Fecal microbiota transplantation alters gut phage communities in a clinical trial for obesity.

BACKGROUND: Fecal microbiota transplantation (FMT) is a therapeutic intervention used to treat diseases associated with the gut microbiome. In the human gut microbiome, phages have been implicated in influencing human health, with successful engraftment of donor phages correlated with FMT treatment efficacy. The impact that gastrointestinal phages exert on human health has primarily been connected to their ability to modulate the bacterial communities in the gut. Nonetheless, how FMT affects recipients' phage populations, and in turn, how this influences the gut environment, is not yet fully understood. In this study, we investigated the effects of FMT on the phageome composition of participants within the Gut Bugs Trial (GBT), a double-blind, randomized, placebo-controlled trial that investigated the efficacy of FMT in treating obesity and comorbidities in adolescents. Stool samples collected from donors at the time of treatment and recipients at four time points (i.e., baseline and 6 weeks, 12 weeks, and 26 weeks post-intervention), underwent shotgun metagenomic sequencing. Phage sequences were identified and characterized in silico to examine evidence of phage engraftment and to assess the extent of FMT-induced alterations in the recipients' phageome composition. RESULTS: Donor phages engrafted stably in recipients following FMT, composing a significant proportion of their phageome for the entire course of the study (33.8 ± 1.2% in females and 33.9 ± 3.7% in males). Phage engraftment varied between donors and donor engraftment efficacy was positively correlated with their phageome alpha diversity. FMT caused a shift in recipients' phageome toward the donors' composition and increased phageome alpha diversity and variability over time. CONCLUSIONS: FMT significantly altered recipients' phage and, overall, microbial populations. The increase in microbial diversity and variability is consistent with a shift in microbial population dynamics. This proposes that phages play a critical role in modulating the gut environment and suggests novel approaches to understanding the efficacy of FMT in altering the recipient's microbiome. TRIAL REGISTRATION: The Gut Bugs Trial was registered with the Australian New Zealand Clinical Trials Registry (ACTR N12615001351505). Trial protocol: the trial protocol is available at https://bmjopen.bmj.com/content/9/4/e026174 . Video Abstract.

Characterizing the gut phageome and phage-borne antimicrobial resistance genes in pigs.

BACKGROUND: Mammalian intestine harbors a mass of phages that play important roles in maintaining gut microbial ecosystem and host health. Pig has become a common model for biomedical research and provides a large amount of meat for human consumption. However, the knowledge of gut phages in pigs is still limited. RESULTS: Here, we investigated the gut phageome in 112 pigs from seven pig breeds using PhaBOX strategy based on the metagenomic data. A total of 174,897 non-redundant gut phage genomes were assembled from 112 metagenomes. A total of 33,487 gut phage genomes were classified and these phages mainly belonged to phage families such as Ackermannviridae, Straboviridae, Peduoviridae, Zierdtviridae, Drexlerviridae, and Herelleviridae. The gut phages in seven pig breeds exhibited distinct communities and the gut phage communities changed with the age of pig. These gut phages were predicted to infect a broad range of 212 genera of prokaryotes, such as Candidatus Hamiltonella, Mycoplasma, Colwellia, and Lactobacillus. The data indicated that broad KEGG and CAZy functions were also enriched in gut phages of pigs. The gut phages also carried the antimicrobial resistance genes (ARGs) and the most abundant antimicrobial resistance genotype was diaminopyrimidine resistance. CONCLUSIONS: Our research delineates a landscape for gut phages in seven pig breeds and reveals that gut phages serve as a key reservoir of ARGs in pigs. Video Abstract.

Metagenomic analysis reveals ecological and functional signatures of oral phageome associated with severe early childhood caries.

OBJECTIVES: Severe early childhood caries (S-ECC) is highly prevalent, affecting children's oral health. S-ECC development is closely associated with the complex oral microbial microbiome and its microorganism interactions, such as the imbalance of bacteriophages and bacteria. Till now, little is known about oral phageome on S-ECC. Therefore, this study aimed to investigate the potential role of the oral phageome in the pathogenesis of S-ECC. METHODS: Unstimulated saliva (2 mL) was collected from 20 children with and without S-ECC for metagenomics analysis. Metagenomics sequencing and bioinformatic analysis were performed to determine the two groups' phageome diversity, taxonomic and functional annotations. Statistical analysis and visualization were performed with R and SPSS Statistics software. RESULTS: 85.7 % of the extracted viral sequences were predicted from phages, in which most phages were classified into Myoviridae, Siphoviridae, and Podoviridae. Alpha diversity decreased, and Beta diversity increased in the S-ECC phageome compared to the healthy group. The abundance of Podoviridae phages increased, and the abundance of Inoviridae, Herelleviridae, and Streptococcus phages decreased in the S-ECC group. Functional annotation revealed increased annotation on glycoside hydrolases and nucleotide metabolism, decreased glycosyl transferases, carbohydrate-binding modules, and biogenic metabolism in the S-ECC phageome. CONCLUSIONS: Metagenomic analysis revealed reduced Streptococcus phages and significant changes in functional annotations within the S-ECC phageome. These findings suggest a potential weakening of the regulatory influence of oral bacteria, which may indicate the development of innovative prevention and treatment strategies for S-ECC. These implications deserve further investigation and hold promise for advancing our understanding and management of S-ECC. CLINICAL SIGNIFICANCE: The findings of this study indicate that oral phageomes are associated with bacterial genomes and metabolic processes, affecting the development of S-ECC. The reduced modulatory effect of the oral phageome in counteracting S-ECC's cariogenic activity suggests a new avenue for the prevention and treatment of S-ECC.

The bacteriome-coupled phage communities continuously contract and shift to orchestrate the traditional rice vinegar fermentation.

Amounts of microbiome studies have uncovered the microbial communities of traditional food fermentations, while in which the phageome development with time is poorly understood. Here, we conducted a study to decipher both phageome and bacteriome of the traditional rice vinegar fermentation. The vinegar phageomes showed significant differences in the alpha diversity, network density and clustering coefficient over time. Peduoviridae had the highest relative abundance. Moreover, the phageome negatively correlated to the cognate bacteriome in alpha diversity, and undergone constantly contracting and shifting across the temporal scale. Nevertheless, 257 core virial clusters (VCs) persistently occurred with time whatever the significant impacts imposed by the varied physiochemical properties. Glycoside hydrolase (GH) and glycosyltransferase (GT) families genes displayed the higher abundances across all samples. Intriguingly, diversely structuring of toxin-antitoxin systems (TAs) and CRISPR-Cas arrays were frequently harbored by phage genomes. Their divergent organization and encoding attributes underlie the multiple biological roles in modulation of network and/or contest of phage community as well as bacterial host community. This phageome-wide mapping will fuel the current insights of phage community ecology in other traditional fermented ecosystems that are challenging to decipher.

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.

Bacteriophages-Dangerous Viruses Acting Incognito or Underestimated Saviors in the Fight against Bacteria?

The steadily increasing number of drug-resistant bacterial species has prompted the search for alternative treatments, resulting in a growing interest in bacteriophages. Although they are viruses infecting bacterial cells, bacteriophages are an extremely important part of the human microbiota. By interacting with eukaryotic cells, they are able to modulate the functioning of many systems, including the immune and nervous systems, affecting not only the homeostasis of the organism, but potentially also the regulation of pathological processes. Therefore, the aim of this review is to answer the questions of (i) how animal/human immune systems respond to bacteriophages under physiological conditions and under conditions of reduced immunity, especially during bacterial infection; (ii) whether bacteriophages can induce negative changes in brain functioning after crossing the blood-brain barrier, which could result in various disorders or in an increase in the risk of neurodegenerative diseases; and (iii) how bacteriophages can modify gut microbiota. The crucial dilemma is whether administration of bacteriophages is always beneficial or rather if it may involve any risks.

CrAss-Like Phages: From Discovery in Human Fecal Metagenome to Application as a Microbial Source Tracking Marker.

CrAss-like phages are a diverse group of bacteriophages genetically similar to the prototypical crAssphage (p-crAssphage), which was discovered in the human gut microbiome through a metagenomics approach. It was identified as a ubiquitous and highly abundant bacteriophage group in the gut microbiome. Initial co-occurrence analysis postulated Bacteroides spp. as the prospective bacterial host. Subsequent studies have confirmed multiple host species under Phylum Bacteroidetes and some Firmicutes. Detection of crAss-like phages in sewage-contaminated environmental water and robust correlation with enteric viruses and bacteria has culminated in their adoption as a microbial source tracking (MST) marker. Polymerase chain reaction (PCR) and real-time PCR assays have been developed utilizing the conserved genes in the p-crAssphage genome to detect human fecal contamination of different water sources, with high specificity. Numerous investigations have examined the implications of crAss-like phages in diverse disease conditions, including ulcerative colitis, obesity and metabolic syndrome, autism spectrum disorders, rheumatoid arthritis, atopic eczema, and other autoimmune disorders. These studies have unveiled associations between certain diseases and diminished abundance and diversity of crAss-like phages. This review offers insights into the diverse aspects of research on crAss-like phages, including their discovery, genomic characteristics, structure, taxonomy, isolation, molecular detection, application as an MST marker, and role as a gut microbiome modulator with consequential health implications.

Phage therapy: Targeting intestinal bacterial microbiota for the treatment of liver diseases.

Phage therapy has been overshadowed by antibiotics for decades. However, it is being revisited as a powerful approach against antimicrobial-resistant bacteria. As bacterial microbiota have been mechanistically linked to gastrointestinal and liver diseases, precise editing of the gut microbiota via the selective bactericidal action of phages has prompted renewed interest in phage therapy. In this review, we summarise the basic virological properties of phages and the latest findings on the composition of the intestinal phageome and the changes associated with liver diseases. We also review preclinical and clinical studies assessing phage therapy for the treatment of gastrointestinal and liver diseases, as well as future prospects and challenges.

Gut Phageome-An Insight into the Role and Impact of Gut Microbiome and Their Correlation with Mammal Health and Diseases.

The gut microbiota, including bacteria, archaea, fungi, and viruses, compose a diverse mammalian gut environment and are highly associated with host health. Bacteriophages, the viruses that infect bacteria, are the primary members of the gastrointestinal virome, known as the phageome. However, our knowledge regarding the gut phageome remains poorly understood. In this review, the critical role of the gut phageome and its correlation with mammalian health were summarized. First, an overall profile of phages across the gastrointestinal tract and their dynamic roles in shaping the surrounding microorganisms was elucidated. Further, the impacts of the gut phageome on gastrointestinal fitness and the bacterial community were highlighted, together with the influence of diets on the gut phageome composition. Additionally, new reports on the role of the gut phageome in the association of mammalian health and diseases were reviewed. Finally, a comprehensive update regarding the advanced phage benchwork and contributions of phage-based therapy to prevent/treat mammalian diseases was provided. This study provides insights into the role and impact of the gut phagenome in gut environments closely related to mammal health and diseases. The findings provoke the potential applications of phage-based diagnosis and therapy in clinical and agricultural fields. Future research is needed to uncover the underlying mechanism of phage-bacterial interactions in gut environments and explore the maintenance of mammalian health via phage-regulated gut microbiota.

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Metagenomics revealed a correlation of gut phageome with autism spectrum disorder.

The human gut bacteriome is believed to have pivotal influences on human health and disease while the particular roles associated with the gut phageome have not been fully characterized yet with few exceptions. It is argued that gut microbiota can have a potential role in autism spectrum disorders (ASD). The public microbiota database of ASD and typically developing (TD) Chinese individuals were analyzed for phage protein-coding units (pPCU) to find any link between the phageome and ASD. The gut phageome of ASD individuals showed a wider diversity and higher abundance compared to TD individuals. The ASD phageome was associated with a significant expansion of Caudoviricetes bacteriophages. Phages infecting Bacteroidaceae and prophages encoded within Faecalibacterium were more frequent in ASD than in TD individuals. The expansion and diversification of ASD phageome can influence the bacterial homeostasis by imposing pressure on the bacterial communities. In conclusion, the differences of phages community in in ASD and TD can be used as potential diagnosis biomarkers of ASD. Further investigations are needed to verify the role of gut phage communities in the pathogenesis of ASD.

The human gut phageome: composition, development, and alterations in disease.

The human gastrointestinal tract is colonized by a large number of microorganisms, including bacteria, archaea, viruses, and eukaryotes. The bacterial community has been widely confirmed to have a significant impact on human health, while viruses, particularly phages, have received less attention. Phages are viruses that specifically infect bacteria. They are abundant in the biosphere and exist in a symbiotic relationship with their host bacteria. Although the application of high-throughput sequencing and bioinformatics technology has greatly improved our understanding of the genomic diversity, taxonomic composition, and spatio-temporal dynamics of the human gut phageome, there is still a large portion of sequencing data that is uncharacterized. Preliminary studies have predicted that the phages play a crucial role in driving microbial ecology and evolution. Prior to exploring the function of phages, it is necessary to address the obstacles that hinder establishing a comprehensive sequencing database with sufficient biological properties and understanding the impact of phage-bacteria interactions on human health. In this study, we provide an overview of the human gut phageome, including its composition, structure, and development. We also explore the various factors that may influence the phageome based on current research, including age, diet, ethnicity, and geographical location. Additionally, we summarize the relationship between the phageome and human diseases, such as IBD, IBS, obesity, diabetes, and metabolic syndrome.

Phage Interactions with the Nervous System in Health and Disease.

The central nervous system manages all of our activities (e.g., direct thinking and decision-making processes). It receives information from the environment and responds to environmental stimuli. Bacterial viruses (bacteriophages, phages) are the most numerous structures occurring in the biosphere and are also found in the human organism. Therefore, understanding how phages may influence this system is of great importance and is the purpose of this review. We have focused on the effect of natural bacteriophages in the central nervous system, linking them to those present in the gut microbiota, creating the gut-brain axis network, as well as their interdependence. Importantly, based on the current knowledge in the field of phage application (e.g., intranasal) in the treatment of bacterial diseases associated with the brain and nervous system, bacteriophages may have significant therapeutic potential. Moreover, it was indicated that bacteriophages may influence cognitive processing. In addition, phages (via phage display technology) appear promising as a targeted therapeutic tool in the treatment of, among other things, brain cancers. The information collected and reviewed in this work indicates that phages and their impact on the nervous system is a fascinating and, so far, underexplored field. Therefore, the aim of this review is not only to summarize currently available information on the association of phages with the nervous system, but also to stimulate future studies that could pave the way for novel therapeutic approaches potentially useful in treating bacterial and non-bacterial neural diseases.

Gut phageome of the giant panda (Ailuropoda melanoleuca) reveals greater diversity than relative species.

The gut flora is a treasure house of diverse bacteriophages maintaining a harmonious and coexistent relationship with their hosts. The giant panda (Ailuropoda melanoleuca), as a vulnerable endemic species in China, has existed for millions of years and is regarded as a flagship species for biodiversity conservation. And yet, limited studies have analyzed the phage communities in the gut of giant pandas. Using viral metagenomic analysis, the phageomes of giant pandas and other relative species were investigated. Our study explored and compared the composition of phage communities from different animal sources. Giant pandas possessed more diverse and abundant phage communities in the gut compared with other relevant animals. Phylogenetic analyses based on the phage terminase large subunit (TerL) showed that the Caudovirales phages in giant pandas also presented highly genetic diversity. Our study revealed the diversity of phage communities in giant pandas and other relative species, contributing to the health maintenance of giant pandas and laying the groundwork for molecular evolution research of bacteriophages in mammals. IMPORTANCE Gut phageome plays an important role in shaping gut microbiomes by direct interactions with bacteria or indirect influences on the host immune system, potentially regulating host health and disease status. The giant panda (Ailuropoda melanoleuca) is a vulnerable and umbrella species for biodiversity conservation. Our work explored and compared the gut phageome of giant pandas and relative species, contributing to the health maintenance of giant pandas.

Gut commensal Enterocloster species host inoviruses that are secreted in vitro and in vivo.

BACKGROUND: Bacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized. RESULTS: In this study, we utilized in silico, in vitro, and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. (formerly Clostridium spp.). We confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated bacterial growth dynamics, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives. CONCLUSION: In this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria. Video Abstract.

Multiomic spatial analysis reveals a distinct mucosa-associated virome.

The human gut virome has been increasingly explored in recent years. However, nearly all virome-sequencing efforts rely solely on fecal samples and few studies leverage multiomic approaches to investigate phage-host relationships. Here, we combine metagenomics, metaviromics, and metatranscriptomics to study virome-bacteriome interactions at the colonic mucosal-luminal interface in a cohort of three individuals with inflammatory bowel disease; non-IBD controls were not included in this study. We show that the mucosal viral population is distinct from the stool virome and houses abundant crAss-like phages that are undetectable by fecal sampling. Through viral protein prediction and metatranscriptomic analysis, we explore viral gene transcription, prophage activation, and the relationship between the presence of integrase and temperate phages in IBD subjects. We also show the impact of deep sequencing on virus recovery and offer guidelines for selecting optimal sequencing depths in future metaviromic studies. Systems biology approaches such as those presented in this report will enhance our understanding of the human virome and its interactions with our microbiome and our health.

Bacteriophage-mediated manipulations of microbiota in gastrointestinal diseases.

Although some gastrointestinal diseases could be managed using various antibiotics regimen, this therapeutic approach lacks precision and damages the microbiota. Emerging literature suggests that phages may play a key role in restoring the gut microbiome balance and controlling disease progression either with exogenous phage intervention or filtered fecal transplantation or even engineered phages. In this review, we will discuss the current phage applications aiming at controlling the bacterial population and preventing infection, inflammation, and cancer progression in the context of gastrointestinal diseases.

Comparing In Vitro Faecal Fermentation Methods as Surrogates for Phage Therapy Application.

The human microbiome and its importance in health and disease have been the subject of numerous research articles. Most microbes reside in the digestive tract, with up to 1012 cells per gram of faecal material found in the colon. In terms of gene number, it has been estimated that the gut microbiome harbours >100 times more genes than the human genome. Several human intestinal diseases are strongly associated with disruptions in gut microbiome composition. Less studied components of the gut microbiome are the bacterial viruses called bacteriophages that may be present in numbers equal to or greater than the prokaryotes. Their potential to lyse their bacterial hosts, or to act as agents of horizontal gene transfer makes them important research targets. In this study in vitro faecal fermentation systems were developed and compared for their ability to act as surrogates for the human colon. Changes in bacterial and viral composition occurred after introducing a high-titre single phage preparation both with and without a known bacterial host during the 24 h-long fermentation. We also show that during this timeframe 50 mL plastic tubes can provide data similar to that generated in a sophisticated faecal fermenter system. This knowledge can guide us to a better understanding of the short-term impact of bacteriophage transplants on the bacteriomes and viromes of human recipients.

Multiomics signatures of type 1 diabetes with and without albuminuria.

Aims/hypothesis: To identify novel pathophysiological signatures of longstanding type 1 diabetes (T1D) with and without albuminuria we investigated the gut microbiome and blood metabolome in individuals with T1D and healthy controls (HC). We also mapped the functional underpinnings of the microbiome in relation to its metabolic role. Methods: One hundred and sixty-one individuals with T1D and 50 HC were recruited at the Steno Diabetes Center Copenhagen, Denmark. T1D cases were stratified based on levels of albuminuria into normoalbuminuria, moderate and severely increased albuminuria. Shotgun sequencing of bacterial and viral microbiome in stool samples and circulating metabolites and lipids profiling using mass spectroscopy in plasma of all participants were performed. Functional mapping of microbiome into Gut Metabolic Modules (GMMs) was done using EggNog and KEGG databases. Multiomics integration was performed using MOFA tool. Results: Measures of the gut bacterial beta diversity differed significantly between T1D and HC, either with moderately or severely increased albuminuria. Taxonomic analyses of the bacterial microbiota identified 51 species that differed in absolute abundance between T1D and HC (17 higher, 34 lower). Stratified on levels of albuminuria, 10 species were differentially abundant for the moderately increased albuminuria group, 63 for the severely increased albuminuria group while 25 were common and differentially abundant both for moderately and severely increased albuminuria groups, when compared to HC. Functional characterization of the bacteriome identified 23 differentially enriched GMMs between T1D and HC, mostly involved in sugar and amino acid metabolism. No differences in relation to albuminuria stratification was observed. Twenty-five phages were differentially abundant between T1D and HC groups. Six of these varied with albuminuria status. Plasma metabolomics indicated differences in the steroidogenesis and sugar metabolism and circulating sphingolipids in T1D individuals. We identified association between sphingolipid levels and Bacteroides sp. abundances. MOFA revealed reduced interactions between gut microbiome and plasma metabolome profiles albeit polar metabolite, lipids and bacteriome compositions contributed to the variance in albuminuria levels among T1D individuals. Conclusions: Individuals with T1D and progressive kidney disease stratified on levels of albuminuria show distinct signatures in their gut microbiome and blood metabolome.

Bidirectional Interactions between Arboviruses and the Bacterial and Viral Microbiota in Aedes aegypti and Culex quinquefasciatus.

Mosquitoes are important vectors for many arboviruses. It is becoming increasingly clear that various symbiotic microorganisms (including bacteria and insect-specific viruses; ISVs) in mosquitoes have the potential to modulate the ability of mosquitoes to transmit arboviruses. In this study, we compared the bacteriome and virome (both eukaryotic viruses and bacteriophages) of female adult Aedes aegypti and Culex quinquefasciatus mosquitoes fed with sucrose/water, blood, or blood spiked with Zika virus (ZIKV) or West Nile virus (WNV), respectively. Furthermore, we investigated associations between the microbiota and vector competence. We show that the influence of arboviruses on the mosquito microbiome-and vice versa-is distinct for each combination of arbovirus/mosquito species. The presence of ZIKV resulted in a temporarily increased Aedes ISV diversity. However, this effect was distinct for different ISVs: some ISVs decreased following the blood meal (Aedes aegypti totivirus), whereas other ISVs increased only when the blood contained ZIKV (Guadeloupe mosquito virus). Also, the diversity of the Aedes bacteriome depended on the diet and the presence of ZIKV, with a lower diversity observed for mosquitoes receiving blood without ZIKV. In Cx. quinquefasciatus, some ISVs increased in WNV-infected mosquitoes (Guadeloupe Culex tymo-like virus). Particularly, the presence of Wenzhou sobemo-like virus 3 (WSLV3) was associated with the absence of infectious WNV in mosquito heads, suggesting that WSLV3 might affect vector competence for WNV. Distinct profiles of bacteriophages were identified in Culex mosquitoes depending on diet, despite the lack of clear changes in the bacteriome. Overall, our data demonstrate a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus-mosquito combinations. A better understanding of these interactions may lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures. IMPORTANCE In this study, we first utilized the single mosquito microbiome analysis, demonstrating a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus-mosquito combinations. Some of the previously described "core virus" increased in the mosquitos receiving viral blood meal, like Guadeloupe mosquito virus and Guadeloupe Culex tymo-like virus, suggesting their potential roles in ZIKV and WNV infection. Notably, Wenzhou sobemo-like virus 3 was associated with the absence of infectious WNV in heads of Culex mosquitoes, which might affect vector competence for WNV. A better understanding of these interactions will lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures.