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ABSTRACT Streptomyces, a multifaceted genus of soil-dwelling bacteria belonging to the phylum Actinomycetota, features intricate phage-host interactions shaped by its complex life cycle and the synthesis of a diverse array of specialized metabolites. Here, we describe the isolation and characterization of four novel Streptomyces phages infecting a variety of different host species. While phage Kamino, isolated on Streptomyces kasugaensis, is predicted to be temperate and encodes a serine integrase in its genome, phages Geonosis (isolated on Streptomyces griseus) and Abafar and Scarif, isolated on Streptomyces albidoflavus, are virulent phages. Phages Kamino and Geonosis were shown to amplify well in liquid culture leading to a pronounced culture collapse already at low titers. Determination of the host range by testing >40 different Streptomyces species identified phages Kamino, Abafar, and Scarif as broad host-range phages. Overall, the phages described in this study expand the publicly available portfolio of phages infecting Streptomyces and will be instrumental in advancing the mechanistic understanding of the intricate antiviral strategies employed by these multicellular bacteria.IMPORTANCEThe actinobacterial genus Streptomyces is characterized by multicellular, filamentous growth and the synthesis of a diverse range of bioactive molecules. These characteristics also play a role in shaping their interactions with the most abundant predator in the environment, bacteriophages—viruses infecting bacteria. In this study, we characterize four new phages infecting Streptomyces. Out of those, three phages feature a broad host range infecting up to 15 different species. The isolated phages were characterized with respect to plaque and virion morphology, host range, and amplification in liquid culture. In summary, the phages reported in this study contribute to the broader collection of publicly available phages infecting Streptomyces, playing a crucial role in advancing our mechanistic understanding of phage-host interactions of these multicellular bacteria.

ABSTRACTThe species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

ABSTRACT In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.

Hierarchical structures are abundant in almost all tissues of the human body. Therefore, it is highly important for tissue engineering approaches to mimic such structures if a gain of function of the new tissue is intended. Here, the hierarchical structures of the so-called enthesis, a gradient tissue located between tendon and bone, were in focus. Bridging the mechanical properties from soft to hard secures a perfect force transmission from the muscle to the skeleton upon locomotion. This study aimed at a novel method of bioprinting to generate gradient biomaterial constructs with a focus on the evaluation of the gradient printing process. First, a numerical approach was used to simulate gradient formation by computational flow as a prerequisite for experimental bioprinting of gradients. Then, hydrogels were printed in a single cartridge printing set-up to transfer the findings to biomedically relevant materials. First, composites of recombinant spider silk hydrogels with fluorapatite rods were used to generate mineralized gradients. Then, fibroblasts were encapsulated in the recombinant spider silk-fluorapatite hydrogels and gradually printed using unloaded spider silk hydrogels as the second component. Thereby, adjustable gradient features were achieved, and multimaterial constructs were generated. The process is suitable for the generation of gradient materials, e.g., for tissue engineering applications such as at the tendon/bone interface.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day −55 and −27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day −70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient’s respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains.

Worldwide, poultry industry suffers from infections caused by avian pathogenic Escherichia coli. Therapeutic failure due to resistant bacteria is of increasing concern and poses a threat to human and animal health. This causes a high demand to find alternatives to fight bacterial infections in animal farming. Bacteriophages are being especially considered for the control of multi-drug resistant bacteria due to their high specificity and lack of serious side effects. Therefore, the study aimed on characterizing phages and composing a phage cocktail suitable for the prevention of infections with E. coli. Six phages were isolated or selected from our collections and characterized individually and in combination with regard to host range, stability, reproduction, and efficacy in vitro. The cocktail consisting of six phages was able to inhibit formation of biofilms by some E. coli strains but not by all. Phage-resistant variants arose when bacterial cells were challenged with a single phage but not when challenged by a combination of four or six phages. Resistant variants arising showed changes in carbon metabolism and/or motility. Genomic comparison of wild type and phage-resistant mutant E28.G28R3 revealed a deletion of several genes putatively involved in phage adsorption and infection.

The aim of this study was to gain further insight into the diversity of Escherichia coli phages followed by enhanced work on taxonomic issues in that field. Therefore, we present the genomic characterization and taxonomic classification of 50 bacteriophages against E. coli isolated from various sources, such as manure or sewage. All phages were examined for their host range on a set of different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms. Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and 8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb. Annotation and comparison with databases showed similarities in particular to T4- and T5-like phages, but also to less-known groups. Though various phages against E. coli are already described in literature and databases, we still isolated phages that showed no or only few similarities to other phages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny and classification of the newly isolated phages using VICTOR resulted in the proposal of new genera and led to an enhanced taxonomic classification of E. coli phages.