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.

The Potential of Bacteriophage Therapy as an Alternative Treatment Approach for Antibiotic-Resistant Infections.

OBJECTIVE: This study aimed to provide a comprehensive overview of the current state of the literature on the therapeutical application of bacteriophages. METHODS: First, a bibliometric analysis was performed using the database Web of Science to determine annual number of publications and citations. Second, a systematic literature review was conducted on randomized-controlled trials (RCTs) of phage therapy in PubMed. RESULTS: Over the past decade, the number of publications on bacteriophage therapy increased more than fourfold with 212 articles in 2011 and 739 in 2022. The systematic search in PubMed yielded 7 RCTs eligible for inclusion, reporting on a total of 418 participants. Identified indications in this study included bacterial diarrhea, urinary tract infections, infected burn wounds, chronic otitis, chronic venous leg ulcers, and chronic rhinosinusitis. In three studies, mild to moderate adverse events were reported in 10/195 participants (5.1%). Three of the studies reported a statistically significant difference in outcomes comparing phage therapy with standard of care or placebo. CONCLUSION: Phage therapy has gained increasing interest over the years. RCTs on different indications suggest the safety of phage therapy; however, reasons why phage therapy is not yet well accepted are limitations in the study designs. For a successful translation into clinical practice researchers and clinicians should learn from the earlier experiences and consider issues such as the quality of phage preparation, sensitivity testing, titer and dosages, as well as access to the infection site and stability for standardized protocols and future trials.

Gut virome profiling identifies an association between temperate phages and colorectal cancer promoted by Helicobacter pylori infection.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. While a close correlation between chronic Helicobacter pylori infection and CRC has been reported, the role of the virome has been overlooked. Here, we infected Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive metagenomics analysis of H. pylori-induced changes in lower gastrointestinal tract bacterial and viral communities. We observed an expansion of temperate phages in H. pylori infected Apc+/1638N mice at the early stage of carcinogenesis. Some of the temperate phages were predicted to infect bacteria associated with CRC, including Enterococcus faecalis. We also observed a high prevalence of virulent genes, such as flgJ, cwlJ, and sleB, encoded by temperate phages. In addition, we identified phages associated with pre-onset and onset of H. pylori-promoted carcinogenesis. Through co-occurrence network analysis, we found strong associations between the viral and bacterial communities in infected mice before the onset of carcinogenesis. These findings suggest that the expansion of temperate phages, possibly caused by prophage induction triggered by H. pylori infection, may have contributed to the development of CRC in mice by interacting with the bacterial community.

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.

A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria.

The alpha-Gal epitope (α-Gal) with the determining element galactose-α1,3-galactose can lead to clinically relevant allergic reactions and rejections in xenotransplantation. These immune reactions can develop because humans are devoid of this carbohydrate due to evolutionary loss of the enzyme α1,3-galactosyltransferase (GGTA1). In addition, up to 1% of human IgG antibodies are directed against α-Gal, but the stimulus for the induction of anti-α-Gal antibodies is still unclear. Commensal bacteria have been suggested as a causal factor for this induction as α-Gal binding tools such as lectins were found to stain cultivated bacteria isolated from the intestinal tract. Currently available tools for the detection of the definite α-Gal epitope, however, are cross-reactive, or have limited affinity and, hence, offer restricted possibilities for application. In this study, we describe a novel monoclonal IgG1 antibody (27H8) specific for the α-Gal epitope. The 27H8 antibody was generated by immunization of Ggta1 knockout mice and displays a high affinity towards synthetic and naturally occurring α-Gal in various applications. Using this novel tool, we found that intestinal bacteria reported to be α-Gal positive cannot be stained with 27H8 questioning whether commensal bacteria express the native α-Gal epitope at all.

Transplantation of bacteriophages from ulcerative colitis patients shifts the gut bacteriome and exacerbates the severity of DSS colitis.

BACKGROUND: Inflammatory bowel diseases (IBDs) including Crohn's disease (CD) and ulcerative colitis (UC) are characterized by chronic and debilitating gut inflammation. Altered bacterial communities of the intestine are strongly associated with IBD initiation and progression. The gut virome, which is primarily composed of bacterial viruses (bacteriophages, phages), is thought to be an important factor regulating and shaping microbial communities in the gut. While alterations in the gut virome have been observed in IBD patients, the contribution of these viruses to alterations in the bacterial community and heightened inflammatory responses associated with IBD patients remains largely unknown. RESULTS: Here, we performed in vivo microbial cross-infection experiments to follow the effects of fecal virus-like particles (VLPs) isolated from UC patients and healthy controls on bacterial diversity and severity of experimental colitis in human microbiota-associated (HMA) mice. Shotgun metagenomics confirmed that several phages were transferred to HMA mice, resulting in treatment-specific alterations in the gut virome. VLPs from healthy and UC patients also shifted gut bacterial diversity of these mice, an effect that was amplified during experimental colitis. VLPs isolated from UC patients specifically altered the relative abundance of several bacterial taxa previously implicated in IBD progression. Additionally, UC VLP administration heightened colitis severity in HMA mice, as indicated by shortened colon length and increased pro-inflammatory cytokine production. Importantly, this effect was dependent on intact VLPs. CONCLUSIONS: Our findings build on recent literature indicating that phages are dynamic regulators of bacterial communities in the gut and implicate the intestinal virome in modulating intestinal inflammation and disease. Video Abstract.

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.

Differences in Gut Virome Related to Barrett Esophagus and Esophageal Adenocarcinoma.

The relationship between viruses (dominated by bacteriophages or phages) and lower gastrointestinal (GI) tract diseases has been investigated, whereas the relationship between gut bacteriophages and upper GI tract diseases, such as esophageal diseases, which mainly include Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), remains poorly described. This study aimed to reveal the gut bacteriophage community and their behavior in the progression of esophageal diseases. In total, we analyzed the gut phage community of sixteen samples from patients with esophageal diseases (six BE patients and four EAC patients) as well as six healthy controls. Differences were found in the community composition of abundant and rare bacteriophages among three groups. In addition, the auxiliary metabolic genes (AMGs) related to bacterial exotoxin and virulence factors such as lipopolysaccharides (LPS) biosynthesis proteins were found to be more abundant in the genome of rare phages from BE and EAC samples compared to the controls. These results suggest that the community composition of gut phages and functional traits encoded by them were different in two stages of esophageal diseases. However, the findings from this study need to be validated with larger sample sizes in the future.

Sewage and sewage-contaminated environments are the most prominent sources to isolate phages against Pseudomonas aeruginosa.

BACKGROUND: P. aeruginosa is the primary source of hospital-acquired infections. Unfortunately, antibiotic resistance is growing to precariously high levels, making the infections by this pathogen life-threatening and hard to cure. One possible alternative to antibiotics is to use phages. However, the isolation of phages suitable for phage therapy- be lytic, be efficient, and have a broad host range -against some target bacteria has proven difficult. To identify the best places to look for these phages against P. aeruginosa we screened hospital sewages, soils, and rivers in two cities. RESULTS: We isolated eighteen different phages, determined their host range, infection property, and plaque morphology. We found that the sewage and sewage-contaminated environments are the most reliable sources for the isolation of Pseudomonas phages. In addition, phages isolated from hospital sewage showed the highest efficiency in lysing the bacteria used for host range determination. In contrast, phages from the river had larger plaque size and lysed bacteria with higher levels of antibiotic resistance. CONCLUSIONS: Our findings provided additional support for the importance of sewage as the source of phage isolation.

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.