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Bacteria and their viral predators (phages) are constantly evolving to subvert one another. Many bacterial immune systems that inhibit phages are encoded on mobile genetic elements that can be horizontally transmitted to diverse bacteria. Despite the pervasive appearance of immune systems in bacteria, it is not often known if these immune systems function against phages that the host encounters in nature. Additionally, there are limited examples demonstrating how these phages counter-adapt to such immune systems. Here, we identify clinical isolates of the global pathogen Vibrio cholerae harboring a novel genetic element encoding the bacterial immune system DarTG and reveal the immune systems impact on the co-circulating lytic phage ICP1. We show that DarTG inhibits ICP1 genome replication, thus preventing ICP1 plaquing. We further characterize the conflict between DarTG-mediated defense and ICP1 by identifying an ICP1-encoded protein that counters DarTG and allows ICP1 progeny production. Finally, we identify this protein, AdfB, as a functional antitoxin that abrogates the toxin DarT likely through direct interactions. Following the detection of the DarTG system in clinical V. cholerae isolates, we observed a rise in ICP1 isolates with the functional antitoxin. These data highlight the use of surveillance of V. cholerae and its lytic phages to understand the co-evolutionary arms race between bacteria and their phages in nature.IMPORTANCEThe global bacterial pathogen Vibrio cholerae causes an estimated 1 to 4 million cases of cholera each year. Thus, studying the factors that influence its persistence as a pathogen is of great importance. One such influence is the lytic phage ICP1, as once infected by ICP1, V. cholerae is destroyed. To date, we have observed that the phage ICP1 shapes V. cholerae evolution through the flux of anti-phage bacterial immune systems. Here, we probe clinical V. cholerae isolates for novel anti-phage immune systems that can inhibit ICP1 and discover the toxin-antitoxin system DarTG as a potent inhibitor. Our results underscore the importance of V. cholerae and ICP1 surveillance to elaborate novel means by which V. cholerae can persist in both the human host and aquatic reservoir in the face of ICP1.

ICP1, a lytic bacteriophage of Vibrio cholerae, is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs dependence on ICP1s DNA replication machinery and virion components results in inhibition of ICP1s lifecycle. PLEs are expected to depend on ICP1 factors for genome packaging, but the mechanism(s) PLEs use to inhibit ICP1 genome packaging is currently unknown. Here, we identify and characterize Gpi, PLEs indiscriminate genome packaging inhibitor. Gpi binds to ICP1s large terminase (TerL), the packaging motor, and blocks genome packaging. To overcome Gpis negative effect on TerL, a component PLE also requires, PLE uses two genome packaging specifiers, GpsA and GpsB, that specifically allow packaging of PLE genomes. Surprisingly, PLE also uses mimicry of ICP1s pac site as a backup strategy to ensure genome packaging. PLEs pac site mimicry, however, is only sufficient if PLE can inhibit ICP1 at other stages of its lifecycle, suggesting an advantage to maintaining Gpi, GpsA and GpsB. Collectively, these results provide mechanistic insights into another stage of ICP1s lifecycle that is inhibited by PLE, which is currently the most inhibitory of the documented phage satellites. More broadly, Gpi represents the first satellite-encoded inhibitor of a phage TerL.

The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence1. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database2,3. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus-host interactions that are common across the virome.

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.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Viruses are often studied using metagenome-assembled sequences, but genome incompleteness hampers comprehensive and accurate analyses. Contig Overlap Based Re-Assembly (COBRA) resolves assembly breakpoints based on the de Bruijn graph and joins contigs. Here we benchmarked COBRA using ocean and soil viral datasets. COBRA accurately joined the assembled sequences and achieved notably higher genome accuracy than binning tools. From 231 published freshwater metagenomes, we obtained 7,334 bacteriophage clusters, ~83% of which represent new phage species. Notably, ~70% of these were circular, compared with 34% before COBRA analyses. We expanded sampling of huge phages (≥200 kbp), the largest of which was curated to completion (717 kbp). Improved phage genomes from Rotsee Lake provided context for metatranscriptomic data and indicated the in situ activity of huge phages, whiB-encoding phages and cysC- and cysH-encoding phages. COBRA improves viral genome assembly contiguity and completeness, thus the accuracy and reliability of analyses of gene content, diversity and evolution.

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1s DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

BACKGROUND: Environmental surveillance, using detection of Salmonella Typhi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whether S. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays. METHODOLOGY: We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay with S. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes. FINDINGS: S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) river water samples contained phages. All S. Typhi phages were unable to infect other Salmonella and non-Salmonella strains, nor a Vi-knockout S. Typhi strain. Representative strains from S. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed that S. Typhi phages belonged to the class Caudoviricetes and clustered in three distinct groups. CONCLUSIONS: S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific for S. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost (~$2) plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance.

Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells. Surprisingly, the Acb2 hexamer also binds with high affinity to CBASS cyclic trinucleotides (CTNs) 333-cyclic AMP-AMP-AMP and 333-cAAG at a distinct site from CDNs. One Acb2 hexamer can simultaneously bind two CTNs and three CDNs. Phage-encoded Acb2 provides protection from type III-C CBASS that uses cA3 signaling molecules. Moreover, phylogenetic analysis of >2,000 Acb2 homologs encoded by diverse phages and prophages revealed that most are expected to bind both CTNs and CDNs. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

Antibodies and antibody-based immunotherapeutics are the mainstays of cancer immunotherapy. Expanding the repertoire of cancer-specific and cancer-associated epitopes targetable with antibodies represents an important area of research. Phage display is a powerful approach allowing the use of diverse antibody libraries to be screened for binding to a wide range of targets. In this review, we summarize the basics of phage display technology and highlight the advances in anticancer antibody identification and modification via phage display platform. Finally, we describe phage display-derived anticancer monoclonal antibodies that have been approved to date or are in clinical development. • Concise review of principles of phage display technology. • Various approaches for library construction and selection strategies. • Antibody engineering via phage display technology. • Ab formats, guided selection, humanization, affinity maturation, Ab developability. • Current landscape of anticancer antibodies derived from phage display technology. [ABSTRACT FROM AUTHOR]

Due to the overuse of antibiotics, the number of multidrug-resistant pathogen bacteria is rising in recent years posing a serious threat to human health. One promising alternative for treatment is the application of phage therapy using highly selective bacteriophages. Because of their selectivity, individual screens called phagograms for each patient are required to select phages from a phage library. Phagograms are mostly performed via bacterial cultivation on double layer agar plates and phage addition causing bacterial lysis. However, these assays are work-intensive and have a low ability for parallelization and automation. Hence, highly parallelizable and automatable microbioreactors in the lowest microliter scale could offer an economic solution increasing the throughput of phagograms. This paper demonstrates the applicability of a novel capillary-wave microbioreactor (cwMBR) to perform phagograms. Due to its small volume of only 7 µL and the open-droplet design, it can be easily automated and parallelized in future. Furthermore, the ability of online biomass measurement makes the cwMBR a perfect phagogram platform in the future. Herein, phagograms with E. coli and different concentrations of the phages MM02 and EASG3 were performed as proof of concept for phagograms in the cwMBR. Thereby, the cwMBR was able to measure differences in lysis kinetics of different phages. Furthermore, the phagograms were compared to those in conventional microtiter plate readers revealing the cwMBR as ideal alternative for phagograms as it combines favorable mixing conditions and a phage repellent hydrophilic glass surface with online biomass measurement in an open-droplet design for future parallelization and automation. [Display omitted] • Phage sensitivity tests called phagograms are mandatory for phage therapy. • State of the art phagograms are not highly parallelizable and automatable. • Novel capillary-wave microbioreactor (cwMBR) can perform parallelized phagograms. • cwMBR combines parallelizability and automatability with online biomass measurement. • cwMBR is able to measure lysis kinetics of phages. [ABSTRACT FROM AUTHOR]

Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNATyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNATyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNALys, to counteract a tRNA anticodon defense system (PrrC170). Retrons are bacterial genetic elements that, in combination with toxic effector proteins, can serve as antiphage defense systems. Here, the authors show that some phages can evade retrons and other defense systems by expressing specific tRNAs. [ABSTRACT FROM AUTHOR]

IS630/Tc1/mariner (ITm) represents the most widely distributed superfamily of DNA transposons in nature. Currently, bioinformatics research on ITm members primarily involves collecting data of existing and emerging members and organizing them into new groups or families. In the present study, our survey revealed that Tc1 and IS630 members have a broad host range, spanning across all six biological kingdoms (bacteria, fungi, plantae, animalia, archaea and protista) and viruses. The primary discoveries include the first Tn630 member—Tn630-NC1 and the closest homolog of IS630 from viruses—Tc1-C#1. By incorporating our discoveries into existing knowledge, we proposed a model to elucidate the formation of composite transposons. Organization of Tc1 and IS630 members into groups across biological kingdoms facilitates data collection for future research, particularly on their horizontal transfer between different kingdoms. The formation of composite transposons may result from asymmetric of terminal inverted repeats. IS630 should be merged with Tc1 into a single family IS630/Tc1. Furthermore, IS630 and its homologs constitute a valuable resource for studying horizontal gene transfer between gut bacteria and phages, opening up new avenues for research in this field. [ABSTRACT FROM AUTHOR]

Streptococcus (S.) equi subspecies (subsp.) zooepidemicus is an important facultative pathogen in horses and can cause severe infections in other species including humans. Facing the post-antibiotic era, novel antimicrobials are needed for fighting bacterial infections. Bacteriophages (phages) are the natural predators of bacteria and discussed as a promising antimicrobial treatment option. The objective of this study was to isolate and characterize S. equi subsp. zooepidemicus- specific phages for the first time and to evaluate their efficacy in vitro and ex vivo. In total, 13 phages with lytic activity were isolated and host ranges were determined. Two phages with broad host ranges and high efficiency of plating (vB_SeqZP_LmqsRe26-2 (lytic activity: 30/37 bacterial isolates) and vB_SeqZP_LmqsRe26-3 (lytic activity: 29/37 bacterial isolates)) and one phage with relatively low efficiency of plating (vB_SeqZP_LmqsRe26-1) were selected for further characterization, including electron microscopy and whole genome sequencing. In in vitro planktonic killing assays at two tested multiplicities of infection (MOI 1 and MOI 10), significant bacterial growth reduction was observed when the phages vB_SeqZP_LmqsRe26-2 and vB_SeqZP_LmqsRe26-3 were added. These phages were subsequently co-incubated with clinical S. equi subsp. zooepidemicus isolates in an equine endometrial explant model but did not achieve bacterial growth reduction at MOI 1 and MOI 10. However, helium ion microscopy revealed presence of particles adherent to the bacteria on the explant after incubation (25 h), suggesting possible phage-bacteria interactions. In conclusion, phages against S. equi subsp. zooepidemicus were successfully isolated and characterized. Promising results were observed in in vitro but no significant reduction was detected in ex vivo experiments, requiring additional investigations. However, after further adaptations (e.g., optimization of MOIs and phage administration or use of phage-antibiotic combination), phages could be a potential antimicrobial tool for future therapeutic use in S. equi subsp. zooepidemicus infections, although the available results do not currently support the therapeutic usage. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii , a predominant nosocomial pathogen, represents a grave threat to public health due to its multiple antimicrobial resistance. Managing patients afflicted with severe infections caused by multiple drug-resistant A. baumannii is particularly challenging, given the associated high mortality rates and unfavorable prognoses. The diminishing efficacy of antibiotics against this superbug underscores the urgent necessity for novel treatments or strategies to address this formidable issue. Bacteriophage-derived polysaccharide depolymerase enzymes present a potential approach to combating this pathogen. These enzymes target and degrade the bacterial cell's exopolysaccharide, capsular polysaccharide, and lipopolysaccharide, thereby disrupting biofilm formation and impairing the bacteria's defense mechanisms. Nonetheless, the narrow host range of phage depolymerases limits their therapeutic efficacy. Despite the benefits of these enzymes, phage-resistant strains have been identified, highlighting the complexity of phage-host interactions and the need for further investigation. While preliminary findings are encouraging, current investigations are limited, and clinical trials are imperative to advance this treatment approach for broader clinical applications. This review explores the potential of phage-derived depolymerase enzymes against A. baumannii infections. [ABSTRACT FROM AUTHOR]

Deep infection is the second most common complication of arthroplasty following loosening of the implant. Antibiotic-loaded bone cements (ALBCs) and high concentrations of systemic broad-spectrum antibiotics are commonly used to prevent infections following injury and surgery. However, clinical data fails to show that ALBCs are effective against deep infection, and negative side effects can result following prolonged administration of antibiotics. Additionally, the rise of multidrug resistant (MDR) bacteria provides an urgent need for alternatives to broad-spectrum antibiotics. Phage therapy, or the use of bacteriophages (viruses that infect bacteria) to target pathogenic bacteria, might offer a safe alternative to combat MDR bacteria. Application of phage therapy in the setting of deep infections requires formulation strategies that would stabilize bacteriophage against chemical and thermal stress during bone-cement polymerization, that maintain bacteriophage activity for weeks or months at physiological temperatures, and that allow for sustained release of phage to combat slow-growing, persistent bacteria. Here, we demonstrate the formulation of three phages that target diverse bacterial pathogens, which includes spray-drying of the particles for enhanced thermal stability at 37 °C and above. Additionally, we use atomic layer deposition (ALD) to coat spray-dried powders with alumina to allow for delayed release of phage from the dry formulations, and potentially protect phage against chemical damage during bone cement polymerization. Together, these findings present a strategy to formulate phages that possess thermal stability and sustained release properties for use in deep infections. [ABSTRACT FROM AUTHOR]

The growing threat of antimicrobial resistance has made the quest for antibiotic alternatives or synergists one of the most pressing priorities of the 21st century. The emergence of multidrug-resistance in most of the common wound pathogens has amplified the risk of antibiotic-resistant wound infections. Bacteriophages, with their self-replicating ability and targeted specificity, can act as suitable antibiotic alternatives. Nevertheless, targeted delivery of phages to infection sites remains a crucial issue, specifically in the case of topical infections. Hence, different phage delivery systems have been studied in recent years. However, there have been no recent reviews of phage delivery systems focusing exclusively on phage application on wounds. This review provides a compendium of all the major delivery systems that have been used to deliver phages to wound infection sites. Special focus has also been awarded to phage-embedded hydrogels with a discussion on the different aspects to be considered during their preparation. [ABSTRACT FROM AUTHOR]

Phage therapy is a highly promising approach to address the challenge that is presented by the global burden of antimicrobial resistance. Given the natural specificity of phages, phage susceptibility testing (PST) is a prerequisite for successful personalized therapy, allowing the selection of active phages from large and diverse collections. However, the issue of an easy-to-use and standardized technique remains. In this review, we describe the principles, advantages and drawbacks of two routinely used PST techniques: plaque and growth kinetic assays. These are labour-intensive and time-consuming methods that require automation of one or more steps, including preparation of test panels, incubation, reading and analysis of results. In addition to automation, there is an urgent need to establish a reference method to enable efficient of PST techniques selection of therapeutic phages. We discuss knowledge gaps and parameters that need to be investigated to work towards this goal. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa, an antibiotic‐resistant opportunistic pathogen, poses significant challenges in treating infections, particularly in immunocompromised individuals. This review explores current and future innovative approaches to suppress its growth and virulence. We delve into the bacterium's virulence factors, discussing existing strategies like antibiotics, bacteriophages, probiotics, and small‐molecule inhibitors. Additionally, novel approaches, including RNA interference, CRISPR‐Cas systems, and nanotechnology, show promise in preclinical studies. Despite advancements, challenges persist, prompting the need for a multifaceted approach targeting various aspects of P. aeruginosa pathogenesis for effective infection management. This review provides a comprehensive perspective on the status and future directions of innovative strategies against P. aeruginosa. [ABSTRACT FROM AUTHOR]

To determine invasive group A Streptococcus trends in Canada, we characterized emm1 isolates collected during 2018-2023. The percentage of hypervirulent M1UK lineage isolates increased significantly, from 22.1% in 2018 to 60.2% in 2023. Genomic analysis identified geographically and temporally associated clusters and genes associated with virulent bacteriophage acquisition. [ABSTRACT FROM AUTHOR]

Ensuring food safety measures are essential to minimize the risk of foodborne diseases linked to raw food products. Here, we investigated the efficacy of an innovative approach for the control of Salmonella Typhimurium found in fresh produces. Plasma activated water (PAW) and bacteriophages are emerging effective and valuable alternative methods for microbiological decontamination. The efficacy of PAW and a lytic bacteriophage (109 PFU/mL), both separately and sequentially, against S. Typhimurium in fresh produce was investigated. S. Typhimurium (105–107 CFU/g) were inoculated on lettuce leaves and treated with PAW, S. Typhimurium phage SK-T2 or their combination. PAW or bacteriophage inactivated S. Typhimurium, on lettuce leaves at different initial populations, by 2.90–3.46 or 1.45–3.25 log CFU/g, respectively. After sequential treatments of PAW and bacteriophage, S. Typhimurium populations, initially applied at ~ 105 CFU/g reduced by 4.47 log CFU/g, but when the order of application was changed (i.e., bacteriophage followed by PAW), the combination synergistically decreased the Salmonella numbers below the detection limit of the method used for the enumeration (i.e., < 101 CFU/g). At the high-level inoculum (~ 7 log CFU/g), consecutive treatments of PAW and phage decreased the S. Typhimurium population by 3.28 log CFU/g, and a reduction of 6.20 log CFU/g was achieved after reversing the order of treatment. Regardless of the bacterial inoculum level, sequential applications of bacteriophage and PAW resulted in a higher level of inactivation. This study proved that the bacteriophage–PAW combination constitutes a promising alternative approach to the conventional washing process in fresh produce wash waters in the food industry. [ABSTRACT FROM AUTHOR]

Bacterial pathogens in water, food, and the environment are spreading diseases around the world. According to a World Health Organization (WHO) report, waterborne pathogens pose the most significant global health risks to living organisms, including humans and animals. Conventional bacterial detection approaches such as colony counting, microscopic analysis, biochemical analysis, and molecular analysis are expensive, time-consuming, less sensitive, and require a pre-enrichment step. However, the bacteriophage-based detection of pathogenic bacteria is a robust approach that utilizes bacteriophages, which are viruses that specifically target and infect bacteria, for rapid and accurate detection of targets. This review shed light on cutting-edge technologies about the novel structure of phages and the immobilization process on the surface of electrodes to detect targeted bacterial cells. Similarly, the purpose of this study was to provide a comprehensive assessment of bacteriophage-based biosensors utilized for pathogen detection, as well as their trends, outcomes, and problems. This review article summaries current phage-based pathogen detection strategies for the development of low-cost lab-on-chip (LOC) and point-of-care (POC) devices using electrochemical and optical methods such as surface-enhanced Raman spectroscopy (SERS). [ABSTRACT FROM AUTHOR]

Bacteriophage ϕX174 has been widely used as a model organism to study fundamental processes in molecular biology. However, several aspects of ϕX174 gene regulation are not fully resolved. Here we construct a computational model for ϕX174 and use the model to study gene regulation during the phage infection cycle. We estimate the relative strengths of transcription regulatory elements (promoters and terminators) by fitting the model to transcriptomics data. We show that the specific arrangement of a promoter followed immediately by a terminator, which occurs naturally in the ϕX174 genome, poses a parameter identifiability problem for the model, since the activity of one element can be partially compensated for by the other. We also simulate ϕX174 gene expression with two additional, putative transcription regulatory elements that have been proposed in prior studies. We find that the activities of these putative elements are estimated to be weak, and that variation in ϕX174 transcript abundances can be adequately explained without them. Overall, our work demonstrates that ϕX174 gene regulation is well described by the canonical set of promoters and terminators widely used in the literature. [ABSTRACT FROM AUTHOR]

Lytic bacteriophages (phages) are promising clinically viable therapeutic options against carbapenem-resistant Klebsiella pneumoniae (CRKP). In China, the predominant strains are those assigned to sequence type 11 and capsular type 64 (ST11-KL64). The emergence of phage resistance is a major bottleneck hindering effective phage therapy, requiring more new phages to provide the flexibility for creating different phage cocktails. However, the majority of phages against ST11-KL64 CRKP belong to the genus Przondovirus of the family Autographiviridae, which limits the options for constructing cocktails. We recovered a novel lytic phage of the genus Taipeivirus within the family Ackermannviridae against ST11-KL64 CRKP from a river in China. We phenotypically characterized this phage and obtained its genome sequence for analysis. This phage can inhibit the growth of ST11-KL64 CRKP for 6.5  h at a 0.1 multiplicity of infection and exhibits a narrow host range, being unable to attack CRKP strains of the other 30 capsular types. This phage carries no genes encoding antimicrobial resistance, virulence, or lysogeny. It is stable across a wide range of temperatures and pH values, making it suitable for phage therapy. Unlike other Taipeivirus phages, P01 has two tail spike proteins and a unique tail fiber protein. The distinct tail composition of this phage contributes to its activity against ST11-KL64 CRKP and its narrow host range. Taken together, we recovered a phage of a novel viral species with the potential for therapy, which expands the phage biobank against CRKP. [ABSTRACT FROM AUTHOR]

Introduction: Accurate taxonomic profiling of the human microbiome composition is crucial for linking microbial species to health outcomes. Therefore, we created the Clinical Microbiomics Human Microbiome Profiler (CHAMP), a comprehensive tool designed for the profiling of prokaryotes, eukaryotes, and viruses across all body sites. Methods: CHAMP uses a reference database derived from 30,382 human microbiome samples, covering 6,567 prokaryotic and 244 eukaryotic species, as well as 64,003 viruses. We benchmarked CHAMP against established profiling tools (MetaPhlAn 4, Bracken 2, mOTUs 3, and Phanta) using a diverse set of in silico metagenomes and DNA mock communities. Results: CHAMP demonstrated unparalleled species recall, F1 score, and significantly reduced false positives compared to all other tools benchmarked. The false positive relative abundance (FPRA) for CHAMP was, on average, 50-fold lower than the second-best performing profiler. CHAMP also proved to be more robust than other tools at low sequencing depths, highlighting its application for low biomass samples. Discussion: Taken together, this establishes CHAMP as a best-in-class human microbiome profiler of prokaryotes, eukaryotes, and viruses in diverse and complex communities across low and high biomass samples. [ABSTRACT FROM AUTHOR]

Background: The extensively drug-resistant (XDR) strains of Acinetobacter baumannii have become a major cause of nosocomial infections, increasing morbidity and mortality worldwide. Many different treatments, including phage therapy, are attractive ways to overcome the challenges of antibiotic resistance. Methods: This study investigates the biofilm formation ability of 30 XDR A. baumannii isolates and the efficacy of a cocktail of four tempetate bacteriophages (SA1, Eve, Ftm, and Gln) and different antibiotics (ampicillin/sulbactam, meropenem, and colistin) in inhibiting and degrading the biofilms of these strains. Results: The majority (83.3%) of the strains exhibited strong biofilm formation. The bacteriophage cocktail showed varying degrees of effectiveness against A. baumannii biofilms, with higher concentrations generally leading to more significant inhibition and degradation rates. The antibiotics-bacteriophage cocktail combinations also enhanced the inhibition and degradation of biofilms. Conclusion: The findings suggested that the bacteriophage cocktail is an effective tool in combating A. baumannii biofilms, with its efficacy depending on the concentration. Combining antibiotics with the bacteriophage cocktail improved the inhibition and removal of biofilms, indicating a promising strategy for managing A. baumannii infections. These results contribute to our understanding of biofilm dynamics and the potential of bacteriophage cocktails as a novel therapeutic approach to combat antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

Background: Klebsiella quasipneumoniae (previously known as K. pneumoniae K6) strains are among the multidrug-resistant hypervirulent bacterial pathogens. Phage therapy can help treat infections caused by such pathogens. Here we report some aspects of virology and therapeutic potentials of vB_KquU_ 9K11K6, a bacteriophage that infects Klebsiella quasipneumoniae. Methods: K. quasipneumoniae (ATCC 700603) was used to screen wastewater lytic phages. The isolate vB_KquU_çKuK6 that consistently created large clear plaques was characterized using standard virological and molecular methods. Results: vB_KquU_çKuK6 has a complex capsid with an icosahedral head (~60 nm) and a slender tail (~140 nm x 10 nm). The phage has a 51% AT-rich linear dsDNA genome (51,251 bp) containing 121 open reading frames. The genome contains genes encoding spanin, endolysin, and holin proteins necessary for lytic infection and a recombinase gene possibly involved in lysogenic infection. vB_KquU_çKuK6 is stable at -80 to +67°C, pH 4-9, and brief exposure to one volume percent of chloroform. vB_KquU_çKuK6 has a narrow host range. Its lytic infection cycle involves a latency of 20 min and a burst size of 435 plaqueforming units. The phage can cause lysogenic infection, and the resulting lysogens are resistant to lytic infection by vB_KquU_çKuK6. vB_KquU_çKuK6 reduces the host cells' ability to form biofilm but fails to eliminate that ability. vB_KquU_çKuK6 demonstrates phage-antibiotic synergy and reduces the minimum inhibitory concentration of chloramphenicol and neomycin sulfate by about 8 folds. Conclusion: vB_KquU_çKuK6 cannot be directly used for phage therapy because it is a temperate bacteriophage. However, genetically modified strains of vB_KquU_çKuK6 alone or combined with antibiotics or other lytic Klebsiella phages can have therapeutic utilities in treating K. quasipneumoniae infections. [ABSTRACT FROM AUTHOR]

Small single-stranded DNA phages of the Microviridae family are diverse and prevalent in oceans. Our understanding of Microviridae phages that infect the ecologically important marine Roseobacter is currently limited, comprising few isolates. Here, we report six roseophages that infect Roseobacter RCA strains. Genomic and phylogenetic analyses revealed that they were new members of the previously identified subfamily Occultatumvirinae. Additionally, 232 marine uncultivated virus genomes (UViGs) affiliated to Occultatumvirinae were obtained from environmental genome datasets. Phylogenomic analysis revealed that marine Occultatumvirinae phages could be further grouped into 11 subgroups. Moreover, meta-omics based read-mapping analysis showed that Occultatumvirinae phages were globally distributed, with two low G + C subgroups showing the most prevalent distribution. Furthermore, one phage in subgroup 2 was found to be extremely ubiquitous. Overall, this study expands our understanding of the diversity and ecology of the Occultatumvirinae microviruses in the ocean and highlights their ecological impacts. Six roseophages that infect Roseobacter RCA are described as new Occultatumvirinae, to which further 232 marine uncultivated virus genomes obtained from environmental genome datasets could be assigned; these are globally distributed in 11 subgroups. [ABSTRACT FROM AUTHOR]

Klebsiella variicola is considered an emerging pathogen, which may colonize a variety of hosts, including environmental sources. Klebsiella variicola investigated in this study was obtained from an influent wastewater treatment plant in the North-West Province, South Africa. Whole genome sequencing was conducted to unravel the genetic diversity and antibiotic resistance patterns of K. variicola. Whole genome core SNP phylogeny was employed on publicly available 170 genomes. Furthermore, capsule types and antibiotic resistance genes, particularly beta-lactamase and carbapenems genes were investigated from the compared genomes. A 38 099 bp bacteriophage was uncovered alongside with K. variicola genome. Whole genome sequencing revealed that the extended beta-lactamase blaLEN (75.3%) of the beta-lactamase is dominant among compared K. variicola strains. The identified IncF plasmid AA035 confers resistance genes of metal and heat element subtypes, i.e., silver, copper, and tellurium. The capsule type KL107-D1 is a predominant capsule type present in 88.2% of the compared K. variicola genomes. The phage was determined to be integrase-deficient consisting of a fosB gene associated with fosfomycin resistance and clusters with the Wbeta genus Bacillus phage group. In silico analysis showed that the phage genome interacts with B. cereus as opposed to K. variicola strain T2. The phage has anti-repressor proteins involved in the lysis-lysogeny decision. This phage will enhance our understanding of its impact on bacterial dissemination and how it may affect disease development and antibiotic resistance mechanisms in wastewater treatment plants. This study highlights the need for ongoing genomic epidemiological surveillance of environmental K. variicola isolates. [ABSTRACT FROM AUTHOR]

Biofilms are complex microbial communities in which planktonic and dormant bacteria are enveloped in extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, lipids, and DNA. These multicellular structures present resistance to conventional antimicrobial treatments, including antibiotics. The formation of biofilms raises considerable concern in healthcare settings, biofilms can exacerbate infections in patients and compromise the integrity of medical devices employed during treatment. Similarly, certain bacterial species contribute to bulking, foaming, and biofilm development in water environments such as wastewater treatment plants, water reservoirs, and aquaculture facilities. Additionally, food production facilities provide ideal conditions for establishing bacterial biofilms, which can serve as reservoirs for foodborne pathogens. Efforts to combat antibiotic resistance involve exploring various strategies, including bacteriophage therapy. Research has been conducted on the effects of phages and their individual proteins to assess their potential for biofilm removal. However, challenges persist, prompting the examination of refined approaches such as drug-phage combination therapies, phage cocktails, and genetically modified phages for clinical applications. This review aims to highlight the progress regarding bacteriophage-based approaches for biofilm eradication in different settings. [ABSTRACT FROM AUTHOR]

Dental infections, such as apical Periodontitis, periodontitis, and peri-implantitis (PI), are closely associated with specific bacterial species, including Streptococcus mutans (S. mutans), Porphyromonas gingivalis (P. gingivalis), and Fusobacterium nucleatum (F. nucleatum), among others. Antibiotics are extensively utilized for prophylactic and therapeutic purposes in the treatment of dental infections and other dental-related issues. Unfortunately, the rapid emergence of antimicrobial resistance has accompanied the increased use of antibiotics in recent years. Specific bacterial pathogens have reached a critical stage of antibiotic resistance, characterized by the proliferation of pan-resistant strains and the scarcity of viable therapeutic alternatives. Therapeutic use of particular bacteriophage (phage) particles that target bacterial pathogens is one potential alternative to antibiotics that are now being seriously considered for treating bacterial illnesses. A kind of virus known as a phage is capable of infecting and eliminating bacteria. Because they can't infect cells in plants and animals, phages might be a harmless substitute for antibiotics. To control oral disorders including periodontitis and dental caries, several research have been conducted in this area to study and identify phages from human saliva and dental plaque. The capacity of these agents to disturb biofilms expands their effectiveness against dental plaque biofilms and oral pathogens in cases of periodontitis, PI, and apical periodontitis. This review summarizes the current antibacterial properties of phages used to treat a variety of dental infections, such as periodontitis, peri-implantitis, infected dentin, and apical periodontitis. [ABSTRACT FROM AUTHOR]

Bacteria at different growth stages usually coordinate capsular polysaccharide (CPS) formation and may affect their susceptibility to phage. In this study, we evaluated the infection efficacy of phage vB_VspM_VS2 in V. splendidus AJ01 at different growth stages and explored the role of growth stage-related CPS translocon Wza in the susceptibility of V. splendidus to phage vB_VspM_VS2. The results showed that V. splendidus locked in the stationary growth stage (SGS) or early exponential stage (EES) infected with phage (EES-P) has a low susceptibility to phage vB_VspM_VS and exhibit a pronounced reduction in phage adsorption rate as compared to the EES bacteria. The expression of wza of CPS transport gene was significantly increased in EES bacteria compared to that bacteria locked in the SGS or EES-P. Bacteria with deleted wza (Δwza mutant) escaped phage adsorption due to absence of Wza mediated down-regulation of CPS expression, otherwise. Our results reveal that the Wza of V. splendidus can promotes phage to infect these bacteria via increasing the phage absorption, which provides important implications for using phages therapeutically target pathogenic bacteria in dynamics communities. Vibrio splendidus with deleted wza (Δwza mutant) escaped phage adsorption due to absence of Wza mediated down-regulation of CPS expression. [ABSTRACT FROM AUTHOR]

Bacteriophages (phages) are viruses that infect bacteria and can be classified into two different lifecycles. Virulent phages (or lytic phages) have a lytic cycle that can lyse the bacteria host after their infection. Temperate phages (or lysogenic phages) can integrate their phage genomes into bacterial chromosomes and replicate with bacterial hosts via the lysogenic cycle. Identifying phage lifecycles is a crucial step in developing suitable applications for phages. Compared to the complicated traditional biological experiments, several tools have been designed for predicting phage lifecycle using different algorithms, such as random forest (RF), linear support-vector classifier (SVC), and convolutional neural network (CNN). In this study, we developed a natural language processing (NLP)-based tool—DeepPL—for predicting phage lifecycles via nucleotide sequences. The test results showed that our DeepPL had an accuracy of 94.65% with a sensitivity of 92.24% and a specificity of 95.91%. Moreover, DeepPL had 100% accuracy in lifecycle prediction on the phages we isolated and biologically verified previously in the lab. Additionally, a mock phage community metagenomic dataset was used to test the potential usage of DeepPL in viral metagenomic research. DeepPL displayed a 100% accuracy for individual phage complete genomes and high accuracies ranging from 71.14% to 100% on phage contigs produced by various next-generation sequencing technologies. Overall, our study indicates that DeepPL has a reliable performance on phage lifecycle prediction using the most fundamental nucleotide sequences and can be applied to future phage and metagenomic research. Author summary: Bacteriophages are viruses that infect bacteria and play a critical role in the microbial community within different environments via phage-bacterial evolutionary interactions. The classification of phage lifecycles is of great importance in deploying the potential applications of phages and better understanding complex microbial interactions. However, the traditional biological methods for phage lifecycle identification are complicated and time-consuming. In this study, we proposed a deep learning-based tool—DeepPL—for predicting phage lifecycles using the phage nucleotide genome. Compared with other bioinformatic tools, DeepPL was developed from the pre-trained transformers model—DNABERT—designed for fundamental nucleotide language combined with representative phage complete genomes. Our in-house biological results were further used to verify the output of DeepPL. Overall, DeepPL performs with high precision for phage lifecycle prediction and could contribute to the genomic data-driven direction of phage research and applications. [ABSTRACT FROM AUTHOR]

Three‐dimensional (3D) hierarchical wrinkled materials built with biological entities have so far remained exclusive to nature. Herein, multiscale functional ultraporous 3D bio‐networks of bioprinted phage‐built wrinkled microarrays are created through establishing a universal heat‐ and solvent‐independent substrate‐shrinkage method induced by high‐pressure carbon dioxide (HPCD). This method results in diverse wrinkled patterns on soft materials and is particularly powerful for solvent‐ and heat‐sensitive biomaterials, for which other methods have failed. The phage nanofilaments (7 nm width) self‐assemble into orderly‐aligned submicron bundles (100 nm width), which crimp into tunable microscale wrinkles (0.7–5.0 µm width) on size‐controllable micro‐arrays (200–600 µm width) exhibiting a four‐level hierarchical nano‐reticular structure. The HPCD method also protects the bioactivity of biorecognition molecules loaded into the microarrays, leading to the design of bacteria‐sensing chips, made with in‐house deoxyribozyme‐loaded 3D phage microarrays. The developed bacteria‐sensing chips achieve a limit of detection that is 100 × more sensitive with greater reproducibility compared to two‐dimensional (2D) microdot arrays and correctly identify Legionella pneumophila in contaminated water samples collected from industrial cooling towers, highlighting phage‐built wrinkled networks as a platform for bottom‐up assembly of biological building blocks into biofunctional material. [ABSTRACT FROM AUTHOR]

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii , a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using in vitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy. [Display omitted] • A few CRAB types dominate infections in individual world regions • Phylogeography reveals stable strain composition for countries over 6 years • Spatiotemporal distribution informs the development of region-specific phage collections • Phage cocktails are effective against the most prevalent strain types in Eastern Europe By mapping the genetic diversity and geographic distribution of carbapenem-resistant Acinetobacter baumannii , bacteriophage cocktails were developed and designed to treat the maximum number of patients within a specific geographic region. [ABSTRACT FROM AUTHOR]

Bacteriophages are influential within the human gut microbiota, yet they remain understudied relative to bacteria. This is a limitation of studies on fecal microbiota transplantation (FMT) where bacteriophages likely influence outcome. Here, using metagenomics, we profile phage populations - the phageome - in individuals recruited into two double-blind randomized trials of FMT in ulcerative colitis. We leverage the trial designs to observe that phage populations behave similarly to bacterial populations, showing temporal stability in health, dysbiosis in active disease, modulation by antibiotic treatment and by FMT. We identify a donor bacteriophage putatively associated with disease remission, which on genomic analysis was found integrated in a bacterium classified to Oscillospiraceae, previously isolated from a centenarian and predicted to produce vitamin B complex except B12. Our study provides an in-depth assessment of phage populations during different states and suggests that bacteriophage tracking has utility in identifying determinants of disease activity and resolution. Here, the authors profile the gut phageome of individuals recruited into two double-blind randomized trials of Fecal Microbial Transplantation for ulcerative colitis, showing that phage communities are stable in health, dysbiotic in ulcerative colitis, modulated by antibiotics and by fecal transplants, with one Oscillospiraceae phage being associated with disease remission. [ABSTRACT FROM AUTHOR]

The global dissemination of multi-drug resistant (MDR) pathogenic bacteria requires the rapid research and development of alternative therapies that can support or replace conventional antibiotics. Among MDR pathogens, carbapenem-resistant Klebsiella pneumoniae (CR-Kp) are of particular concern due to their extensive resistance profiles, global dissemination in hospital environments, and their major role in some life-threatening infections. Phages, or some of their components, are recognized as one of the potential alternatives that might be helpful to treat bacterial infections. In this study, we have isolated and characterized four lytic bacteriophages targeting K. pneumoniae strains of Sequence Type (ST) 307 or ST147, two predominant high-risk clones of CR-Kp. Phages, designated vB_KpS_GP-1, vB_KpP_GP-2, vB_KpP_GP-4, and vB_KpP_GP-5, were isolated from sewage wastewater samples. The vB_KpS_GP-1 phage was a siphovirus unable to establish lysogeny with its host, while the other three were podoviruses. While 85.7% of K. pneumoniae strains of ST307 were selectively lysed by the phages vB_KpS_GP-1 or vB_KpP_GP-5, the other two phages were able to lyse all the tested strains of ST147 (n = 12). Phages were stable over a broad pH and temperature range and were characterized by burst sizes of 10-100 plaque forming units and latency periods of 10-50 minutes. Genome sequencing confirmed the absence of antibiotic resistance genes, virulence factors or toxins and revealed that two phages were likely members of new genera. Given their strictly lytic nature and high selectivity towards two of the major high-risk clones of K. pneumoniae, cocktails of these phages could represent promising candidates for further evaluation in in vivo experimental models of K. pneumoniae infection. [ABSTRACT FROM AUTHOR]

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems. [ABSTRACT FROM AUTHOR]

The food industry has incurred substantial losses from contamination by Pseudomonas fluorescens, emphasizing the critical importance of implementing effective control strategies. Phages are potential sterilizers due to their specific killing abilities and the difficulty bacteria face in developing resistance. However, a significant barrier to their development is the lack of diversity among phage types. In this study, we characterized a novel lytic P. fluorescens phage, named vB_PF_Y1-MI. Phage vB_PF_Y1-MI displayed a latent period of nearly 10 min and a high burst size of 1493 PFU/cell. This phage showed good activity over a wide range of temperature (up to 70 °C) and pH (3–12). The genome of phage vB_PF_Y1-MI spans 93,233 bp with a GC content of 45%. It encompasses 174 open-reading frames and 19 tRNA genes, while no lysogeny or virulence-associated genes were detected. Phylogenetic analysis positions it as a novel unassigned evolutionary lineage within the Caudoviricetes class among related dsDNA phages. Our study provides foundational insights into vB_PF_Y1-MI and emphasizes its potential as an effective biological control agent against P. fluorescens. This research offers crucial theoretical groundwork and technical support for subsequent efforts in preventing and controlling P. fluorescens contamination. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus has multiple mechanisms to evade the host's immune system and antibiotic treatment. One such mechanism is the invasion of the osteocyte lacuno‐canalicular network (OLCN), which may be particularly important in recurrence of infection after debridement and antibiotic therapy. The aim of this study was to develop an ex vivo model to facilitate further study of S. aureus invasion of the OLCN and early‐stage testing of antibacterial strategies against bacteria in this niche. The diameter of the canaliculi of non‐infected human, sheep, and mouse bones was measured microscopically on Schmorl's picrothionin stained sections, showing a large overlap in canalicular diameter. S. aureus successfully invaded the OLCN in all species in vitro as revealed by presence in osteocyte lacunae in Brown and Brenn‐stained sections and by scanning electron microscopy. Murine bones were then selected for further experiments, and titanium pins with either a wild‐type or ΔPBP4 mutant S. aureus USA300 were placed trans‐cortically and incubated for 2 weeks in tryptic soy broth. Wild‐type S. aureus readily invaded the osteocyte lacunae in mouse bones while the ΔPBP4 showed a significantly lower invasion of the OLCN (p = 0.0005). Bone specimens were then treated with gentamicin, sitafloxacin, R14 bacteriophages, or left untreated. Gentamicin (p = 0.0027) and sitafloxacin (p = 0.0280) significantly reduced the proportion of S. aureus‐occupied lacunae, whilst bacteriophage treatment had no effect. This study shows that S. aureus is able to invade the OLCN in an ex vivo model. This ex vivo model can be used for future early‐stage studies before proceeding to in vivo studies. [ABSTRACT FROM AUTHOR]

The symbiotic relationship between the gut microbiome and the human body is a concept that has grown in popularity in recent years. Bacteriophages (phages) are components of the gut microbiota and their imbalance plays a role in the pathogenesis of numerous intestinal disorders. Meanwhile, as a new antimicrobial agent, phage therapy (PT) offers unique advantages when compared with antibiotics and brings a new dawn for treatment of multidrugresistant bacteria in intestinal and extraintestinal disorders. In this review, we provide a brief introduction to the characterization of phages, particularly focusing on newly discovered phages. Additionally, we outline the involvement of gut phages in disease pathogenesis and discuss the status and challenges of utilizing phages as therapeutic targets for treatment of enteric infection. [ABSTRACT FROM AUTHOR]

The transcriptional architecture of phages can deepen our understanding of the phage‐host infection process and can be of key importance for phage engineering and biotechnological applications. Here, we applied ONT‐cappable‐sequencing, a long‐read RNA‐sequencing technique, to study the regulatory mechanisms of Pseudomonas infecting giant phage 201ϕ2‐1. We identified 67 promoters and 132 terminators that together represent 92 transcriptional units. A full comparison of these data to the transcriptome of model Pseudomonas phage ϕKZ confirmed that the transcriptional programs of these prototypes of the Serwervirus and Phikzvirus genera are largely conserved, despite some subtle regulatory differences. Evidence supporting these shared mechanisms include the identification of highly similar sequence motifs for regulatory elements in both phages and the conservation of regulatory elements loci relative to homologous genes in each phage. Moreover, we discovered a sRNA in 201ϕ2‐1 that is highly conserved among prototype members of different giant phage genera. Sequencing of the 201ϕ2‐1 host genome resulted in its reclassification as Pseudomonas atacamensis, a close relative of the important agricultural biocontrol agent Pseudomonas chlororaphis. Finally, we conducted in vivo assays of eight 201ϕ2‐1 terminators and found them to strongly terminate transcription in P. chlororaphis. Control elements from phage transcriptional programs have a rich history for applications in biotechnology. In these studies, we demonstrate new insight into the transcriptional program of 201ϕ2‐1 and demonstrate the potential of its regulatory elements for novel and useful tools for synthetic biology circuitry. [ABSTRACT FROM AUTHOR]

Agricultural crop yield losses and food destruction due to infections by phytopathogenic bacteria such as Burkholderia gladioli, which causes devastating diseases in onion, mushroom, corn, and rice crops, pose major threats to worldwide food security and cause enormous damage to the global economy. Biocontrol using bacteriophages has emerged as a promising strategy against a number of phytopathogenic species but has never been attempted against B. gladioli due to a lack of quantitative infection models and a scarcity of phages targeting this specific pathogen. In this study, we present a novel, procedurally straightforward, and highly generalizable fully quantitative ex planta maceration model and an accompanying quantitative metric, the ex planta maceration index (xPMI). In utilizing this model to test the ex planta virulence of a panel of 12 strains of B. gladioli in Allium cepa and Agaricus bisporus, we uncover substantial temperature-, host-, and strain-dependent diversity in the virulence of this fascinating pathogenic species. Crucially, we demonstrate that Burkholderia phages KS12 and AH2, respectively, prevent and reduce infection-associated onion tissue destruction, measured through significant (P < 0.0001) reductions in xPMI, by phytopathogenic strains of B. gladioli, thereby demonstrating the potential of agricultural phage biocontrol targeting this problematic microorganism. IMPORTANCE Agricultural crop destruction is increasing due to infections caused by bacteria such as Burkholderia gladioli, which causes plant tissue diseases in onion, mushroom, corn, and rice crops. These bacteria pose a major threat to worldwide food production, which, in turn, damages the global economy. One potential solution being investigated to prevent bacterial infections of plants is "biocontrol" using bacteriophages (or phages), which are bacterial viruses that readily infect and destroy bacterial cells. In this article, we demonstrate that Burkholderia phages KS12 and AH2 prevent or reduce infection-associated plant tissue destruction caused by strains of B. gladioli, thereby demonstrating the inherent potential of agricultural phage biocontrol. [ABSTRACT FROM AUTHOR]

Traditional antibiotics have been effective in many cases. However, the rise in multidrug-resistant bacteria has diminished their therapeutic efficacy, signaling the dawn of an era beyond antibiotics. The challenge of multidrug resistance in Klebsiella pneumoniae is particularly critical, with increasing global mortality and resistance rates. Therefore, the development of alternative therapies to antibiotics is urgently needed. Phages, which are natural predators of bacteria, have inherent advantages. However, comprehensive information on K. pneumoniae phages is lacking in current literature. This review aims to analyze and summarize relevant studies, focusing on the present state of phage therapy for K. pneumoniae infections. This includes an examination of treatment methodologies, associated challenges, strategies, new phage technologies, clinical trial safety and efficacy, regulatory issues, and future directions for phage therapy development. Enhancing phage technology is crucial for addressing the evolving threat of multidrug-resistant K. pneumoniae. [ABSTRACT FROM AUTHOR]

Background: Infections following shoulder surgery, particularly periprosthetic joint infection (PJI), are challenging to treat. Cutibacterium acnes is the causative pathogen in 39% to 76% of these cases. This study explores the efficacy of bacteriophage therapy as an alternative to conventional antibiotics for treating such infections. Methods: Nine phages with lytic activity were isolated from the skin of humans using C. acnes ATCC 6919 as the indicator host. These phages were tested individually or in combination to assess host range and antibiofilm activity against clinical strains of C. acnes associated with PJIs. The phage cocktail was optimized for broad-spectrum activity and tested in vitro against biofilms formed on titanium discs to mimic the prosthetic environment. Results: The isolated phages displayed lytic activity against a range of C. acnes clinical isolates. The phage cocktail significantly reduced the bacterial load of C. acnes strains 183, 184, and GG2A, as compared with untreated controls (p < 0.05). Individual phages, particularly CaJIE7 and CaJIE3, also demonstrated significant reductions in bacterial load with respect to specific strains. Moreover, phages notably disrupted the biofilm structure and reduced biofilm biomass, confirming the potential of phage therapy in targeting biofilm-associated infections. Conclusions: Our preclinical findings support the potential of phage therapy as a viable adjunct to traditional antibiotics for treating C. acnes infections in orthopedic device-related infections. The ability of phages to disrupt biofilms may be particularly beneficial for managing infections associated with prosthetic implants. [ABSTRACT FROM AUTHOR]

Bacteriophage (phage) studies established the field of molecular biology and continue to propel life science research forward due to their diversity, abundance, and potential applications. In this Gem article, we orient newcomers to four common ways phages are currently classified: infection cycle, morphology, taxonomy, and supergroup. By using these classifications, researchers can determine where any novel phage fits into the scheme of the known "phage-verse". [ABSTRACT FROM AUTHOR]

Bacteria exposed to bactericidal treatment, such as antibiotics or bacteriophages (phages), often develop resistance. While phage therapy is proposed as a solution to the antibiotic resistance crisis, the bacterial resistance emerging during phage therapy remains poorly characterized. In this study, we examined a large population of phage-resistant extra-intestinal pathogenic Escherichia coli 536 clones that emerged from both in vitro (non-limited liquid medium) and in vivo (murine pneumonia) conditions. Genome sequencing uncovered a convergent mutational pattern in phage resistance mechanisms under both conditions, particularly targeting two cell-wall components, the K15 capsule and the lipopolysaccharide (LPS). This suggests that their identification in vivo could be predicted from in vitro assays. Phage-resistant clones exhibited a wide range of fitness according to in vitro tests, growth rate, and resistance to amoeba grazing, which could not distinguish between the K15 capsule and LPS mutants. In contrast, K15 capsule mutants retained virulence comparable to the wild-type strain, whereas LPS mutants showed significant attenuation in the murine pneumonia model. Additionally, we observed that resistance to the therapeutic phage through an onspecific mechanism, such as capsule overproduction, did not systematically lead to co-resistance to other phages that were initially capable or incapable of infecting the wild-type strain. Our findings highlight the importance of incorporating a diverse range of phages in the design of therapeutic cocktails to target potential future phage-resistant clones effectively. [ABSTRACT FROM AUTHOR]