecal viral transplants and phage therapy in relation to cardiometabolic diseases. We then summarize the current state of phageome research in cardiometabolic diseases and propose how the phageome might indirectly influence cardiometabolic health through gut bacteria and their metabolites.

Targeted Single-Phage Isolation Reveals Phage-Dependent Heterogeneous Infection Dynamics.

Due to rising antibiotic resistance, there is an urgent need for different treatment options for multidrug-resistant infections. One alternative under investigation is phage therapy, which uses phages to treat bacterial infections. Although phages are highly abundant in the environment, not all phages are suitable for phage therapy, and finding efficient phages that lack undesirable traits such as bacterial virulence factors is challenging. Here, we developed a targeted single-phage isolation method to detect and isolate phages of interest and to characterize their kinetics in a high-throughput manner. This assay has also revealed cell-to-cell variations at a single-cell level among cells infected with the same phage species, as well as among cells infected with different phage species. IMPORTANCE The spread of multidrug-resistant bacteria is a global human health threat, and without immediate action we are fast approaching a postantibiotic era. One possible alternative to antibiotics is the use of phages, that is, bacterial viruses. However, the isolation of phages that effectively kill their target bacteria has proven challenging. In addition, isolated phages must go through significant characterization before their efficacy is measured. The method developed in this work can isolate single phage particles on the basis of their similarity to previously characterized phages while excluding those with known undesirable traits, such as bacterial toxins, as well as characterizing their kinetics. Using this method, we revealed significant cell-to-cell variations in phage kinetics at a single-cell level among highly virulent phages. These results shed some light on unknown phage-bacterium interactions at the single-cell level.

ViroProfiler: a containerized bioinformatics pipeline for viral metagenomic data analysis.

Bacteriophages play central roles in the maintenance and function of most ecosystems by regulating bacterial communities. Yet, our understanding of their diversity remains limited due to the lack of robust bioinformatics standards. Here we present ViroProfiler, an in-silico workflow for analyzing shotgun viral metagenomic data. ViroProfiler can be executed on a local Linux computer or cloud computing environments. It uses the containerization technique to ensure computational reproducibility and facilitate collaborative research. ViroProfiler is freely available at https://github.com/deng-lab/viroprofiler.

Gut bacteria, bacteriophages, and probiotics: Tripartite mutualism to quench the SARS-CoV2 storm.

Patients with SARS-CoV-2 infection, exhibit various clinical manifestations and severity including respiratory and enteric involvements. One of the main reasons for death among covid-19 patients is excessive immune responses directed toward cytokine storm with a low chance of recovery. Since the balanced gut microbiota could prepare health benefits by protecting against pathogens and regulating immune homeostasis, dysbiosis or disruption of gut microbiota could promote severe complications including autoimmune disorders; we surveyed the association between the imbalanced gut bacteria and the development of cytokine storm among COVID-19 patients, also the impact of probiotics and bacteriophages on the gut bacteria community to alleviate cytokine storm in COVID-19 patients. In present review, we will scrutinize the mechanism of immunological signaling pathways which may trigger a cytokine storm in SARS-CoV2 infections. Moreover, we are explaining in detail the possible immunological signaling pathway-directing by the gut bacterial community. Consequently, the specific manipulation of gut bacteria by using probiotics and bacteriophages for alleviation of the cytokine storm will be investigated. The tripartite mutualistic cooperation of gut bacteria, probiotics, and phages as a candidate prophylactic or therapeutic approach in SARS-CoV-2 cytokine storm episodes will be discussed at last.

Emerging technologies in the study of the virome.

Despite the growing interest in the microbiome in recent years, the study of the virome, the major part of which is made up of bacteriophages, is relatively underdeveloped compared with their bacterial counterparts. This is due in part to the lack of a universally conserved marker such as the 16S rRNA gene. For this reason, the development of metagenomic approaches was a major milestone in the study of the viruses in the microbiome or virome. However, it has become increasingly clear that these wet-lab methods have not yet been able to detect the full range of viruses present, and our understanding of the composition of the virome remains incomplete. In recent years, a range of new technologies has been developed to further our understanding. Direct RNA-Seq technologies bypass the need for cDNA synthesis, thus avoiding biases subjected to this step, which further expands our understanding of RNA viruses. The new generation of amplification methods could solve the low biomass issue relevant to most virome samples while reducing the error rate and biases caused by whole genome amplification. The application of long-read sequencing to virome samples can resolve the shortcomings of short-read sequencing in generating complete viral genomes and avoid the biases introduced by the assembly. Novel experimental methods developed to measure viruses' host range can help overcome the challenges of assigning hosts to many phages, specifically unculturable ones.

The role of virome in the gastrointestinal tract and beyond.

The human gut virome is comprised of diverse commensal and pathogenic viruses. The colonization by these viruses begins right after birth through vaginal delivery, then continues through breastfeeding, and broader environmental exposure. Their constant interaction with their bacterial hosts in the body shapes not only our microbiomes but us. In addition, these viruses interact with the immune cells, trigger a broad range of immune responses, and influence different metabolic pathways. Besides its key role in regulating the human gut homeostasis, the intestinal virome contributes to disease development in distant organs, both directly and indirectly. In this review, we will describe the changes in the gut virome through life, health, and disease, followed by discussing the interactions between the virome, the microbiome, and the human host as well as providing an overview of their contribution to gut disease and disease of distant organs.

New technologies for developing phage-based tools to manipulate the human microbiome.

Gut bacteria play an essential role in the human body by regulating multiple functions, producing essential metabolites, protecting against pathogen invasion, and much more. Conversely, changes in their community structure are linked to several gastrointestinal (GI) and non-GI conditions. Fortunately, these bacteria are amenable to external perturbations, but we need specific tools for their safe manipulation as nonspecific changes can cause unpredicted long-term consequences. Here, we mainly discuss recent advances in cultivation-independent technologies and argue their relevance to different key steps, that is, identifying the modulation targets and developing phage-based tools to precisely modulate gut bacteria and restore a sustainable microbiome in humans. We finally suggest multiple modulating strategies for different dysbiosis-associated diseases.

Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics.

Antibiotic resistance causes around 700,000 deaths a year worldwide. Without immediate action, we are fast approaching a post-antibiotic era in which common infections can result in death. Pseudomonas aeruginosa is the leading cause of nosocomial infection and is also one of the three bacterial pathogens in the WHO list of priority bacteria for developing new antibiotics against. A viable alternative to antibiotics is to use phages, which are bacterial viruses. Yet, the isolation of phages that efficiently kill their target bacteria has proven difficult. Using a combination of phages and antibiotics might increase treatment efficacy and prevent the development of resistance against phages and/or antibiotics, as evidenced by previous studies. Here, in vitro populations of a Pseudomonas aeruginosa strain isolated from a burn patient were treated with a single phage, a mixture of two phages (used simultaneously and sequentially), and the combination of phages and antibiotics (at sub-minimum inhibitory concentration (MIC) and MIC levels). In addition, we tested the stability of these phages at different temperatures, pH values, and in two burn ointments. Our results show that the two-phages-one-antibiotic combination had the highest killing efficiency against the P. aeruginosa strain. The phages tested showed low stability at high temperatures, acidic pH values, and in the two ointments. This work provides additional support for the potential of using combinations of phage-antibiotic cocktails at sub-MIC levels for the treatment of multidrug-resistant P. aeruginosa infections.

Challenges of Studying the Human Virome - Relevant Emerging Technologies.

In this review we provide an overview of current challenges and advances in bacteriophage research within the growing field of viromics. In particular, we discuss, from a human virome study perspective, the current and emerging technologies available, their limitations in terms of de novo discoveries, and possible solutions to overcome present experimental and computational biases associated with low abundance of viral DNA or RNA. We summarize recent breakthroughs in metagenomics assembling tools and single-cell analysis, which have the potential to increase our understanding of phage biology, diversity, and interactions with both the microbial community and the human body. We expect that these recent and future advances in the field of viromics will have a strong impact on how we develop phage-based therapeutic approaches.

Sustainable Microbiome: a symphony orchestrated by synthetic phages.

We are surrounded by microbes, mostly bacteria and their viruses or phages, on the inside and outside of our bodies. These bacteria in constant interactions with phages are regulating multiple functions critical to our health. Luckily, they are amenable, but we need precise tools for their safe manipulation and improving human health. Here, we argue that recent advances in single-cell technologies, culturomics and synthetic biology offer exciting opportunities to create these tools as well as revealing specific phages-bacteria interactions in the body.

Bacteriophages Isolated from Stunted Children Can Regulate Gut Bacterial Communities in an Age-Specific Manner.

Stunting, a severe and multigenerational growth impairment, globally affects 22% of children under the age of 5 years. Stunted children have altered gut bacterial communities with higher proportions of Proteobacteria, a phylum with several known human pathogens. Despite the links between an altered gut microbiota and stunting, the role of bacteriophages, highly abundant bacterial viruses, is unknown. Here, we describe the gut bacterial and bacteriophage communities of Bangladeshi stunted children younger than 38 months. We show that these children harbor distinct gut bacteriophages relative to their non-stunted counterparts. In vitro, these gut bacteriophages are infectious and can regulate bacterial abundance and composition in an age-specific manner, highlighting their possible role in the pathophysiology of child stunting. Specifically, Proteobacteria from non-stunted children increased in the presence of phages from younger stunted children, suggesting that phages could contribute to the bacterial community changes observed in child stunting.

The prevalence of CMY-2, OXA-48 and KPC-2 genes in clinical isolates of Klebsiella spp.

Klebsiella pneumoniae is a Gram-negative bacterium which causes several human infections. Treatment of infections related to K. pneumoniae has become problematic, because of increasing trend of extended spectrum ß-lactamases producing (ESBLs) strains. The present study was aimed to detect the prevalence of ESBL-producing Klebsiella spp. and KPC-2, CMY-2 and OXA-48 ß-lactamase encoding genes in clinical isolates of Klebsiella spp. isolated from hospitalized patients. In this cross-sectional study carried out from February to August 2014, 144 isolates of Klebsiella spp. were collected from different clinical specimens in hospitals in the North of Iran. Klebsiella isolates were identified using standard microbiological procedure. Antimicrobial susceptibility patterns were determined using disk diffusion method in accordance with CLSI recommendation. The presence of antibiotic resistance genes were investigated by PCR method. Among 144 Klebsiella spp., 118 isolates were identified as K. pneumoniae and 26 isolates as Klebsiella oxytoca. Antibiotic susceptibility test showed the most resistance rates were against amoxicillin (96.5%) and ampicillin (95.8%). On the other hand, the most effective antibiotic was revealed to be imipenem with resistance rate of 4.9% (Table 2). Among 144 isolated Klebsiella strains, 57 cases (39.6%) were ESBL producers. The OXA-48 and KPC-2 genes were not detected among Klebsiella spp. during the present study, but 21.1% of isolates contained CMY-2 gene. This is the first report of CMY-2 gene detection in Klebsiella spp. in Iran. The homology between CMY-2 genes identified in isolates from Northern Iran and in other countries showed the wide dispersion of this gene around the world.

The Mammalian Gut as a Matchmaker.

Dynamics of phages and bacteria in the gut may play key roles in human health. In this issue of Cell Host & Microbe, De Sordi et al. (2017) provide insights into phage-bacteria interactions, finding that microbial communities contribute to phage persistence in the mammalian gut by supplying new hosts.

Adapting Drug Approval Pathways for Bacteriophage-Based Therapeutics.

The global rise of multi-drug resistant bacteria has resulted in the notion that an "antibiotic apocalypse" is fast approaching. This has led to a number of well publicized calls for global funding initiatives to develop new antibacterial agents. The long clinical history of phage therapy in Eastern Europe, combined with more recent in vitro and in vivo success, demonstrates the potential for whole phage or phage based antibacterial agents. To date, no whole phage or phage derived products are approved for human therapeutic use in the EU or USA. There are at least three reasons for this: (i) phages possess different biological, physical, and pharmacological properties compared to conventional antibiotics. Phages need to replicate in order to achieve a viable antibacterial effect, resulting in complex pharmacodynamics/pharmacokinetics. (ii) The specificity of individual phages requires multiple phages to treat single species infections, often as part of complex cocktails. (iii) The current approval process for antibacterial agents has evolved with the development of chemically based drugs at its core, and is not suitable for phages. Due to similarities with conventional antibiotics, phage derived products such as endolysins are suitable for approval under current processes as biological therapeutic proteins. These criteria render the approval of phages for clinical use theoretically possible but not economically viable. In this review, pitfalls of the current approval process will be discussed for whole phage and phage derived products, in addition to the utilization of alternative approval pathways including adaptive licensing and "Right to try" legislation.

Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

Corrigendum: Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

[This corrects the article on p. 437 in vol. 7, PMID: 27065990.].

Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy.

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.