An autonomous plasmid as an inovirus phage satellite.

Bacterial viruses (phages) are potent agents of lateral gene transfer and thus are important drivers of evolution. A group of mobile genetic elements, referred to as phage satellites, exploits phages to disseminate their own genetic material. Here, we isolated a novel member of the family Inoviridae, Shewanella phage Dolos, along with an autonomously replicating plasmid, pDolos. Dolos causes a chronic infection in its host Shewanella oneidensis by phage production with only minor effects on the host cell proliferation. When present, plasmid pDolos hijacks Dolos functions to be predominantly packaged into phage virions and released into the environment and, thus, acts as a phage satellite. pDolos can disseminate further genetic material encoding, e.g., resistances or fluorophores to host cells sensitive to Dolos infection. Given the rather simple requirements of a plasmid for takeover of an inovirus and the wide distribution of phages of this group, we speculate that similar phage-satellite systems are common among bacteria.IMPORTANCEPhage satellites are mobile genetic elements, which hijack phages to be transferred to other host cells. The vast majority of these phage satellites integrate within the host's chromosome, and they all carry remaining phage genes. Here, we identified a novel phage satellite, pDolos, which uses an inovirus for dissemination. pDolos (i) remains as an autonomously replicating plasmid within its host, (ii) does not carry recognizable phage genes, and (iii) is smaller than any other phage satellites identified so far. Thus, pDolos is the first member of a new class of phage satellites, which resemble natural versions of phagemids.

Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The 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.

Bacteriophages targeting protective commensals impair resistance against Salmonella Typhimurium infection in gnotobiotic mice.

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Flow Simulation and Gradient Printing of Fluorapatite- and Cell-Loaded Recombinant Spider Silk Hydrogels.

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.

Effects of atherogenic diet supplemented with fermentable carbohydrates on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model.

Fermentable carbohydrates are gaining interest in the field of human nutrition because of their benefits in obesity-related comorbidities. The aim of this study was to investigate the influence of fermentable carbohydrates, such as pectin and inulin, in an atherogenic diet on metabolic responses and plaque formation in coronary arteries using a Saddleback pig model. Forty-eight healthy pigs aged five months were divided into four feeding groups (n = 10) and one baseline group (n = 8). Three feeding groups received an atherogenic diet (38% crisps, 10% palm fat, and 2% sugar with or without supplementation of 5% pectin or inulin), and one group received a conventional diet over 15 weeks. Feed intake, weight gain, body condition score, and back fat thickness were monitored regularly. Blood and fecal samples were collected monthly to assess the metabolites associated with high cardiovascular risk and fat content, respectively. At the end of 15 weeks, the coronary arteries of the pigs were analyzed for atherosclerotic plaque formation. Independent of supplementation, significant changes were observed in lipid metabolism, such as an increase in triglycerides, bile acids, and cholesterol in serum, in all groups fed atherogenic diets in comparison to the conventional group. Serum metabolome analysis showed differentiation of the feeding groups by diet (atherogenic versus conventional diet) but not by supplementation with pectin or inulin. Cardiovascular lesions were found in all feeding groups and in the baseline group. Supplementation of pectin or inulin in the atherogenic diet had no significant impact on cardiovascular lesion size. Saddleback pigs can develop naturally occurring plaques in coronary arteries. Therefore, this pig model offers potential for further research on the effects of dietary intervention on obesity-related comorbidities, such as cardiovascular lesions, in humans.

A cytoskeletal vortex drives phage nucleus rotation during jumbo phage replication in E. coli.

Nucleus-forming jumbo phages establish an intricate subcellular organization, enclosing phage genomes within a proteinaceous shell called the phage nucleus. During infection in Pseudomonas, some jumbo phages assemble a bipolar spindle of tubulin-like PhuZ filaments that positions the phage nucleus at midcell and drives its intracellular rotation. This facilitates the distribution of capsids on its surface for genome packaging. Here we show that the Escherichia coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of a mutant PhuZ protein strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes for cytoplasmic streaming and nucleus alignment, this work identifies a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.

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.

Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.

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.

Natural Bred ε2-Phages Have an Improved Host Range and Virulence against Uropathogenic Escherichia coli over Their Ancestor Phages.

Alternative treatments for Escherichia coli infections are urgently needed, and phage therapy is a promising option where antibiotics fail, especially for urinary tract infections (UTI). We used wastewater-isolated phages to test their lytic activity against a panel of 47 E. coli strains reflecting the diversity of strains found in UTI, including sequence type 131, 73 and 69. The plaquing host range (PHR) was between 13 and 63%. In contrast, the kinetic host range (KHR), describing the percentage of strains for which growth in suspension was suppressed for 24 h, was between 0% and 19%, substantially lower than the PHR. To improve the phage host range and their efficacy, we bred the phages by mixing and propagating cocktails on a subset of E. coli strains. The bred phages, which we termed evolution-squared ε2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae ε2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε2-phages with the broadest host range reveals that they are mosaic intercrossings of 2-3 ancestor phages. The recombination sites are distributed over the whole length of the genome. All ε2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε2-phages are remarkably more suitable than the wild-type phages for phage therapy.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

ε2-Phages Are Naturally Bred and Have a Vastly Improved Host Range in Staphylococcus aureus over Wild Type Phages.

Due to the rapid spread of antibiotic resistance, and the difficulties of treating biofilm-associated infections, alternative treatments for S. aureus infections are urgently needed. We tested the lytic activity of several wild type phages against a panel of 110 S. aureus strains (MRSA/MSSA) composed to reflect the prevalence of S. aureus clonal complexes in human infections. The plaquing host ranges (PHR) of the wild type phages were in the range of 51% to 60%. We also measured what we called the kinetic host range (KHR), i.e., the percentage of strains for which growth in suspension was suppressed for 24 h. The KHR of the wild type phages ranged from 2% to 49%, substantially lower than the PHRs. To improve the KHR and other key pharmaceutical properties, we bred the phages by mixing and propagating cocktails on a subset of S. aureus strains. These bred phages, which we termed evolution-squared (ε2) phages, have broader KHRs up to 64% and increased virulence compared to the ancestors. The ε2-phages with the broadest KHR have genomes intercrossed from up to three different ancestors. We composed a cocktail of three ε2-phages with an overall KHR of 92% and PHR of 96% on 110 S. aureus strains and called it PM-399. PM-399 has a lower propensity to resistance formation than the standard of care antibiotics vancomycin, rifampicin, or their combination, and no resistance was observed in laboratory settings (detection limit: 1 cell in 1011). In summary, ε2-phages and, in particular PM-399, are promising candidates for an alternative treatment of S. aureus infections.

A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient.

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.

In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli.

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.

Phage Diversity for Research and Application.

Bacteriophages (in short, phages) are viruses that specifically recognize and infect bacteria; theyare the most abundant forms of life in the biosphere outnumbering bacteria by an estimated factor ofone order of magnitude [1]. Logically, lytic phages have an enormous potential in human andveterinary medicine as "intelligent antibiotics". In contrast to static antibacterial drugs, phagesself-control their numbers at the locus of bacterial infection as they replicate on the unwantedpathogen and disintegrate into their subunits when the bacterial host is eliminated. [...].

From Orphan Phage to a Proposed New Family-the Diversity of N4-Like Viruses.

Escherichia phage N4 was isolated in 1966 in Italy and has remained a genomic orphan for a long time. It encodes an extremely large virion-associated RNA polymerase unique for bacterial viruses that became characteristic for this group. In recent years, due to new and relatively inexpensive sequencing techniques the number of publicly available phage genome sequences expanded rapidly. This revealed new members of the N4-like phage group, from 33 members in 2015 to 115 N4-like viruses in 2020. Using new technologies and methods for classification, the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) has moved the classification and taxonomy of bacterial viruses from mere morphological approaches to genomic and proteomic methods. The analysis of 115 N4-like genomes resulted in a huge reassessment of this group and the proposal of a new family "Schitoviridae", including eight subfamilies and numerous new genera.

Kinetic Fingerprinting Links Bacteria-Phage Interactions with Emergent Dynamics: Rapid Depletion of Klebsiella pneumoniae Indicates Phage Synergy.

The specific temporal evolution of bacterial and phage population sizes, in particular bacterial depletion and the emergence of a resistant bacterial population, can be seen as a kinetic fingerprint that depends on the manifold interactions of the specific phage-host pair during the course of infection. We have elaborated such a kinetic fingerprint for a human urinary tract Klebsiella pneumoniae isolate and its phage vB_KpnP_Lessing by a modeling approach based on data from in vitro co-culture. We found a faster depletion of the initially sensitive bacterial population than expected from simple mass action kinetics. A possible explanation for the rapid decline of the bacterial population is a synergistic interaction of phages which can be a favorable feature for phage therapies. In addition to this interaction characteristic, analysis of the kinetic fingerprint of this bacteria and phage combination revealed several relevant aspects of their population dynamics: A reduction of the bacterial concentration can be achieved only at high multiplicity of infection whereas bacterial extinction is hardly accomplished. Furthermore the binding affinity of the phage to bacteria is identified as one of the most crucial parameters for the reduction of the bacterial population size. Thus, kinetic fingerprinting can be used to infer phage-host interactions and to explore emergent dynamics which facilitates a rational design of phage therapies.

Characterization of Clostridioides difficile DSM 101085 with A-B-CDT+ Phenotype from a Late Recurrent Colonization.

During the last decades, hypervirulent strains of Clostridioides difficile with frequent disease recurrence and increased mortality appeared. Clostridioides difficile DSM 101085 was isolated from a patient who suffered from several recurrent infections and colonizations, likely contributing to a fatal outcome. Analysis of the toxin repertoire revealed the presence of a complete binary toxin locus and an atypical pathogenicity locus consisting of only a tcdA pseudogene and a disrupted tcdC gene sequence. The pathogenicity locus shows upstream a transposon and has been subject to homologous recombination or lateral gene transfer events. Matching the results of the genome analysis, neither TcdA nor TcdB production but the expression of cdtA and cdtB was detected. This highlights a potential role of the binary toxin C. difficile toxin in this recurrent colonization and possibly further in a host-dependent virulence. Compared with the C. difficile metabolic model strains DSM 28645 (630Δerm) and DSM 27147 (R20291), strain DSM 101085 showed a specific metabolic profile, featuring changes in the threonine degradation pathways and alterations in the central carbon metabolism. Moreover, products originating from Stickland pathways processing leucine, aromatic amino acids, and methionine were more abundant in strain DSM 101085, indicating a more efficient use of these substrates. The particular characteristics of strain C. difficile DSM 101085 may represent an adaptation to a low-protein diet in a patient with recurrent infections.

Impact of Bacteriophage-Supplemented Drinking Water on the E. coli Population in the Chicken Gut.

Among intestinal coliform microbes in the broiler gut, there are potentially pathogenic Escherichia (E.) coli that can cause avian colibacillosis. The treatment with antibiotics favors the selection of multidrug-resistant bacteria and an alternative to this treatment is urgently required. A chicken model of intestinal colonization with an apathogenic model strain of E. coli was used to test if oral phage application can prevent or reduce the gut colonization of extraintestinal pathogenic E. coli variants in two individual experiments. The E. coli strain E28 was used as a model strain, which could be differentiated from other E. coli strains colonizing the broiler gut, and was susceptible to all cocktail phages applied. In the first trial, a mixture of six phages was continuously applied via drinking water. No reduction of the model E. coli strain E28 occurred, but phage replication could be demonstrated. In the second trial, the applied mixture was limited to the four phages, which showed highest efficacy in vitro. E. coli colonization was reduced in this trial, but again, no reduction of the E. coli strain E28 was observed. The results of the trials presented here can improve the understanding of the effect of phages on single strains in the multi-strain microbiota of the chicken gut.

Taxonomy of prokaryotic viruses: 2018-2019 update from the ICTV Bacterial and Archaeal Viruses Subcommittee.

This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.