Phage Group Research Articles

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.

Staphylococcal scalded skin syndrome in neonate: Another face of CA-MRSA.

Staphylococcal scalded skin syndrome (SSSS), also known as Ritter's disease, in its severe form occurs predominantly in infants and children. It is caused by infection with group II (often phage group 71) Staphylococcus aureus. The foci of infection include nasopharynx, less commonly umbilicus, urinary tract, superficial abrasion, conjunctivae, and blood. Staphylococci are non-motile, non-spore-forming, catalase-positive, gram-positive cocci that appear predominantly as grape-like clusters. Although this organism is frequently a part of normal human microbial flora, it can cause significant opportunistic infections under certain conditions such as when extremes of age groups are involved, the presence of indwelling medical devices, and intravenous (iv) drug abuse. Staphylococcus aureus may cause a variety of infectious manifestations ranging from relatively benign skin infections to life-threatening systemic illnesses. SSSS caused by S. aureus strains produces exfoliative toxins which result in the development of blisters, erythema, and desquamation. Here, we present a case of an 11-day-old neonate who was diagnosed with SSSS. The causative agent responsible for this syndrome was identified as methicillin-resistant Staphylococcus aureus (MRSA). The molecular characterization of the gene Panton-Valentine leukocidin (PVL) was done by polymerase chain reaction (PCR) and was detected positive for PVL which is a distinctive virulence factor seen almost in all of the community-acquired MRSA strains. The patient was discharged after parenteral clindamycin therapy with almost complete resolution of symptoms.

The Role of Prophage ϕSa3 in the Adaption of Staphylococcus aureus ST398 Sublineages from Human to Animal Hosts.

Staphylococcus aureus sequence type (ST) 398 is a lineage affecting both humans and livestock worldwide. However, the mechanisms underlying its clonal evolution are still not clearly elucidated. We applied whole-genome sequencing (WGS) typing to 45 S. aureus strains from China and Canada between 2005 and 2014, in order to gain insight into their evolutionary pathway. Based on WGS phylogenetic analysis, 42 isolates were assigned to the human-associated clade (I/II-GOI) and 3 isolates to livestock-associated clade (IIa). Phylogeny of ϕSa3 sequences revealed five phage groups (Groups 1-5), with Group 1 carrying ϕSa3-Group 1 (ϕSa3-G1), Group 2 carrying ϕSa3-G2, Group 3 carrying ϕSa3-G3, Group 4 carrying ϕSa3-G4 and Group 5 lacking ϕSa3. ϕSa3-G1 was only found in strains that accounted for the most ancestral human clade I, while ϕSa3-G2, ϕSa3-G3 and ϕSa3-G4 were found restricted to sublineages within clade II-GOI. Some isolates of clade II-GOI were also found to be ϕSa3-negative or resistant to methicillin which are unusual characteristics for human-adapted isolates. This study demonstrated a strong association between phylogenetic grouping and phage type, suggesting an important role of ϕSa3 prophage in the evolution of human-adapted ST398 subclones. In addition, our results suggest that this subclone slowly began to adapt to animal hosts by losing ϕSa3 and acquiring methicillin resistance, which was observed in some strains of human-associated clade II-GOI, an intermediate human to livestock transmission clade.

A Randomized, Placebo-controlled, Double-blind Clinical Trial of Bacteriophage Cocktails in Chronic Wound Infections.

Chronic wounds are prevalent globally at endemic proportions. The common features associated with chronic wounds are prolonged inflammatory phase, infection with multidrug-resistant (MDR) bacteria, and subsequent biofilm formation. The present randomized-controlled trial (RCT) study was undertaken on chronic wounds of ≥6 weeks longer duration using customized phages to evaluate the efficacy and safety of bacteriophage therapy. The study was conducted from December 2021 to August 2023. Thirty patients in each of the arms (placebo and bacteriophage) were recruited with chronic wounds. The patients, both arms, received the conventional treatment of wound debridement, local antiseptics, and local and systemic antibiotics at the discretion of the treating surgeon. However, before applying the customized bacteriophage cocktail or placebo, the wound surface was thoroughly washed to remove the residual antiseptics. The phage cocktails or placebo were applied on alternate days. The wounds were evaluated using the Bates-Jensen Wound Assessment Tool for the progress of wound healing. A total of 93.3% of the wound became sterile in 39 days (median sterility time), followed by complete healing by the end of 90 days in the phage group. Contrary to this, 83.3% of those on placebo therapy remained colonized by original bacteria or additional new bacteria without healing for up to 90 days. With the well-designed RCT, we could conclude that customized bacteriophage therapy using bacteriophage cocktails will definitely cure the chronic wound, irrespective of age, sex, diabetes status, and infection by MDR bacteria.

Recruitment of complete crAss-like phage genomes reveals their presence in chicken viromes, few human-specific phages, and lack of universal detection.

The order Crassvirales, which includes the prototypical crAssphage (p-crAssphage), is predominantly associated with humans, rendering it the most abundant and widely distributed group of DNA phages in the human gut. The reported human specificity and wide global distribution of p-crAssphage makes it a promising human fecal marker. However, the specificity for the human gut as well as the geographical distribution around the globe of other members of the order Crassvirales remains unknown. To determine this, a recruitment analysis using 91 complete, non-redundant genomes of crAss-like phages in human and animal viromes revealed that only 13 crAss-like phages among the 91 phages analyzed were highly specific to humans, and p-crAssphage was not in this group. Investigations to elucidate whether any characteristic of the phages was responsible for their prevalence in humans showed that the 13 human crAss-like phages do not share a core genome. Phylogenomic analysis placed them in three independent families, indicating that within the Crassvirales group, human specificity is likely not a feature of a common ancestor but rather was introduced on separate/independent occasions in their evolutionary history. The 13 human crAss-like phages showed variable geographical distribution across human metagenomes worldwide, with some being more prevalent in certain countries than in others, but none being universally identified. The varied geographical distribution and the absence of a phylogenetic relationship among the human crAss-like phages are attributed to the emergence and dissemination of their bacterial host, the symbiotic human strains of Bacteroides, across various human populations occupying diverse ecological niches worldwide.

Active prophages in coral-associated Halomonas capable of lateral transduction.

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

Oral phages prophylaxis against mixed Escherichia coli O157:H7 and Salmonella Typhimurium infections in weaned piglets

Escherichia coli and Salmonella Typhimurium are the main pathogens of diarrhea in weaned piglets. The prevention of bacterial diarrhea in weaned piglets by phage is rarely reported. We conducted this study to evaluate the preventive effect of phages on mixed Escherichia coli and Salmonella Typhimurium infections in weaned piglets. A novel phage named NJ12 was isolated by using Salmonella Typhimurium SM022 as host bacteria and characterized by electron microscopy, genomic analysis and in vitro bacteriostatic activity. Phage NJ12 and a previously reported phage EP01 were microencapsulated with sodium alginate to make phage cocktail. Microencapsulated phage cocktail and PBS (Phosphate buffer solution) were used to piglets the phage and phage-free group through oral administration before bacterial infection 2 h, respectively. Piglets of the phage and phage-free group were consumed with feed contaminated with 6 mL (108CFU/mL) Escherichia coli O157:H7 GN07 (GXEC-N07) and 6 mL (108CFU/mL) SM022 every day for seven consecutive days. The results showed that piglets in the phage-free group had more severe diarrhea, larger decreased average weight gain and higher levels of neutrophils compared with piglets in phage group. Meanwhile, piglets in the phage-free group had higher load of SM022 and GN07 in jejunal tissue and more severe intestinal damage compared with piglets in group phage in vivo. In addition, oral administration phage can significant decreased the relative abundance of Enterobacteriaceae but hardly repaired the changes of diversity and composition of gut microbiota caused by the mixed infection of SM022 and GN07. This implies that phage used as a feed additive have a marvelous preventive effect on bacterial diarrhea during weaning of piglets.

Detection and Quantification of Bacteriophages in Wastewater Samples by Culture and Molecular Methods.

Bacteriophages are promising tools for the detection of fecal pollution in water bodies and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages are perfect surrogates for the study of the fate and transport of enteric viruses, generally better than any other group of indicators.Different groups of bacteriophages, such as somatic coliphages, F-specific RNA bacteriophages, and bacteriophages infecting selected strains of Bacteroides, have been comprehensively tested as indicators of fecal pollution. Somatic coliphages and F-specific RNA bacteriophages can be used as indicators of general fecal contamination, whereas Bacteroides phages can be used to detect a particular fecal source, for instance, human, bovine, porcine, or poultry fecal contamination.Feasible and cost-effective protocols standardized by the International Standardization Organization and the United States Environmental Protection Agency for the detection of infectious bacteriophages belonging to these three groups are available. Molecular methods for the detection of some particular phages have also been developed. Here we introduce those methods for the detection, enumeration, and isolation of bacteriophages in wastewater samples.

Analysis of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Revealed Novel Groups of Filamentous Bacteriophages.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.

Comparative analysis of Actinobacteria phage-plasmids and their transduction potential

The genus Streptomyces is the most extensively studied group within actinobacteria, which are widely used for the production of antibiotics and other metabolites. Streptomyces bacteriophages attract a lot of interest to study phage-host coevolution. One of the probable interactions is a bacteriophage-mediated horizontal transfer of genes encoding bacterial metabolites, which are important for humans. In this study, a search for Streptomyces bacteriophages having capacities necessary for efficient horizontal transfer was performed. Groups of bacteriophages of actinobacteria and their plasmids with relatively long GC-rich sequences containing both genes related to viral morphogenesis and plasmid-associated genes were revealed by comparative genome analysis. In one of these groups, homologs of proteins of DNA packaging into a capsid of Punavirus genus phages, that representatives are capable to perform a horizontal transfer of genes with high frequency were identified. Taking it into account, as well as the peculiarities of their genomic structure, horizontal gene transfer by this group of viruses and plasmids was assumed to occur. Considering the genome length and GC-content, it is thought that these viruses could be useful for producers construction. Moreover, some given examples showing the chimerical origin of phage-plasmids allow suggesting that recombination events between considered phages-plasmids occur at high frequency. New representatives of viruses with low similarity to known phage genomes were also detected among plasmids. A wide diversity of actinobacterial phage-plasmids that is related to their variability and a probable existence of a great number of sequences of this type of viruses in databases were found out.

Reductionism Debate in Molecular Biology: Max Delbrück’s Complementarity Approach

In this paper, I address Max Delbrück’s conceptual and experimental importance for molecular biology (henceforth MB) origins. In particular, his complementarity approach and its anti-reductive implications on the (epistemic) reductionism debate in MB. Regarding Delbrück’s conceptual and experimental importance, I examine his influence on the development of MB by exploring a shift of his interests from physics to biology. Particularly, I outline his central role in “The Phage Group”, the informal group of scientists examining the origin of hereditary life using bacteriophages as their experimental model of choice. Delbrück and “The Phage Group” greatly influenced the development of MB, which culminated with the shared 1969 Nobel Prize for the discoveries regarding replication mechanism and genetic structure of viruses. Moreover, I examine Delbrück’s complementarity approach towards biological explanations. The complementarity in biology assumes that “biological phenomena might require the employment of descriptions that are mutually exclusive yet jointly necessary for understanding life processes” (McKaughan 2011, p. 11). I explore Delbrück’s complementarity approach, in particular the debate between the reductive and anti-reductive interpretations of it. I argue for the latter interpretation by suggesting that Delbrück advanced an anti-reductive view towards biological explanations by advocating for independent status of explanations of various biological disciplines. Furthermore, I address the complementarity approach in the light of the anti-reductive interpretation in the recent developments in MB, particularly, the potentiality of finding the complementarity approach in systems biology, epigenetics, and boundary selection.

PhamClust: a phage genome clustering tool using proteomic equivalence.

Bacteriophage comparative genomics is complex due to the mosaic nature of the genomes, and an underlying continuum of diversity confounds the identification of clear taxonomic boundaries. Nucleotide sequence comparison methods have been described for phage taxonomy, but they are computationally intensive and scale poorly as the number of sequenced phage genomes increases. Here, we describe PhamClust as a new bioinformatic approach for grouping phages according to their inter-genome relatedness. PhamClust calculates a proteomic equivalence quotient (PEQ) for each pair of phages based on amino acid sequence identity for those genes that are shared among phages. PEQ values span from 0% (no shared genes) to 100% (all genes shared at 100% identity), and using a large mycobacteriophage genome data set, we show that two-step clustering down to a PEQ of 25% constructs genome groupings (clusters) closely mirroring those constructed manually over time, with the differences attributable to historically arising incongruities rather than illogicalities in PhamClust. PEQ values can also faithfully divide clusters into subclusters, although the relationships are highly heterogeneous, with different PEQ values needed for the subdivision of different clusters. PhamClust can be used to assort any group of phages, including the RefSeq phage collection. IMPORTANCE Bacteriophage genomes are pervasively mosaic, presenting challenges to describing phage relatedness. Here, we describe PhamClust, a bioinformatic approach for phage genome comparisons that uses a new metric of proteomic equivalence quotient for comparative genomics. PhamClust reliably assorts genomes into groups or clusters of related phages and can subdivide clusters into subclusters. PhamClust is computationally efficient and can readily process thousands of phage genomes. It is also a useful analytic tool for exploring the different types of inter-genome relatedness characteristic of phages in different clusters.

The Novel Yersinia enterocolitica Telomere Phage vB_YenS_P840 Is Closely Related to PY54, but Reveals Some Striking Differences.

Telomere phages are a small group of temperate phages, whose prophages replicate as a linear plasmid with covalently closed ends. They have been isolated from some Enterobacteriaceae and from bacterial species living in aquatic environments. Phage PY54 was the first Yersinia (Y.) enterocolitica telomere phage isolated from a nonpathogenic O:5 strain, but recently a second telomeric Yersinia phage (vB_YenS_P840) was isolated from a tonsil of a wild boar in Germany. Both PY54 and vB_YenS_P840 (P840) have a siphoviridal morphology and a similar genome organization including the primary immunity region immB and telomere resolution site telRL. However, whereas PY54 only possesses one prophage repressor for the lysogenic cycle, vB_YenS_P840 encodes two. The telRL region of this phage was shown to be processed by the PY54 protelomerase under in vivo conditions, but unlike with PY54, a flanking inverted repeat was not required for processing. A further substantial difference between the phages is their host specificity. While PY54 infects Y. enterocolitica strains belonging to the serotypes O:5 and O:5,27, vB_YenS_P840 exclusively lyses O:3 strains. As the tail fiber and tail fiber assembly proteins of the phages differ significantly, we introduced the corresponding genes of vB_YenS_P840 by transposon mutagenesis into the PY54 genome and isolated several mutants that were able to infect both serotypes, O:5,27 and O:3.

Novel T4 bacteriophage and royal jelly infused disinfectant versus E. coli compared to QACs

Antimicrobial resistance is an increasingly worsening phenomenon in the 21st century having resulted in thousands of deaths per year. Increases in the diffusion of antimicrobial resistance in gram-negative bacteria and prevalence of specific genes leading to resistance have been linked to an excessive usage of quaternary ammonium compounds (QACs) in gram-negative bacteria such as Escherichia coli. This experiment aims to develop a novel disinfectant solution (T4MRJP) that utilizes the entero-bacteriophage t4 and nine major royal jelly proteins (MRJPs) to inhibit the growth of E. coli on a MHA (Mueller Hinton Agar) growth medium. In the experiment, the Kirby Bauer Disk Diffusion Assay was first applied on six MHA plates inoculated with 36 evenly distributed susceptibility disks containing different concentrations of solution for three QAC groups, a positive t4 bacteriophage group, and two T4 groups at 90% and 98% dilution respectively. Results demonstrated a strong positive correlation between the increases of concentration to demonstrated inhibition but a lack of statistical significance between the T4 and QAC groups in regards to the proposed hypothesis. However, the diluted T4 group was just as effective as the QAC groups at inhibiting bacterial growth, especially at higher concentrations of the administered solution. In a second trial, however, the T4MRJP (Major royal jelly protein) cocktail was significantly less effective than the experimented QAC groups compared to the bacteriophage alone.

Genomic diversity and biogeographic distributions of a novel lineage of bacteriophages that infect marine OM43 bacteria.

The marine methylotrophic OM43 clade is considered an important bacterial group in coastal microbial communities. OM43 bacteria, which are closely related to phytoplankton blooms, have small cell sizes and streamlined genomes. Bacteriophages profoundly shape the evolutionary trajectories, population dynamics, and physiology of microbes. The prevalence and diversity of several phages that infect OM43 bacteria have been reported. In this study, we isolated and sequenced two novel OM43 phages, MEP401 and MEP402. These phages share 90% of their open reading frames (ORFs) and are distinct from other known phage isolates. Furthermore, a total of 99 metagenomic viral genomes (MVGs) closely related to MEP401 and MEP402 were identified. Phylogenomic analyses suggest that MEP401, MEP402, and these identified MVGs belong to a novel subfamily in the family Zobellviridae and that they can be separated into two groups. Group I MVGs show conserved whole-genome synteny with MEP401, while group II MVGs possess the MEP401-type DNA replication module and a distinct type of morphogenesis and packaging module, suggesting that genomic recombination occurred between phages. Most members in these two groups were predicted to infect OM43 bacteria. Metagenomic read-mapping analysis revealed that the phages in these two groups are globally ubiquitous and display distinct biogeographic distributions, with some phages being predominant in cold regions, some exclusively detected in estuarine stations, and others displaying wider distributions. This study expands our knowledge of the diversity and ecology of a novel phage lineage that infects OM43 bacteria by describing their genomic diversity and global distribution patterns. IMPORTANCE OM43 phages that infect marine OM43 bacteria are important for host mortality, community structure, and physiological functions. In this study, two OM43 phages were isolated and characterized. Metagenomic viral genome (MVG) retrieval using these two OM43 phages as baits led to the identification of two phage groups of a new subfamily in the family Zobellviridae. We found that group I MVGs share similar genomic content and arrangement with MEP401 and MEP402, whereas group II MVGs only possess the MEP401-type DNA replication module. Metagenomic mapping analysis suggests that members in these two groups are globally ubiquitous with distinct distribution patterns. This study provides important insights into the genomic diversity and biogeography of the OM43 phages in the global ocean.

Impact of multizonal decontamination of upper respiratory tract on incidence of ventilator-associated pneumonia: multicenter randomized pilot study

INTRODUCTION: Ventilator-associated pneumonia (VAP) is the one of nosocomial infections in intensive care units. VAP can result in prolonged duration of hospitalization and invasive mechanical ventilation (MV) that is associated with attributive mortality. OBJECTIVE: The aim of our pilot multicenter randomized study was to assess effect of the multizonal decontamination of upper airway including subglottic space on the VAP incidence and time of onset, as well as colonization upper airway and clinical outcomes. MATERIALS AND METHODS: Sixty patients requiring the prolonged MV were included in the prospective study. All participants were randomized into three groups: the control, the local antiseptic (LA) and the bacteriophage (BP). All patients were managed using similar VAP protective bundle. Infection-related ventilator-associated complications (IVAC) were registered based on clinical, laboratory, and instrumental examination. Diagnosis of VAP was confirmed if CPIS was ≥ 6 points. RESULTS: A total incidence of IVAC did nоt differ between groups: 15 (75 %), 14 (70 %), and 17 (85 %) cases in the control, LA, and BP groups, respectively. The VAP incidence was lower in the LA and BP groups compared with the controls: 3 (15 %) and 3 (15 %) vs 10 (50 %) cases (χ2 = 8.35; p = 0.015). The overall mortality was 30 % and did not differ between the groups. Multi-selective decontamination resulted in a trend to increased ventilator-associated tracheobronchitis incidence in the BP group and to silent colonization in both LA and BP groups (p = 0.07). No differences in the duration of MV, ICU and hospital stay were observed. CONCLUSIONS: The combined multizonal upper airway decontamination involving subglottic space is associated with a reduction of the VAP incidence but did not change the overall IVAC rate. Multizonal decontamination based on subglottic bacteriophage instillation might have a potential to influence the microbial heterogeneity of upper airways.

Characterization of crAss-like phage isolates highlights Crassvirales genetic heterogeneity and worldwide distribution

Crassvirales (crAss-like phages) are an abundant group of human gut-specific bacteriophages discovered in silico. The use of crAss-like phages as human fecal indicators is proposed but the isolation of only seven cultured strains of crAss-like phages to date has greatly hindered their study. Here, we report the isolation and genetic characterization of 25 new crAss-like phages (termed crAssBcn) infecting Bacteroides intestinalis, belonging to the order Crassvirales, genus Kehishuvirus and, based on their genomic variability, classified into six species. CrAssBcn phage genomes are similar to ΦCrAss001 but show genomic and aminoacidic differences when compared to other crAss-like phages of the same family. CrAssBcn phages are detected in fecal metagenomes around the world at a higher frequency than ΦCrAss001. This study increases the known crAss-like phage isolates and their abundance and heterogeneity open the question of what member of the Crassvirales group should be selected as human fecal marker.

Metatranscriptome Analysis of Nasopharyngeal Swabs across the Varying Severity of COVID-19 Disease Demonstrated Unprecedented Species Diversity.

The recent global emergence of the SARS-CoV-2 pandemic has accelerated research in several areas of science whose valuable outputs and findings can help to address future health challenges in the event of emerging infectious agents. We conducted a comprehensive shotgun analysis targeting multiple aspects to compare differences in bacterial spectrum and viral presence through culture-independent RNA sequencing. We conducted a comparative analysis of the microbiome between healthy individuals and those with varying degrees of COVID-19 severity, including a total of 151 participants. Our findings revealed a noteworthy increase in microbial species diversity among patients with COVID-19, irrespective of disease severity. Specifically, our analysis revealed a significant difference in the abundance of bacterial phyla between healthy individuals and those infected with COVID-19. We found that Actinobacteria, among other bacterial phyla, showed a notably higher abundance in healthy individuals compared to infected individuals. Conversely, Bacteroides showed a lower abundance in the latter group. Infected people, regardless of severity and symptoms, have the same proportional representation of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteriales. In addition to SARS-CoV-2 and numerous phage groups, we identified sequences of clinically significant viruses such as Human Herpes Virus 1, Human Mastadenovirus D, and Rhinovirus A in several samples. Analyses were performed retrospectively, therefore, in the case of SARS-CoV-2 various WHO variants such as Alpha (B.1.1.7), Delta (B.1.617.2), Omicron (B.1.1.529), and 20C strains are represented. Additionally, the presence of specific virus strains has a certain effect on the distribution of individual microbial taxa.

Long-Read Sequencing Reveals Extensive DNA Methylations in Human Gut Phagenome Contributed by Prevalently Phage-Encoded Methyltransferases.

DNA methylation plays a crucial role in the survival of bacteriophages (phages), yet the understanding of their genome methylation remains limited. In this study, DNA methylation patterns are analyzed in 8848 metagenome-assembled high-quality phages from 104 fecal samples using single-molecule real-time sequencing. The results demonstrate that 97.60% of gut phages exhibit methylation, with certain factors correlating with methylation densities. Phages with higher methylation densities appear to have potential viability advantages. Strikingly, more than one-third of the phages possess their own DNA methyltransferases (MTases). Increased MTase copies are associated with higher genome methylation densities, specific methylation motifs, and elevated prevalence of certain phage groups. Notably, the majority of these MTases share close homology with those encoded by gut bacteria, suggesting their exchange during phage-bacterium interactions. Furthermore, these MTases can be employed to accurately predict phage-host relationships. Overall, the findings indicate the widespread utilization of DNA methylation by gut DNA phages as an evasion mechanism against host defense systems, with a substantial contribution from phage-encoded MTases.