ABSTRACT
As the world moves toward a green economy and sustainable agriculture, bacterial viruses or bacteriophages (phages) become attractive biocontrol agents for controlling crop diseases. Effective utilization of phages in farms requires integrated knowledge of crops, pathogens, phages, and surroundings. Phages must encounter environmental fluctuations, including temperature, and must remain infectious for successful bacteria lysis. This work studied a soilborne RSJ2 phage discovered in Thailand, which can eliminate Ralstonia solanacearum, causing bacterial wilt disease in chili. We investigated how phage infectivity and nanomechanics responded to thermal changes. The plaque-based assay showed that the infectivity of the RSJ2 phage was stable within 24-40 °C, an average temperature fluctuation in tropical regions. The structural examination also showed that the phage remained intact. The nanomechanical property of the phage was inspected by the atomic force microscopy-based nanoindentation. The result revealed that the phage stiffness within 24-40 °C was statistically similar (0.05-0.06 N/m). Upon heating at 40 °C for 1, 5, and 10 h and resting at 25 °C, the stiffness of the phage particles increased to 0.09-0.11 N/m (54-83% increase). The stiffness results suggest structural adaptation of the protein subunits as a response to thermal alteration. The study exhibits that the phage structure is highly dynamic and can nanomechanically respond to varying temperatures. The phage stiffness may reveal insight into phage adaptation to environmental factors. Equipped with the knowledge of phage infectivity, structure, and nanomechanics, we can design practical guidelines for effective phage usage in farming and propelling green and safe agriculture.
Subject(s)
Bacteriophages , Bacteriophages/physiology , Temperature , Ralstonia solanacearum/virology , Microscopy, Atomic Force , ThailandABSTRACT
Fecal filtrate transfer (FFT) is emerging as a safer alternative to traditional fecal microbiota transplantation (FMT) - particularly in the context of necrotizing enterocolitis (NEC), a severe gastrointestinal condition affecting preterm infants. Using a preterm piglet model, FFT has demonstrated superiority over FMT in safety and NEC prevention. Since FFT is virtually devoid of bacteria, prokaryotic viruses (bacteriophages) are assumed to mediate the beneficial effects. However, this assumption remains unproven. To address this gap, we separated virus-like particles (30 kDa to 0.45 µm) of donor feces from the residual postbiotic fluid. We then compared clinical and gut microbiota responses to these fractions with the parent FFT solution after transferring them to NEC-susceptible preterm piglets. Virome transfer was equally effective as FFT in reducing the severity of NEC-like pathology. The bacterial compositional data corroborated clinical findings as virome transfer reduced the relative abundance of several NEC-associated pathogens e.g. Klebsiella pneumoniae and Clostridium perfringens. Virome transfer diversified gut viral communities with concomitant constraining effects on the bacterial composition. Unexpectedly, virome transfer, but not residual postbiotic fluid, led to earlier diarrhea. While diarrhea may be a minor concern in human infants, future work should identify ways of eliminating this side effect without losing treatment efficacy.
Subject(s)
Enterocolitis, Necrotizing , Fecal Microbiota Transplantation , Feces , Gastrointestinal Microbiome , Enterocolitis, Necrotizing/prevention & control , Enterocolitis, Necrotizing/therapy , Animals , Feces/virology , Feces/microbiology , Fecal Microbiota Transplantation/methods , Swine , Humans , Bacteria/classification , Bacteria/isolation & purification , Bacteria/genetics , Animals, Newborn , Disease Models, Animal , Virome , Clostridium perfringens , Bacteriophages/genetics , Bacteriophages/physiology , Diarrhea/therapy , Diarrhea/virology , Diarrhea/prevention & control , Diarrhea/microbiologyABSTRACT
Staphylococcus aureus, a major foodborne pathogen, is frequently detected in fresh produce. It often causes food poisoning accompanied by abdominal pain, diarrhea, and vomiting. Additionally, the abuse of antibiotics to control S. aureus has resulted in the emergence of antibiotics-resistant bacteria, such as methicillin resistant S. aureus. Therefore, bacteriophage, a natural antimicrobial agent, has been suggested as an alternative to antibiotics. In this study, a lytic phage SSP49 that specifically infects S. aureus was isolated from a sewage sample, and its morphological, biological, and genetic characteristics were determined. We found that phage SSP49 belongs to the Straboviridae family (Caudoviricetes class) and maintained host growth inhibition for 30 h in vitro. In addition, it showed high host specificity and a broad host range against various S. aureus strains. Receptor analysis revealed that phage SSP49 utilized cell wall teichoic acid as a host receptor. Whole genome sequencing revealed that the genome size of SSP49 was 137,283 bp and it contained 191 open reading frames. The genome of phage SSP49 did not contain genes related to lysogen formation, bacterial toxicity, and antibiotic resistance, suggesting its safety in food application. The activity of phage SSP49 was considerably stable under various high temperature and pH conditions. Furthermore, phage SSP49 effectively inhibited S. aureus growth on baby spinach leaves both at 4 °C and 25 °C while maintaining the numbers of active phage during treatments (reductions of 1.2 and 2.1 log CFU/cm2, respectively). Thus, this study demonstrated the potential of phage SSP49 as an alternative natural biocontrol agent against S. aureus contamination in fresh produce.
Subject(s)
Host Specificity , Plant Leaves , Spinacia oleracea , Staphylococcus aureus , Spinacia oleracea/microbiology , Staphylococcus aureus/virology , Plant Leaves/microbiology , Food Microbiology , Genome, Viral , Bacteriophages/isolation & purification , Bacteriophages/physiology , Food Contamination/prevention & control , Staphylococcus Phages , Whole Genome Sequencing , Sewage/virology , Sewage/microbiologyABSTRACT
Vibrio parahaemolyticus, a prevalent foodborne pathogen found in both water and seafood, poses substantial risks to public health. The conventional countermeasure, antibiotics, has exacerbated the issue of antibiotic resistance, increasing the difficulty of controlling this bacterium. Phage lysins, as naturally occurring active proteins, offer a safe and reliable strategy to mitigate the impact of V. parahaemolyticus on public health. However, there is currently a research gap concerning bacteriophage lysins specific to Vibrio species. To address this, our study innovatively and systematically evaluates 37 phage lysins sourced from the NCBI database, revealing a diverse array of conserved domains and notable variations in similarity among Vibrio phage lysins. Three lysins, including Lyz_V_pgrp, Lyz_V_prgp60, and Lyz_V_zlis, were successfully expressed and purified. Optimal enzymatic activity was observed at 45â, 800 mM NaCl, and pH 8-10, with significant enhancements noted in the presence of 1 mM membrane permeabilizers such as EDTA or organic acids. These lysins demonstrated effective inhibition against 63 V. parahaemolyticus isolates from clinical, food, and environmental sources, including the reversal of partial resistance, synergistic interactions with antibiotics, and disruption of biofilms. Flow cytometry analyses revealed that the combination of Lyz_V_pgp60 and gentamicin markedly increased bacterial killing rates. Notably, Lyz_V_pgrp, Lyz_V_pgp60, and Lyz_V_zlis exhibited highly efficient biofilm hydrolysis, clearing over 90 % of preformed V. parahaemolyticus biofilms within 48 h. Moreover, these lysins significantly reduced bacterial loads in various food samples and environmental sources, with reductions averaging between 1.06 and 1.29 Log CFU/cm2 on surfaces such as stainless-steel and bamboo cutting boards and approximately 0.87 CFU/mL in lake water and sediment samples. These findings underscore the exceptional efficacy and versatile application potential of phage lysins, offering a promising avenue for controlling V. parahaemolyticus contamination in both food and environmental contexts.
Subject(s)
Bacteriophages , Vibrio parahaemolyticus , Vibrio parahaemolyticus/virology , Vibrio parahaemolyticus/drug effects , Viral Proteins/metabolism , Viral Proteins/genetics , Food Microbiology , Seafood/microbiology , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Biofilms/growth & developmentABSTRACT
Bacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognise and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognises cognate motifs has not been fully understood. In this study we characterise BREX from pathogenic Salmonella and present X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that both methyltransferase activity and defence require larger BREX complexes. Through rational engineering of PglX we broaden both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is used to recognise specific DNA sequences for BREX activity, contributing to motif recognition for both phage defence and host methylation.
Subject(s)
Bacteriophages , Methyltransferases , Methyltransferases/metabolism , Methyltransferases/genetics , Bacteriophages/genetics , Bacteriophages/enzymology , Crystallography, X-Ray , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , DNA Methylation , Salmonella/virology , Salmonella/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , Models, MolecularABSTRACT
BACKGROUND: Despite being among the most abundant biological entities on earth, bacteriophage (phage) remain an understudied component of host-associated systems. One limitation to studying host-associated phage is the lack of consensus on methods for sampling phage communities. Here, we compare paired total metagenomes and viral size fraction metagenomes (viromes) as methods for investigating the dsDNA viral communities associated with the GI tract of two bee species: the European honey bee Apis mellifera and the eastern bumble bee Bombus impatiens. RESULTS: We find that viromes successfully enriched for phage, thereby increasing phage recovery, but only in honey bees. In contrast, for bumble bees, total metagenomes recovered greater phage diversity. Across both bee species, viromes better sampled low occupancy phage, while total metagenomes were biased towards sampling temperate phage. Additionally, many of the phage captured by total metagenomes were absent altogether from viromes. Comparing between bees, we show that phage communities in commercially reared bumble bees are significantly reduced in diversity compared to honey bees, likely reflecting differences in bacterial titer and diversity. In a broader context, these results highlight the complementary nature of total metagenomes and targeted viromes, especially when applied to host-associated environments. CONCLUSIONS: Overall, we suggest that studies interested in assessing total communities of host-associated phage should consider using both approaches. However, given the constraints of virome sampling, total metagenomes may serve to sample phage communities with the understanding that they will preferentially sample dominant and temperate phage. Video Abstract.
Subject(s)
Bacteriophages , Metagenome , Virome , Bees/virology , Bees/microbiology , Animals , Bacteriophages/genetics , Bacteriophages/isolation & purification , Bacteriophages/classification , Gastrointestinal Microbiome/genetics , Metagenomics/methods , Gastrointestinal Tract/microbiology , Gastrointestinal Tract/virologyABSTRACT
Infectious diseases lead to significant morbidity and mortality. Often, resolution of the acute stage of the disease leads to microbial persistence, resulting in chronic debilitating disease. Management of persistent infections frequently requires lifelong therapy with antimicrobial agents. These infections could be chronic viral infections like HIV, hepatitis B or chronic bacterial persistent infections like prosthetic joint infections caused by multi-drug resistant organisms. Bacteriophages have been designed specifically to target recalcitrant bacterial infections, such as prosthetic joint infections with varying success. In this review, we describe the historic evolution of scenarios and risks associated with innovative therapy using infectious agents to treat other persistent infections.
[Box: see text].
Subject(s)
Persistent Infection , Humans , Phage Therapy/methods , Bacterial Infections/drug therapy , Bacterial Infections/therapy , Bacterial Infections/microbiology , Anti-Infective Agents/therapeutic use , Bacteriophages/physiology , Virus Diseases/drug therapy , Virus Diseases/therapy , Virus Diseases/virologyABSTRACT
BACKGROUND: Klebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear. METHODS: To isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis. RESULTS: K. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group. CONCLUSIONS: This study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.
Subject(s)
Anti-Bacterial Agents , Bacteriophages , Carbapenems , Klebsiella pneumoniae , Proteomics , Tigecycline , Tigecycline/pharmacology , Klebsiella pneumoniae/virology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Bacteriophages/genetics , Bacteriophages/physiology , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Klebsiella Infections/microbiology , Humans , Extracellular Vesicles/metabolism , Bacterial Outer Membrane/metabolism , Bacterial Outer Membrane/drug effects , beta-Lactamases/genetics , beta-Lactamases/metabolism , Carbapenem-Resistant Enterobacteriaceae/genetics , Carbapenem-Resistant Enterobacteriaceae/drug effects , Plasmids/genetics , Microbial Sensitivity Tests , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
One of the most common bacteria that cause nosocomial infections is Klebsiella pneumonia (K. pneumoniae), especially in patients who are very sick and admitted to the intensive care unit (ICU). The frequency of multi-drug-resistant Klebsiella pneumoniae (MDRKP) has dramatically increased worldwide in recent decades, posing an urgent threat to public health. The Western world's bacteriophage (phage) studies have been revitalized due to the increasing reports of antimicrobial resistance and the restricted development and discovery of new antibiotics. These factors have also spurred innovation in other scientific domains. The primary agent in phage treatment is an obligately lytic organism (called bacteriophage) that kills the corresponding bacterial host while sparing human cells and lessening the broader effects of antibiotic usage on commensal bacteria. Phage treatment is developing quickly, leading to many clinical studies and instances of life-saving medicinal use. In addition, phage treatment has a few immunological adverse effects and consequences in addition to its usefulness. Since K. pneumoniae antibiotic resistance has made treating multidrug-resistant (MDR) infections challenging, phage therapy (PT) has emerged as a novel therapeutic strategy. The effectiveness of phages has also been investigated in K. pneumoniae biofilms and animal infection models. Compared with antibiotics, PT exhibits numerous advantages, including a particular lysis spectrum, co-evolution with bacteria to avoid the emergence of phage resistance, and a higher abundance and diversity of phage resources than found in antibiotics. Moreover, phages are eliminated in the absence of a host bacterium, which makes them the only therapeutic agent that self-regulates at the sites of infection. Therefore, it is essential to pay attention to the role of PT in treating these infections. This study summarizes the state of knowledge on Klebsiella spp. phages and provides an outlook on the development of phage-based treatments that target K. pneumoniae in clinical trials.
Subject(s)
Anti-Bacterial Agents , Bacteriophages , Drug Resistance, Multiple, Bacterial , Klebsiella Infections , Klebsiella pneumoniae , Phage Therapy , Klebsiella pneumoniae/virology , Klebsiella pneumoniae/drug effects , Bacteriophages/physiology , Klebsiella Infections/therapy , Klebsiella Infections/microbiology , Humans , Animals , Anti-Bacterial Agents/pharmacology , Cross Infection/microbiology , Disease Models, AnimalABSTRACT
BACKGROUND: The composition of the vaginal microbiota during the menstrual cycle is dynamic, with some women remaining eu- or dysbiotic and others transitioning between these states. What defines these dynamics, and whether these differences are microbiome-intrinsic or mostly driven by the host is unknown. To address this, we characterized 49 healthy, young women by metagenomic sequencing of daily vaginal swabs during a menstrual cycle. We classified the dynamics of the vaginal microbiome and assessed the impact of host behavior as well as microbiome differences at the species, strain, gene, and phage levels. RESULTS: Based on the daily shifts in community state types (CSTs) during a menstrual cycle, the vaginal microbiome was classified into four Vaginal Community Dynamics (VCDs) and reported in a classification tool, named VALODY: constant eubiotic, constant dysbiotic, menses-related, and unstable dysbiotic. The abundance of bacteria, phages, and bacterial gene content was compared between the four VCDs. Women with different VCDs showed significant differences in relative phage abundance and bacterial composition even when assigned to the same CST. Women with unstable VCDs had higher phage counts and were more likely dominated by L. iners. Their Gardnerella spp. strains were also more likely to harbor bacteriocin-coding genes. CONCLUSIONS: The VCDs present a novel time series classification that highlights the complexity of varying degrees of vaginal dysbiosis. Knowing the differences in phage gene abundances and the genomic strains present allows a deeper understanding of the initiation and maintenance of permanent dysbiosis. Applying the VCDs to further characterize the different types of microbiome dynamics qualifies the investigation of disease and enables comparisons at individual and population levels. Based on our data, to be able to classify a dysbiotic sample into the accurate VCD, clinicians would need two to three mid-cycle samples and two samples during menses. In the future, it will be important to address whether transient VCDs pose a similar risk profile to persistent dysbiosis with similar clinical outcomes. This framework may aid interdisciplinary translational teams in deciphering the role of the vaginal microbiome in women's health and reproduction. Video Abstract.
Subject(s)
Bacteria , Bacteriophages , Dysbiosis , Menstrual Cycle , Menstruation , Microbiota , Vagina , Humans , Female , Vagina/microbiology , Bacteriophages/genetics , Bacteriophages/physiology , Microbiota/genetics , Bacteria/classification , Bacteria/genetics , Adult , Dysbiosis/microbiology , Young Adult , Genes, Bacterial/genetics , Metagenomics/methodsABSTRACT
The constant arms race between bacteria and their parasites has resulted in a large diversity of bacterial defenses, with many bacteria carrying multiple systems. Here, we report the discovery of a phylogenetically widespread defense system, coined methylation-associated defense system (MADS), which is distributed across gram-positive and gram-negative bacteria. MADS interacts with a CRISPR-Cas system in its native host to provide robust and durable resistance against phages. While phages can acquire epigenetic-mediated resistance against MADS, co-existence of MADS and a CRISPR-Cas system limits escape emergence. MADS comprises eight genes with predicted nuclease, ATPase, kinase, and methyltransferase domains, most of which are essential for either self/non-self discrimination, DNA restriction, or both. The complex genetic architecture of MADS and MADS-like systems, relative to other prokaryotic defenses, points toward highly elaborate mechanisms of sensing infections, defense activation, and/or interference.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , Bacteriophages/genetics , Bacteriophages/physiology , Phylogeny , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/virology , Bacteria/virology , Bacteria/genetics , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/virology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , MethylationABSTRACT
Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.
Subject(s)
Pseudomonas aeruginosa , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunology , Pseudomonas aeruginosa/virology , Immunity, Innate , Bacteriophages/genetics , Bacteriophages/physiology , Host-Pathogen Interactions/immunology , Host-Pathogen Interactions/genetics , Animals , Evolution, Molecular , Inflammasomes/immunology , Inflammasomes/genetics , Eukaryota/virology , Eukaryota/genetics , Eukaryota/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Evolution , Plants/immunology , Plants/virology , Plants/microbiologyABSTRACT
Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases.
Subject(s)
Bacteriophages , Computational Biology , Bacteriophages/genetics , Bacteriophages/enzymology , Computational Biology/methods , Molecular Sequence Annotation , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/chemistry , Neural Networks, Computer , Machine Learning , Software , Protein Domains , Genome, Viral/genetics , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Carboxylic Ester Hydrolases/chemistryABSTRACT
Genetic variations are instrumental for unraveling phage evolution and deciphering their functional implications. Here, we explore the underlying fine-scale genetic variations in the gut phageome, especially structural variations (SVs). By using virome-enriched long-read metagenomic sequencing across 91 individuals, we identified a total of 14,438 nonredundant phage SVs and revealed their prevalence within the human gut phageome. These SVs are mainly enriched in genes involved in recombination, DNA methylation, and antibiotic resistance. Notably, a substantial fraction of phage SV sequences share close homology with bacterial fragments, with most SVs enriched for horizontal gene transfer (HGT) mechanism. Further investigations showed that these SV sequences were genetic exchanged between specific phage-bacteria pairs, particularly between phages and their respective bacterial hosts. Temperate phages exhibit a higher frequency of genetic exchange with bacterial chromosomes and then virulent phages. Collectively, our findings provide insights into the genetic landscape of the human gut phageome.
Subject(s)
Bacteria , Bacteriophages , Gastrointestinal Microbiome , Gene Transfer, Horizontal , Bacteriophages/genetics , Humans , Gastrointestinal Microbiome/genetics , Bacteria/virology , Bacteria/genetics , Metagenomics/methods , Genetic Variation , Virome/genetics , Genome, Viral , High-Throughput Nucleotide SequencingABSTRACT
The constant battle between bacteria and viruses has led to the development of sophisticated antiviral defense strategies by bacteria to defend themselves against phages. This study analyzed a marshland metagenome to identify and characterize bacterial antiviral defense systems and phage interactions. We assembled 210 metagenome-assembled genomes (MAGs) from environmental DNA extracted from Pallikaranai marshland soil and 37 unclassified MAGs were filtered. MIMAG standards were followed, 2 high-quality and 15 medium-quality unclassified MAGs were picked. MINCED was used to identify 137 CRISPR arrays in the quality MAGs, and ViroBLAST was used to identify the phages that interact with the bacteria. About 242 spacer sequences were extracted from the CRISPR arrays, of which 54 had significant matches in the ViroBLAST database. 7 unverified bacteriophage species were also detected in the MAGs. The viral group of Caudoviricetes phage elements were identified as a frequent genome terminal repeat. The PADLOC identified 11 genes involved as a defense system in the MAGs. The PD-T4-6 defense system was found to be prevalent in 15 different unclassified MAGs. This study presents valuable insights intothe adaptations of unclassified bacteria to bacteriophages, as well as the genes used by these bacteria as a defense mechanism.
Subject(s)
Bacteria , Bacteriophages , Clustered Regularly Interspaced Short Palindromic Repeats , Genome, Bacterial , Bacteriophages/genetics , Bacteria/genetics , Bacteria/virology , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Soil Microbiology , Metagenome , Phylogeny , CRISPR-Cas SystemsABSTRACT
Phages are pivotal in shaping microbial communities and biogeochemical cycles, while our understanding of the diversity, functions potential, and resistance gene carriage of phages in hospital wastewater (HWW) remains limited. We collected influent and effluent samples from the 3 hospital wastewater treatment plants (HWTPs) to assess the diversity and fate of phages, the interactions between phages and hosts, and the presence of resistance genes and auxiliary metabolic genes (AMGs) encoded by phages. Compared to influent, effluent showed reduced phage abundance and altered composition, with decreases in Microviridae and Inoviridae. The gene-sharing network highlights that many phages in HWW are not classified in known viral genera, suggesting HWW as a rich source of new viruses. There was a significant association between phages and microorganisms, with approximately 32.57 % of phages expected to be capable of infecting microbial hosts, characterized primarily by lytic activity. A total of 8 unique antibiotic resistance genes, 13 unique metal resistance genes, and 5 mobile genetic elements were detected in 3 HWTPs phageomes. Phage AMGs have the potential to influence carbon, nitrogen, phosphorus, and sulfur metabolism, impacting biogeochemical cycles. This study reveals the genomic diversity and ecological role of phages in HWTPs, highlighting their environmental and ecosystem impact.
Subject(s)
Bacteriophages , Hospitals , Wastewater , Wastewater/microbiology , Wastewater/virology , Bacteriophages/genetics , Bacteriophages/isolation & purification , Genome, Viral , Waste Disposal, Fluid , Genetic Variation , Drug Resistance, Microbial/genetics , Bacteria/genetics , Bacteria/virologyABSTRACT
Morganella psychrotolerans is a histamine-producing bacterium that causes histamine poisoning. In this study, we isolated and characterized a novel phage, MopsHU1, that infects M. psychrotolerans. MopsHU1 is a podovirus with a limited host spectrum. Genomic analysis showed that MopsHU1 belongs to the family Autographiviridae, subfamily Studiervirinae, and genus Kayfunavirus. Comparative analysis revealed that the MopsHU1 genome is similar to those of Citrobacter phage SH3 and Cronobacter phage Dev2. Moreover, the Escherichia coli phage K1F genome is also similar, except for its tailspike gene sequence. These results expand our understanding of the Kayfunavirus phages that infect Morganella spp. Note: The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank database under the accession number LC799501.
Subject(s)
Bacteriophages , Genome, Viral , Morganella , Phylogeny , Bacteriophages/genetics , Bacteriophages/isolation & purification , Bacteriophages/classification , Bacteriophages/physiology , Morganella/virology , Morganella/genetics , Genomics , Host Specificity , Podoviridae/genetics , Podoviridae/isolation & purification , Podoviridae/classification , Sequence Analysis, DNA , Base SequenceABSTRACT
DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.
Subject(s)
DNA, Viral , Genome, Viral , DNA, Viral/genetics , DNA, Viral/metabolism , DNA, Viral/chemistry , Cryoelectron Microscopy , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Models, Molecular , DNA Packaging , Virus Assembly/genetics , Bacteriophages/genetics , Viral Genome PackagingABSTRACT
The mucilage event witnessed in the Sea of Marmara in 2021 has emerged as a prominent environmental concern, capturing public attention due to its detrimental effects on ecological, economic, and aesthetic dimensions. Addressing the multifaceted impacts of mucilage demands a nature-centric scientific approach, given its global ramifications spanning economy, public health, international relations, and tourism. Consequently, this study sought to explore alternative approaches for the removal of pathogenic enteric bacteria associated with mucilage occurrences, diverging from conventional methodologies. Specifically, the primary objective was to assess the efficacy of rhamnolipid and a bacteriophage cocktail in mitigating the proliferation of enteric pathogens within mucilaginous environments. During the study, 91 phage isolations were obtained from 45 water samples taken and 10 phages were selected for the broad host range and because of the efficacy tests, a phage cocktail was created with 5 phages. It was found that the mixture of rhamnolipid, phage cocktail and rhamnolipid-phage cocktail reduced bacterial load by 7-9 log10, 9-12 log10 and 9-11 log10 respectively under laboratory conditions. When the study was carried out in seawater, reductions of 4-5 log10, 3 log10 and 4 log10 were achieved. This study has shown that the combined use of rhamnolipid, phage cocktail and rhamnolipid-phage cocktail can be considered as the most effective natural solution proposal for reducing bacterial load, both in laboratory conditions and in sea surface water.
Subject(s)
Bacteriophages , Glycolipids , Seawater , Bacteriophages/physiology , Seawater/microbiology , Seawater/virology , Enterobacteriaceae/virologyABSTRACT
The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.