Phage Abundance Research Articles

Multiomic spatial analysis reveals a distinct mucosa-associated virome

ABSTRACT 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.

When Plaquing Is Not Possible: Computational Methods for Detecting Induced Phages.

High-throughput sequencing of microbial communities has uncovered a large, diverse population of phages. Frequently, phages found are integrated into their bacterial host genome. Distinguishing between phages in their integrated (lysogenic) and unintegrated (lytic) stage can provide insight into how phages shape bacterial communities. Here we present the Prophage Induction Estimator (PIE) to identify induced phages in genomic and metagenomic sequences. PIE takes raw sequencing reads and phage sequence predictions, performs read quality control, read assembly, and calculation of phage and non-phage sequence abundance and completeness. The distribution of abundances for non-phage sequences is used to predict induced phages with statistical confidence. In silico tests were conducted to benchmark this tool finding that PIE can detect induction events as well as phages with a relatively small burst size (10×). We then examined isolate genome sequencing data as well as a mock community and urinary metagenome data sets and found instances of induced phages in all three data sets. The flexibility of this software enables users to easily include phage predictions from their preferred tool of choice or phage sequences of interest. Thus, genomic and metagenomic sequencing now not only provides a means for discovering and identifying phage sequences but also the detection of induced prophages.

Bacteriophages and the Microbiome in Dermatology: The Role of the Phageome and a Potential Therapeutic Strategy.

Bacteriophages, also known as phages, are viruses that selectively target and infect bacteria. In addition to bacterial dysbiosis, dermatologic conditions such as acne, psoriasis, and atopic dermatitis are characterized by a relative reduction in the abundance of phages and the overgrowth of the corresponding bacteria. Phages often exhibit high specificity for their targeted bacteria, making phage-replacement therapy a promising therapeutic strategy for the control of pathogenic bacteria in dermatologic disease. Novel therapeutic strategies regulating pathogenic bacteria are especially necessary in light of growing antibiotic resistance. In this review, we aimed to review the medical literature assessing phage dysbiosis and therapeutic trials in dermatology. Ultimately, studies have depicted promising results for the treatment of acne, psoriasis, and atopic dermatitis but are limited by low sample sizes and the omission of control groups in some trials. Additional work is necessary to validate the efficacy depicted in proof-of-concept trials and to further determine optimal treatment vehicles, administration mechanisms, and dosing schedules. This review provides the necessary framework for the assessment of phage efficacy in future trials.

Any alcohol use in NAFLD patients is associated with significant changes to the intestinal virome.

The prevalence of alcohol use disorder (AUD) and metabolic dysfunction-associated fatty liver disease (MAFLD) are increasing worldwide, leading to the increasing likelihood of both etiologies contributing to a patient's liver disease. However, the effects of modest alcohol use in NAFLD are controversial and more studies are needed. We compared the intestinal viromes of patients with AUD and NAFLD in order to evaluate the effect of alcohol consumption on the intestinal viromes of NAFLD patients by extracting virus-like particles and performing metagenomic sequencing. Viral nucleic acids were extracted from fecal samples and subjected to metagenomic sequencing. We demonstrate significant differences in the intestinal viromes of NAFLD and AUD patients, and that alcohol use in NAFLD patients reclassified to MAFLD accounted for significant differences in the intestinal viromes. The relative abundance of several Lactococcus phages was more similar between AUD patients and alcohol-consuming MAFLD patients than non-alcohol-consuming MAFLD patients and control subjects, and multivariate modeling using the most discriminating Lactococcus phages could better predict alcohol use in the MAFLD population than the alcohol-associated liver disease/NAFLD Index. Significant differences in the viral composition and diversity were also seen between MAFLD patients with low and moderate alcohol consumption compared with no alcohol consumption. The intestinal virome of MAFLD patients who consume low to moderate amounts of alcohol are significantly different from those who do not, and many features of the intestinal virome of alcohol-consuming MAFLD patients resemble that of AUD patients.

A Genome of Temperate Enterococcus Bacteriophage Placed in a Space of Pooled Viral Dark Matter Sequences.

In the human gut, temperate bacteriophages interact with bacteria through predation and horizontal gene transfer. Relying on taxonomic data, metagenomic studies have associated shifts in phage abundance with a number of human diseases. The temperate bacteriophage VEsP-1 with siphovirus morphology was isolated from a sample of river water using Enterococcus faecalis as a host. Starting from the whole genome sequence of VEsP-1, we retrieved related phage genomes in blastp searches of the tail protein and large terminase sequences, and blastn searches of the whole genome sequences, with matches compiled from several different databases, and visualized a part of viral dark matter sequence space. The genome network and phylogenomic analyses resulted in the proposal of a novel genus "Vespunovirus", consisting of temperate, mainly metagenomic phages infecting Enterococcus spp.

CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice.

Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.

The Emerging Role of the Gut Virome in Health and Inflammatory Bowel Disease: Challenges, Covariates and a Viral Imbalance.

Virome research is a rapidly growing area in the microbiome field that is increasingly associated with human diseases, such as inflammatory bowel disease (IBD). Although substantial progress has been made, major methodological challenges limit our understanding of the virota. In this review, we describe challenges that must be considered to accurately report the virome composition and the current knowledge on the virome in health and IBD. First, the description of the virome shows strong methodological biases related to wetlab (e.g., VLP enrichment) and bioinformatics approaches (viral identification and classification). Second, IBD patients show consistent viral imbalances characterized by a high relative abundance of phages belonging to the Caudovirales and a low relative abundance of phages belonging to the Microviridae. Simultaneously, a sporadic contraction of CrAss-like phages and a potential expansion of the lysogenic potential of the intestinal virome are observed. Finally, despite numerous studies that have conducted diversity analysis, it is difficult to draw firm conclusions due to methodological biases. Overall, we present the many methodological and environmental factors that influence the virome, its current consensus in health and IBD, and a contributing hypothesis called the "positive inflammatory feedback loop" that may play a role in the pathophysiology of IBD.

In silico discovering relationship between bacteriophages and antimicrobial resistance

Bacteriophages and their potential contribution to antimicrobial resistance (AMR) have attracted growing attention in the context of medicine and pharmaceutics. A major objective of the CAMDA challenge is to acquire more knowledge about the relationship between viruses, their hosts and AMR genes in determining if AMR indeed can spread through phages. This study is focused on exploring the relationship and possible dependencies between bacteriophages and AMR based on the data collected from urban environments all over the world. The samples in the data are classified into two categories: high and low, according to the observed levels of AMR genes. The approach used in our analyses consists of several different methods which assess the differential abundance of phages, their diversity across samples, the impact on AMR categories and associations with AMR genes. The relationship between phages, their hosts and AMR is also explored by a Bayesian spatial model, using the AMR category (low vs high) as a factor. We found a higher relative risk for phages known to infect Staphylococcus aureus alone or both S. aureus and Acinetobacter baumannii in the high AMR group, which implies that these phages may have a role in the dissemination of antimicrobial genes.

Tailed bacteriophages (Caudoviricetes) dominate the microbiome of a diseased stingless bee.

Bacteriophages, viruses that infect bacterial hosts, are known to rule the dynamics and diversity of bacterial populations in a number of ecosystems. Bacterial communities residing in the gut of animals, known as the gut microbiome, have revolutionized our understanding of many diseases. However, the gut phageome, while of apparent importance in this context, remains an underexplored area of research. Here we identify for the first time genomic sequences from tailed viruses (Caudoviricetes) that are associated with the microbiome of stingless bees (Melipona quadrifasciata). Both DNA and RNA were extracted from virus particles isolated from healthy and diseased forager bees, the latter showing symptoms from an annual syndrome that only affects M. quadrifasciata. Viral contigs from previously sequenced metagenomes of healthy and diseased forager bees were used for the analyses. Using conserved proteins deduced from their genomes, we found that Caudoviricetes were only present in the worker bee gut microbiome from diseased stingless bees. The most abundant phages are phylogenetically related to phages that infect Gram-positive bacteria from the order Lactobacillales and Gram-negative bacteria from the genus Gilliamella and Bartonella, that are common honey bee symbionts. The potential implication of these viruses in the M. quadrifasciata syndrome is discussed.

Microbial and phage communities as well as their interaction in PO saponification wastewater treatment systems.

Viruses or phages were considered affecting microbial community composition, metabolic process, and biogeochemical cycles. However, phage communities and their potential associations with microbial community are not well understood in the activated sludge (AS) of wastewater treatment plants (WWTPs). In this study, we explored the interactions between phages and microbial community by using propylene oxide (PO) saponification WWTPs as an example. Bacterial, eukaryal and archaeal communities were investigated and 34 phage contigs (>10 kb) were recovered from PO saponification WWTPs. At least 3 complete phage genomes were assembled. In all 34 phages, 21 of them have been predicted to their host. The association network analysis showed that abundant phages were associated with abundant microorganisms. This result conformed to Kill-the-Winner model. Notably, 45 auxiliary metabolic genes (AMGs) were identified from phage genomes (including small contig fragments). They influenced bacterial metabolism through facilitating phages replication and avoiding host death. Collectively, our results suggested that phage community affect microbial community and metabolic pathways by killing their hosts and AMGs transfer in AS of PO saponification WWTPs.

Impact of HIV infection and integrase strand transfer inhibitors-based treatment on the gut virome

Viruses are the most abundant components of the human gut microbiome with a significant impact on health and disease. The effects of human immunodeficiency virus (HIV) infection on gut virome has been scarcely analysed. Several studies suggested that integrase strand transfers inhibitors (INSTIs) are associated with a healthier gut. Thus, the objective of this work was to evaluate the effects of HIV infection and INSTIs on gut virome composition. 26 non-HIV-infected volunteers, 15 naive HIV-infected patients and 15 INSTIs-treated HIV-infected patients were recruited and their gut virome composition was analysed using shotgun sequencing. Bacteriophages were the most abundant and diverse viruses present in gut. HIV infection was accompanied by a decrease in phage richness which was reverted after INSTIs-based treatment. β-diversity of phages revealed that samples from HIV-infected patients clustered separately from those belonging to the control group. Differential abundant analysis showed an increase in phages belonging to Caudoviricetes class in the naive group and a decrease of Malgrandaviricetes class phages in the INSTIs-treated group compared to the control group. Besides, it was observed that INSTIs-based treatment was not able to reverse the increase of lysogenic phages associated with HIV infection or to modify the decrease observed on the relative abundance of Proteobacteria-infecting phages. Our study describes for the first time the impact of HIV and INSTIs on gut virome and demonstrates that INSTIs-based treatments are able to partially restore gut dysbiosis at the viral level, which opens several opportunities for new studies focused on microbiota-based therapies.

Metagenomics analysis reveals potential pathways and drivers of piglet gut phage-mediated transfer of ARGs

The growing prevalence of antibiotic-resistant pathogens has led to a better understanding of the underlying processes that lead to this expansion. Intensive pig farms are considered one of the hotspots for antibiotic resistance gene (ARG) transmission. Phages, as important mobile carriers of ARGs, are widespread in the animal intestine. However, our understanding of phage-associated ARGs in the pig intestine and their underlying drivers is limited. Here, metagenomic sequencing and analysis of viral DNA and total DNA of different intestinal (ileum, cecum and feces) contents in healthy piglets and piglets with diarrhea were separately conducted. We found that phages in piglet ceca are the main repository for ARGs and mobile genetic element (MGE) genes. Phage-associated MGEs are important factors affecting the maintenance and transfer of ARGs. Interestingly, the colocalization of ARGs and MGE genes in piglet gut phages does not appear to be randomly selected but rather related to a specific phage host (Streptococcus). In addition, in the feces of piglets with diarrhea, the abundance of phages carrying ARGs and MGE genes was significantly increased, as was the diversity of polyvalent phages (phages with broad host ranges), which would facilitate the transfection and wider distribution of ARGs in the bacterial community. Moreover, the predicted host spectrum of polyvalent phages in diarrheal feces tended to be potential enteropathogenic genera, which greatly increased the risk of enteropathogens acquiring ARGs. Notably, we also found ARG-homologous genes in the sequences of piglet intestinal mimiviruses, suggesting that the piglet intestinal mimiviruses are a potential repository of ARGs. In conclusion, this study greatly expands our knowledge of the piglet gut microbiome, revealing the underlying mechanisms of maintenance and dissemination of piglet gut ARGs and providing a reference for the prevention and control of ARG pollution in animal husbandry.

Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

Novel findings in context of molecular diversity and abundance of bacteriophages in wastewater environments of Riyadh, Saudi Arabia.

The diversity among bacteriophages depends on different factors like ecology, temperature conditions and genetic pool. Current study focused on isolation, identification and diversity of phages from 34 sewage water samples collected from two different wastewater treatment plants (WWTPs), King Saud University wastewater treatment plants (KSU-WWTP) and Manfoha wastewater treatment plants (MN-WWTP) in Riyadh, Saudi Arabia. Samples were analyzed by PCR and Next Generation Sequencing (NGS). Siphoviridae, Podoviridae and Myoviridae families were detected by family-specific PCR and highest prevalence of Myoviridae 29.40% was found at MN-WWTP followed by 11.76% at KSU-WWTP. Siphoviridae was detected 11.76% at MN-WWTP and 5.88% at KSU-WWTP. Lowest prevalence for Podoviridae family (5.88%) was recorded at MN-WWTP. Significant influence of temporal variations on prevalence of Myoviridae and Siphoviridae was detected in both WWTP and MN-WWTP, respectively. Highest phage prevalence was obtained in August (75%), followed by September (50%). Highest phage prevalence was recorded at a temperature range of 29–33°C. Significant influence of temperature on the prevalence of Myoviridae phages was detected at MN-WWTP. Four bacteriophages with various abundance levels were identified by NGS. Cronobacter virus Esp2949-1 was found first time with highest abundance (4.41%) in wastewater of Riyadh. Bordetella virus BPP1 (4.14%), Dickeya virus Limestone (1.55%) and Ralstonia virus RSA1 (1.04%) were also detected from samples of MN-WWTP. Highest occurrence of Bordetella virus BPP1 (67%) and (33.33%) was recorded at KSU-WWTP and MN-WWTP, respectively. Highest Bordetella virus BPP1 occurrence was recorded in September (50%) followed by August (40%). The findings of study showed new insights of phage diversity from wastewater sources and further large-scale data studies are suggested for comprehensive understanding.

Cyanophage dynamics in a tropical urban freshwater lake

Considerable studies have been conducted to quantify and examine free cyanophage particles across a wide range of aquatic systems, but few studies have analyzed the dynamics of host cell fraction cyanophages, and even less is known about tropical freshwater cyanophages. Here, we employed qPCR to examine the abundances of four ecologically relevant novel cyanophages (PA-SR01, S-SRP01, S-SRP02 and S-SRM01) in a tropical urban lake in Singapore from July to November 2019. Besides quantifying the extracellular free cyanophage particles, host cell fraction cyanophage were also examined to represent the abundance of intracellular and attached phages. Our study revealed differing fate and dynamics of each cyanophage target. Host cell fraction phage abundance was found to be more abundant than free phage abundance, indicating high level of infection and rapid dispersal of phage particles upon lysis. By further investigating the correlations between free phage particle abundance and numerous biotic (total cyanobacteria, Microcystis abundances, etc) as well as abiotic (nutrients and physiochemical water quality) variables, multiple correlations across different sampling sites were obtained, suggesting pH and rainfall to be potential key factors driving shifts in both cyanophage and cyanobacteria population. This study demonstrated the ecological importance and key roles of the cyanophage PA-SR01, S-SRP01, S-SRP02 and S-SRM01 in tropical freshwater lakes not only as regulators of the cyanobacterial community, but also as promoters of certain cyanotoxin production.

Dynamics of nasopharyngeal tract phageome and association with disease severity and age of patients during three waves of COVID-19.

In December 2019, several patients were hospitalized and diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, which subsequently led to a global pandemic. To date, there are no studies evaluating the relationship between the respiratory phageome and the SARS‐CoV‐2 infection. The current study investigated the phageome profiles in the nasopharyngeal swabs collected from 55 patients during the three different waves of coronavirus disease 2019 (COVID‐19) in the Campania Region (Southern Italy). Data obtained from the taxonomic profiling show that phage families belonging to the order Caudovirales have a high abundance in the patient samples. Moreover, the severity of the COVID‐19 infection seems to be correlated with the phage abundance.

A relationship between phages and organic carbon in wastewater treatment plant effluents

With stringent effluent requirements and the implementation of new processes for micropollutant removal, it is increasingly important for wastewater treatment plants (WWTPs) to understand the factors affecting effluent quality. Phages (viruses infecting prokaryotes) are abundant in the biological treatment processes. They can contribute to organic carbon in the treated effluent both because they are organic in nature and occur in the effluent and because they cause lysis of microorganisms. Today very little is known about the effects of phages on effluent quality. The goal of this study was, therefore, to determine the relationship between phages and organic carbon in WWTP effluents. We also examined the diversity, taxonomy, and host-association of DNA phages using metagenomics. Effluent samples were collected from four WWTPs treating municipal wastewater. Significant differences in both organic carbon and virus-like particle concentrations were observed between the plants and there was a linear relationship between the two parameters. The phage communities were diverse with many members being taxonomically unclassified. Putative hosts were dominated by bacteria known to be abundant in activated sludge systems such as Comamonadaceae. The composition of phages differed between the WWTPs, suggesting that local conditions shape the communities. Overall, our findings suggest that the abundance and composition of phages are related to effluent quality. Thus, there is a need for further research clarifying the association between phage dynamics and WWTP function.

Gut virome dysbiosis following focal cerebral ischemia in mice

Stroke leads to gut bacterial dysbiosis that impacts the post-stroke outcome. The gut microbiome also contains a high abundance of viruses which might play a crucial role in disease progression and recovery by modulating the metabolism of both host and host’s gut bacteria. We presently analyzed the virome composition (viruses and phages) by shotgun metagenomics in the fecal samples obtained at 1 day of reperfusion following transient focal ischemia in adult mice. Viral genomes, viral auxiliary metabolic genes, and viral protein networks were compared between stroke and sham conditions (stroke vs sham, exclusive to sham and exclusive to stroke). Following focal ischemia, abundances of 2 viral taxa decreased, and 5 viral taxa increased compared with the sham. Furthermore, the abundance of Clostridia-like phages and Erysipelatoclostridiaceae-like phages were altered in the stroke compared with the sham cohorts. This is the first report to show that the gut virome responds acutely to stroke.

Metagenomic analysis of wastewater phageome from a University Hospital in Turkey.

Phage DNA analysis gives opportunity to understand living ecosystem of the environment where the samples are taken. In the present study, we analyzed phage DNA obtained from wastewater sample of university hospital sewage. After filtration, long high-speed centrifugation was done to collect phages. DNA was extracted from pellet by phenol chloroform extraction and used for NGS sequencing. The host profile, taxonomic and functional analyses were performed using MG-RAST, and ResFinder program was used for resistance gene detection. High amounts of reads belong to bacteriophage groups (~ 95%) from our DNA sample were obtained and all bacteriophage reads were found belonging to Caudovirales order and Myoviridae (56%), Siphoviridae (43%), and Podoviridae (0.02%) families. The most common host genera were Escherichia (88.20%), Salmonella (5.49%) and Staphylococcus (5.19%). SEED subsystems hits were mostly structural parts and KEGG Orthology hits were nucleotide- and carbohydrate metabolism-related genes. No anti-microbial resistance genes were detected. Our bacteriophage DNA purification method is favorable for phage metagenomic studies. Dominance of coliphages may explain infrequent Podoviridae. Dominancy of structural genes and auxiliary genes is probably due to abundance of lytic phages in our sample. Absence of antibiotic resistance genes even in hospital environment phages indicates that phages are not important carrier of resistance genes.

Community structure and function of microbiomes in polluted stretches of river Yamuna in New Delhi, India, using shotgun metagenomics.

The large population residing in the northern region of India surrounding Delhi mostly depends on water of River Yamuna, a tributary of mighty Ganga for agriculture, drinking and various religious activities. However, continuous anthropogenic activities mostly due to pollution mediated by rapid urbanization and industrialization have profoundly affected river microflora and their function thus its health. In this study, potential of whole-genome metagenomics was exploited to unravel the novel consortia of microbiome and their functional potential in the polluted sediments of the river at Delhi. Analysis of high-quality metagenome data from Illumina NextSeq500 revealed substantial differences in composition of microbiota at different sites dominated by Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Chloroflexi phyla. The presence of highly dominant anaerobic bacteria like Dechloromonas aromatica (benzene reducing and denitrifying), Rhodopseudomonas palustris (organic matter reducing), Syntrophus aciditrophicus (fatty acid reducing) and Syntrophobacter fumaroxidans (sulphate reducing) in the polluted river Yamuna signifies the impact of unchecked pollution in declining health of the river ecosystem. A decline in abundance of phages was also noticed along the downstream river Yamuna. Mining of mycobiome reads uncovered plethora of fungal communities (i.e. Nakaseomyces, Aspergillus, Schizosaccharomyces and Lodderomyces) in the polluted stretches due to the availability of higher organic carbon and total nitrogen (%) could be decoded as promising bioindicators of river trophic status. Pathway analysis through KEGG revealed higher abundance of genes involved in energy metabolism (nitrogen and sulphur), methane metabolism, degradation of xenobiotics (Nitrotoluene, Benzoate and Atrazine), two-component system (atoB, cusA and silA) and membrane transport (ABC transporters). Catalase-peroxidase and 4-hydroxybenzoate 3-monooxygenase were the most enriched pollution degrading enzymes in the polluted study sites of river Yamuna. Overall, our results provide crucial insights into microbial dynamics and their function in response to high pollution and could be insightful to the ongoing remediation strategies to clean river Yamuna.