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.

The inaugural mBio Junior Editorial Board-lessons learned and the path forward toward improving the peer review process.

The inaugural Junior Editorial Board (JEB) of mBio consisted of 64 early-career researchers active from 2022 to 2023. The goal of the JEB was to train early-career researchers in the art of peer review under the guidance of experienced editors. JEB members gained hands-on experience in peer review by participating in modules detailing the publishing process through the lenses of the journal, editor, and reviewer. Ultimately, JEB members applied this new knowledge by reviewing mBio manuscripts. Here, we summarize the background, the mission, and the achievements of the first mBio JEB. We also include possible trajectories for the future editions of this important program.

Synthetic Biology to Engineer Bacteriophage Genomes.

Recent advances in the synthetic biology field have enabled the development of new molecular biology techniques used to build specialized bacteriophages with new functionalities. Bacteriophages have been engineered toward a wide range of applications, including pathogen control and detection, targeted drug delivery, or even assembly of new materials.In this chapter, two strategies that have been successfully used to genetically engineer bacteriophage genomes will be addressed: the bacteriophage recombineering of electroporated DNA (BRED) and the yeast-based phage-engineering platform.

A systematic review of the use of bacteriophages for in vitro biofilm control.

Bacteriophages (phages) are very promising biological agents for the prevention and control of bacterial biofilms. However, little is known about the parameters that can influence the efficacy of phages on biofilms. This systematic review provides a summary and analysis of the published data about the use of phages to control pre-formed biofilms in vitro, suggesting recommendations for future experiments in this area. A total of 68 articles, containing data on 605 experiments addressing the efficacy of phages to control biofilms in vitro were included, after a search conducted in Web of Science, Embase, and Medline (PubMed). The data collected from each experiment included information about biofilm growth conditions, phage characteristics, treatment conditions and biofilm reduction. In most cases, biofilms were formed in the surface of microtiter plates (82.5%); the median time for biofilm formation was 24 h, as is the median treatment duration. Quantification of biofilm biomass (52.6%), viable cells (25.5%) and metabolic activity (17.9%) were the most common biofilm assessment methods. Correlation analysis revealed that some phage parameters can influence the treatment outcome: higher phage concentrations were strongly associated with improved biofilm control, leading to higher levels of biofilm reduction, and phages with higher burst sizes and shorter latent periods seem to be the best candidates to control biofilms in vitro. However, the great variability of the methodologies used prompts the need for the development of standardized in vitro methodologies to characterize phage/biofilm interactions and to assess the efficacy of phages to control biofilms.

CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers.

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. KEY POINTS: • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens.

Impact of phage predation on P. aeruginosa adhered to human airway epithelium: major transcriptomic changes in metabolism and virulence-associated genes.

Phage therapy is a promising adjunct therapeutic approach against bacterial multidrug-resistant infections, including Pseudomonas aeruginosa-derived infections. Nevertheless, the current knowledge about the phage-bacteria interaction within a human environment is limited. In this work, we performed a transcriptome analysis of phage-infected P. aeruginosa adhered to a human epithelium (Nuli-1 ATCC® CRL-4011™). To this end, we performed RNA-sequencing from a complex mixture comprising phage-bacteria-human cells at early, middle, and late infection and compared it to uninfected adhered bacteria. Overall, we demonstrated that phage genome transcription is unaltered by bacterial growth and phage employs a core strategy of predation through upregulation of prophage-associated genes, a shutdown of bacterial surface receptors, and motility inhibition. In addition, specific responses were captured under lung-simulating conditions, with the expression of genes related to spermidine syntheses, sulphate acquisition, biofilm formation (both alginate and polysaccharide syntheses), lipopolysaccharide (LPS) modification, pyochelin expression, and downregulation of virulence regulators. These responses should be carefully studied in detail to better discern phage-induced changes from bacterial responses against phage. Our results establish the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, being obvious the phage versatility on bacterial cell invasion.

An overview of the current state of phage therapy for the treatment of biofilm-related infections.

Bacterial biofilms are involved in many chronic and difficult-to-treat infections. Phage therapy against infectious biofilms is becoming a promising strategy, as suggested by the increasing number of publications demonstrating the efficacy of phages against in vitro formed biofilms. However, the translation between in vitro results to in vivo phage therapy outcome is not straightforward due to the complexity of phage-biofilm interactions in clinical contexts. Here, we provide a critical overview of the in vitro studies of phages for biofilm control of clinical pathogens, followed by the major outcomes and lessons learned from the recently reported case studies (between 2018 and 2021) of phage therapy against biofilm-related infections.

Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages.

Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.

Unpuzzling Friunavirus-Host Interactions One Piece at a Time: Phage Recognizes Acinetobacter pittii via a New K38 Capsule Depolymerase.

Acinetobacter pittii is a species that belong to the Acinetobacter calcoaceticus-baumannii complex, increasingly recognized as major nosocomial bacterial pathogens, often associated with multiple drug-resistances. The capsule surrounding the bacteria represents a main virulence factor, helping cells avoid phage predation and host immunity. Accordingly, a better understanding of the phage infection mechanisms is required to efficiently develop phage therapy against Acinetobacter of different capsular types. Here, we report the isolation of the novel A. pittii-infecting Fri1-like phage vB_Api_3043-K38 (3043-K38) of the Podoviridae morphotype, from sewage samples. Its 41,580 bp linear double-stranded DNA genome harbours 53 open reading frames and 302 bp of terminal repeats. We show that all studied Acinetobacter Fri1-like viruses have highly similar genomes, which differentiate only at the genes coding for tailspike, likely to adapt to different host receptors. The isolated phage 3043-K38 specifically recognizes an untapped Acinetobacter K38 capsule type via a novel tailspike with K38 depolymerase activity. The recombinant K38 depolymerase region of the tailspike (center-end region) forms a thermostable trimer, and quickly degrades capsules. When the K38 depolymerase is applied to the cells, it makes them resistant to phage predation. Interestingly, while K38 depolymerase treatments do not synergize with antibiotics, it makes bacterial cells highly susceptible to the host serum complement. In summary, we characterized a novel phage-encoded K38 depolymerase, which not only advances our understanding of phage-host interactions, but could also be further explored as a new antibacterial agent against drug-resistant Acinetobacter.

Exploitation of a Klebsiella Bacteriophage Receptor-Binding Protein as a Superior Biorecognition Molecule.

Klebsiella pneumoniae is a Gram-negative bacterium that has become one of the leading causes of life-threatening healthcare-associated infections (HAIs), including pneumonia and sepsis. Moreover, due to its increasingly antibiotic resistance, K. pneumoniae has been declared a global top priority concern. The problem of K. pneumoniae infections is due, in part, to the inability to detect this pathogen rapidly and accurately and thus to treat patients within the early stages of infections. The success in bacterial detection is greatly dictated by the biorecognition molecule used, with the current diagnostic tools relying on expensive probes often lacking specificity and/or sensitivity. (Bacterio)phage receptor-binding proteins (RBPs) are responsible for the recognition and adsorption of phages to specific bacterial host receptors and thus present high potential as biorecognition molecules. In this study, we report the identification and characterization of a novel RBP from the K. pneumoniae phage KpnM6E1 that presents high specificity against the target bacteria and high sensitivity (80%) to recognize K. pneumoniae strains. Moreover, adsorption studies validated the role of gp86 in the attachment to bacterial receptors, as it highly inhibits (86%) phage adsorption to its Klebsiella host. Overall, in this study, we unravel the role and potential of a novel Klebsiella phage RBP as a powerful tool to be used coupled with analytical techniques or biosensing platforms for the diagnosis of K. pneumoniae infections.

Understanding the Complex Phage-Host Interactions in Biofilm Communities.

Bacteriophages and bacterial biofilms are widely present in natural environments, a fact that has accelerated the evolution of phages and their bacterial hosts in these particular niches. Phage-host interactions in biofilm communities are rather complex, where phages are not always merely predators but also can establish symbiotic relationships that induce and strengthen biofilms. In this review we provide an overview of the main features affecting phage-biofilm interactions as well as the currently available methods of studying these interactions. In addition, we address the applications of phages for biofilm control in different contexts.

Differential transcription profiling of the phage LUZ19 infection process in different growth media.

RNA sequencing of phage-infected bacterial cultures offers a snapshot of transcriptional events occurring during the infection process, providing insights into the phage transcriptional organization as well as the bacterial response. To better mimic real environmental contexts, we performed RNA-seq of Pseudomonas aeruginosa PAO1 cultures infected with phage LUZ19 in a mammalian cell culture medium to better simulate a phage therapy event and the data were compared to lysogeny broth medium. Regardless of the media, phage LUZ19 induces significant transcriptional changes in the bacterial host over time, particularly during early infection (t = 5 min) and gradually shuts down bacterial transcription. In a common response in both media, 56 P. aeruginosa PAO1 genes are differentially transcribed and clustered into several functional categories such as metabolism, translation and transcription. Our data allowed us to tease apart a medium-specific response during infection from the identified infection-associated responses. This reinforces the concept that phages overtake bacterial transcriptome in a strict manner to gain control of the bacterial machinery and reallocate resources for infection, in this case overcoming the nutritional limitations of the mammalian cell culture medium. From a phage therapy perspective, this study contributes towards a better understanding of phage-host interaction in human physiological conditions and demonstrates the versatility of phage LUZ19 to adapt to different environments.

Designing P. aeruginosa synthetic phages with reduced genomes.

In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

Current challenges and future opportunities of phage therapy.

Antibiotic resistance is a major public health challenge worldwide, whose implications for global health might be devastating if novel antibacterial strategies are not quickly developed. As natural predators of bacteria, (bacterio)phages may play an essential role in escaping such a dreadful future. The rising problem of antibiotic resistance has revived the interest in phage therapy and important developments have been achieved over the last years. But where do we stand today and what can we expect from phage therapy in the future? This is the question we set to answer in this review. Here, we scour the outcomes of human phage therapy clinical trials and case reports, and address the major barriers that stand in the way of using phages in clinical settings. We particularly address the potential of phage resistance to hinder phage therapy and discuss future avenues to explore the full capacity of phage therapy.

A different vision of translational research in biomarker discovery: a pilot study on circulatory mitochondrial proteins as Parkinson's disease potential biomarkers.

Background: The identification of circulating biomarkers that closely correlate with Parkinson's Disease (PD) has failed several times in the past. Nevertheless, in this pilot study, a translational approach was conducted, allowing the evaluation of the plasma levels of two mitochondrial-related proteins, whose combination leads to a robust model with potential diagnostic value to discriminate the PD patients from matched controls. Methods: The proposed translational approach was initiated by the analysis of secretomes from cells cultured under control or well-defined oxidative stress conditions, followed by the identification of proteins related to PD pathologic mechanisms that were altered between the two states. This pipeline was further translated into the analysis of undepleted plasma samples from 28 control and 31 PD patients. Results: From the secretome analysis, several mitochondria-related proteins were found to be differentially released between control and stress conditions and to be able to distinguish the two secretomes. Similarly, two mitochondrial-related proteins were found to be significantly changed in a PD cohort compared to matched controls. Moreover, a linear discriminant model with potential diagnostic value to discriminate PD patients was obtained using the combination of these two proteins. Both proteins are associated with apoptotic mitochondrial changes, which may correspond to potential indicators of cell death. Moreover, one of these proteins, the VPS35 protein, was reported in plasma for the first time, and its quantification was only possible due to its previous identification in the secretome analysis. Conclusions: In this work, an adaptation of a translational pipeline for biomarker selection was presented and transposed to neurological diseases, in the present case Parkinson's Disease. The novelty and success of this pilot study may arise from the combination of: i) a translational research pipeline, where plasma samples are interrogated using knowledge previously obtained from the evaluation of cells' secretome under oxidative stress; ii) the combined used of statistical analysis and an informed selection of candidates based on their link with relevant disease mechanisms, and iii) the use of SWATH-MS, an untargeted MS method that allows a complete record of the analyzed samples and a targeted data extraction of the quantitative values of proteins previously identified.

Phage therapy efficacy: a review of the last 10 years of preclinical studies.

Due to the rise of multidrug-resistant infections in humans, phage therapy is gaining renewed attention in Western medicine. Despite the increasing number of publications focussed on the isolation, characterization and in vitro performance of different phages, there is still a lack of concise pre-clinical information to guide the application of phage therapy in clinical practice. Nevertheless, over the last decade, efforts have been made to conduct more detailed studies of the in vivo efficacy of phages. Here, we review the most relevant in vivo studies performed in the last decade covering phage efficacy in both preclinical and clinical trials. We compare different routes of administration, dosage effect and different animal models of distinct types of infections. Moreover, insights into case studies and results from clinical trials are presented. Challenges and limitations of phage use as evidenced by the current state of research are also discussed in order to improve both the trustworthiness and success of the implementation of phage therapy.

Phage Therapy: Going Temperate?

Strictly lytic phages have been consensually preferred for phage therapy purposes. In contrast, temperate phages have been avoided due to an inherent capacity to mediate transfer of genes between bacteria by specialized transduction - an event that may increase bacterial virulence, for example, by promoting antibiotic resistance. Now, advances in sequencing technologies and synthetic biology are providing new opportunities to explore the use of temperate phages for therapy against bacterial infections. By doing so we can considerably expand our armamentarium against the escalating threat of antibiotic-resistant bacteria.

In Vitro Activity of Bacteriophages Against Planktonic and Biofilm Populations Assessed by Flow Cytometry.

The in vitro activity of bacteriophages against planktonic cultures and biofilms is commonly evaluated by culture methods. However, these methods can lead to an underestimation of total bacterial cells when they undergo different physiological states.This chapter describes the methodology used to assess the in vitro activity of bacteriophages against planktonic cultures of bacteria in different metabolic states and biofilm populations by flow cytometry.

Synthetic Biology to Engineer Bacteriophage Genomes.

Recent advances in the synthetic biology field have enabled the development of new molecular biology techniques used to build specialized bacteriophages with new functionalities. Bacteriophages have been engineered towards a wide range of applications including pathogen control and detection, targeted drug delivery, or even assembly of new materials.In this chapter, two strategies that have been successfully used to genetically engineer bacteriophage genomes are addressed: a yeast-based platform and bacteriophage recombineering of electroporated DNA.

A Genotypic Analysis of Five P. aeruginosa Strains after Biofilm Infection by Phages Targeting Different Cell Surface Receptors.

Antibiotic resistance constitutes one of the most serious threats to the global public health and urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to traditional antibiotic therapies. In this study, the efficacy of phages, targeting different cell receptors, against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 h. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. To further investigate the genetic makeup of phage-resistant variants isolated from biofilm infection experiments, some of these bacteria were selected for phenotypic and genotypic characterization. Whole genome sequencing was performed on five phage-resistant variants and all of them carried mutations affecting the galU gene as well as one of pil genes. The sequencing analysis further revealed that three of the P. aeruginosa PAO1 variants carry large deletions (>200 kbp) in their genomes. Complementation of the galU mutants with wild-type galU in trans restored LPS expression on the bacterial cell surface of these bacterial strains and rendered the complemented strains to be sensitive to phages. This provides unequivocal evidence that inactivation of galU function was associated with resistance to the phages that uses LPS as primary receptors. Overall, this work demonstrates that P. aeruginosa biofilms can survive phage attack and develop phage-resistant variants exhibiting defective LPS production and loss of type IV pili that are well adapted to the biofilm mode of growth.