Isolation and characterization of novel Staphylococcus aureus bacteriophage Hesat from dairy origin.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (108 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins.

Refining the transcriptional landscapes for distinct clades of virulent phages infecting Pseudomonas aeruginosa.

The introduction of high-throughput sequencing has resulted in a surge of available bacteriophage genomes, unveiling their tremendous genomic diversity. However, our current understanding of the complex transcriptional mechanisms that dictate their gene expression during infection is limited to a handful of model phages. Here, we applied ONT-cappable-seq to reveal the transcriptional architecture of six different clades of virulent phages infecting Pseudomonas aeruginosa. This long-read microbial transcriptomics approach is tailored to globally map transcription start and termination sites, transcription units, and putative RNA-based regulators on dense phage genomes. Specifically, the full-length transcriptomes of LUZ19, LUZ24, 14-1, YuA, PAK_P3, and giant phage phiKZ during early, middle, and late infection were collectively charted. Beyond pinpointing traditional promoter and terminator elements and transcription units, these transcriptional profiles provide insights in transcriptional attenuation and splicing events and allow straightforward validation of Group I intron activity. In addition, ONT-cappable-seq data can guide genome-wide discovery of novel regulatory element candidates, including noncoding RNAs and riboswitches. This work substantially expands the number of annotated phage-encoded transcriptional elements identified to date, shedding light on the intricate and diverse gene expression regulation mechanisms in Pseudomonas phages, which can ultimately be sourced as tools for biotechnological applications in phage and bacterial engineering.

Phage proteins target and co-opt host ribosomes immediately upon infection.

Bacteriophages must seize control of the host gene expression machinery to replicate. To bypass bacterial anti-phage defence systems, this host takeover occurs immediately upon infection. A general understanding of phage mechanisms for immediate targeting of host transcription and translation processes is lacking. Here we introduce an integrative high-throughput approach to uncover phage-encoded proteins that target the gene expression machinery of Pseudomonas aeruginosa immediately upon infection with the jumbo phage ΦKZ. By integrating biochemical, genetic and structural analyses, we identify an abundant and conserved phage factor ΦKZ014 that targets the large ribosomal subunit by binding the 5S ribosomal RNA, and rapidly promotes replication in several clinical isolates. ΦKZ014 is among the earliest ΦKZ proteins expressed after infection and remains bound to ribosomes during the entire translation cycle. Our study provides a strategy to decipher molecular components of phage-mediated host takeover and argues that phage genomes represent an untapped discovery space for proteins that modulate the host gene expression machinery.

Targeted Genome Editing of Virulent Pseudomonas Phages Using CRISPR-Cas3.

The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.

Obtaining Detailed Phage Transcriptomes Using ONT-Cappable-Seq.

Detailed transcription maps of bacteriophages are not usually explored, limiting our understanding of molecular phage biology and restricting their exploitation and engineering. The ONT-cappable-seq method described here brings phage transcriptomics to the accessible nanopore sequencing platform and provides an affordable and more detailed overview of transcriptional features compared to traditional RNA-seq experiments. With ONT-cappable-seq, primary transcripts are specifically capped, enriched, and prepared for long-read sequencing on the nanopore sequencing platform. This enables end-to-end sequencing of unprocessed transcripts covering both phage and host genome, thus providing insight on their operons. The subsequent analysis pipeline makes it possible to rapidly identify the most important transcriptional features such as transcription start and stop sites. The obtained data can thus provide a comprehensive overview of the transcription by your phage of interest.

Complete genome sequences of two Leuconostoc carnosum strains: 4010 and AMS1.

Leuconostoc carnosum is a bacterial species commonly associated with meat spoilage. However, some strains exhibit preservative effects due to bacteriocin production. Here, we report the complete genome sequences for two strains, L. carnosum 4010 and AMS1. Bacteriocin-related gene clusters were found on the plasmids of both strains.

Meeting Report of the Second Symposium of the Belgian Society for Viruses of Microbes and Launch of the Phage Valley.

The second symposium of the Belgian Society for Viruses of Microbes (BSVoM) took place on 8 September 2023 at the University of Liège with 141 participants from 10 countries. The meeting program covered three thematic sessions opened by international keynote speakers: two sessions were devoted to "Fundamental research in phage ecology and biology" and the third one to the "Present and future applications of phages". During this one day symposium, four invited keynote lectures, nine selected talks and eight student pitches were given along with thirty presented posters. The president of the Belgian Society for Viruses of Microbes, Prof. Yves Briers, took advantage of this symposium to launch the Phage Valley concept that will put the spotlight on the exceptionally high density of researchers investigating viruses of microbes as well as the successful triple helix approach between academia, industry and government in Belgium.

Posttranslational modifications in bacteria during phage infection.

During phage infection, both virus and bacteria attempt to gain and/or maintain control over critical bacterial functions, through a plethora of strategies. These strategies include posttranslational modifications (PTMs, including phosphorylation, ribosylation, and acetylation), as rapid and dynamic regulators of protein behavior. However, to date, knowledge on the topic remains scarce and fragmented, while a more systematic investigation lies within reach. The release of AlphaFold, which advances PTM enzyme discovery and functional elucidation, and the increasing inclusivity and scale of mass spectrometry applications to new PTM types, could significantly accelerate research in the field. In this review, we highlight the current knowledge on PTMs during phage infection, and conceive a possible pipeline for future research, following an enzyme-target-function scheme.

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.

Endolysin NC5 improves early cloxacillin treatment in a mouse model of Streptococcus uberis mastitis.

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 µg) combined with either a low (23.5 µg) or high (235.0 µg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog10; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. KEY POINTS: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.

FLAMS: Find Lysine Acylations and other Modification Sites.

SUMMARY: Today, hundreds of post-translational modification (PTM) sites are routinely identified at once, but the comparison of new experimental datasets to already existing ones is hampered by the current inability to search most PTM databases at the protein residue level. We present FLAMS (Find Lysine Acylations and other Modification Sites), a Python3-based command line and web-tool that enables researchers to compare their PTM sites to the contents of the CPLM, the largest dedicated protein lysine modification database, and dbPTM, the most comprehensive general PTM database, at the residue level. FLAMS can be integrated into PTM analysis pipelines, allowing researchers to quickly assess the novelty and conservation of PTM sites across species in newly generated datasets, aiding in the functional assessment of sites and the prioritization of sites for further experimental characterization. AVAILABILITY AND IMPLEMENTATION: FLAMS is implemented in Python3, and freely available under an MIT license. It can be found as a command line tool at https://github.com/hannelorelongin/FLAMS, pip and conda; and as a web service at https://www.biw.kuleuven.be/m2s/cmpg/research/CSB/tools/flams/.

Exploring the transcriptional landscape of phage-host interactions using novel high-throughput approaches.

In the last decade, powerful high-throughput sequencing approaches have emerged to analyse microbial transcriptomes at a global scale. However, to date, applications of these approaches to microbial viruses such as phages remain scarce. Tailoring these techniques to virus-infected bacteria promises to obtain a detailed picture of the underexplored RNA biology and molecular processes during infection. In addition, transcriptome study of stress and perturbations induced by phages in their infected bacterial hosts is likely to reveal new fundamental mechanisms of bacterial metabolism and gene regulation. Here, we provide references and blueprints to implement emerging transcriptomic approaches towards addressing transcriptome architecture, RNA-RNA and RNA-protein interactions, RNA modifications, structures and heterogeneity of transcription profiles in infected cells that will provide guides for future directions in phage-centric therapeutic applications and microbial synthetic biology.

Exploiting phage-antibiotic synergies to disrupt Pseudomonas aeruginosa PAO1 biofilms in the context of orthopedic infections.

IMPORTANCE: Biofilm-related infections are among the most difficult-to-treat infections in all fields of medicine due to their antibiotic tolerance and persistent character. In the field of orthopedics, these biofilms often lead to therapeutic failure of medical implantable devices and urgently need novel treatment strategies. This forthcoming article aims to explore the dynamic interplay between newly isolated bacteriophages and routinely used antibiotics and clearly indicates synergetic patterns when used as a dual treatment modality. Biofilms were drastically more reduced when both active agents were combined, thereby providing additional evidence that phage-antibiotic combinations lead to synergism and could potentially improve clinical outcome for affected patients.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing.

IMPORTANCE: The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

Distinct mode of action of a highly stable, engineered phage lysin killing Gram-negative bacteria.

IMPORTANCE: Engineered lysins are considered as highly promising alternatives for antibiotics. Our previous screening study using VersaTile technology identified 1D10 as a possible lead compound with activity against Acinetobacter baumannii strains under elevated human serum concentrations. In this manuscript, we reveal an unexpected mode of action and exceptional thermoresistance for lysin 1D10. Our findings shed new light on the development of engineered lysins, providing valuable insights for future research in this field.

The phage-encoded PIT4 protein affects multiple two-component systems of Pseudomonas aeruginosa.

IMPORTANCE: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.

ECOPHAGE: Combating Antimicrobial Resistance Using Bacteriophages for Eco-Sustainable Agriculture and Food Systems.

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors.

Isolation and molecular characterization of the Salmonella Typhimurium orphan phage Arash.

The current threat of multidrug resistant strains necessitates development of alternatives to antibiotics such as bacteriophages. This study describes the isolation and characterization of a novel Salmonella Typhimurium phage 'Arash' from hospital wastewater in Leuven, Belgium. Arash has a myovirus morphology with a 95 nm capsid and a 140 nm tail. The host range of Arash is restricted to its isolation host. Approximately 86% of the phage particles are adsorbed to a host cell within 10 min. Arash has latent period of 65 min and burst size of 425 PFU/cell. Arash has a dsDNA genome of 180,819 bp with GC content of 53.02% with no similarities to any characterized phages, suggesting Arash as a novel species in the novel 'Arashvirus' genus. Arash carries no apparent lysogeny-, antibiotic resistance- nor virulence-related genes. Proteome analysis revealed 116 proteins as part of the mature phage particles of which 27 could be assigned a function. Therefore, the present findings shed light on the morphological, microbiological and genomic characteristics of Arash and suggest its potential application as therapeutic and/or biocontrol agent.

Alginate microbeads and hydrogels delivering meropenem and bacteriophages to treat Pseudomonas aeruginosa fracture-related infections.

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9-30 and MK.R3-15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future.