Bacteriophage defends murine gut from Escherichia coli invasion via mucosal adherence.

Bacteriophage are sophisticated cellular parasites that can not only parasitize bacteria but are increasingly recognized for their direct interactions with mammalian hosts. Phage adherence to mucus is known to mediate enhanced antimicrobial effects in vitro. However, little is known about the therapeutic efficacy of mucus-adherent phages in vivo. Here, using a combination of in vitro gastrointestinal cell lines, a gut-on-a-chip microfluidic model, and an in vivo murine gut model, we demonstrated that a E. coli phage, øPNJ-6, provided enhanced gastrointestinal persistence and antimicrobial effects. øPNJ-6 bound fucose residues, of the gut secreted glycoprotein MUC2, through domain 1 of its Hoc protein, which led to increased intestinal mucus production that was suggestive of a positive feedback loop mediated by the mucus-adherent phage. These findings extend the Bacteriophage Adherence to Mucus model into phage therapy, demonstrating that øPNJ-6 displays enhanced persistence within the murine gut, leading to targeted depletion of intestinal pathogenic bacteria.

Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility.

OBJECTIVES: Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. METHODS: Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. RESULTS: We observed a repertoire of phage resistance mechanisms in K pneumoniae, such as disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin. DISCUSSION: Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.

Universal scaling of the diffusivity of dendrimers in a semidilute solution of linear polymers.

The static and dynamic properties of dendrimers in semidilute solutions of linear chains of comparable size are investigated using Brownian dynamics simulations. The radius of gyration and diffusivity of a wide variety of low generation dendrimers and linear chains in solution follow universal scaling laws independent of their topology. Analysis of the shape functions and internal density of dendrimers shows that they are more spherical than linear chains and have a dense core. At intermediate times, dendrimers become subdiffusive, with an exponent higher than that previously reported for nanoparticles in semidilute polymer solutions. The long-time diffusivity of dendrimers does not follow theoretical predictions for nanoparticles. We propose a new scaling law for the long-time diffusion coefficients of dendrimers which accounts for the fact that, unlike nanoparticles, dendrimers shrink with an increase in background solution concentration. Analysis of the properties of a special case of a higher functionality dendrimer shows a transition from polymer-like to nanoparticle-like behaviour.

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.

Mammalian cells internalize bacteriophages and use them as a resource to enhance cellular growth and survival.

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.

A Mammalian Cell's Guide on How to Process a Bacteriophage.

Bacteriophages are enigmatic entities that defy definition. Classically, they are specialist viruses that exclusively parasitize bacterial hosts. Yet this definition becomes limiting when we consider their ubiquity in the body coupled with their vast capacity to directly interact with the mammalian host. While phages certainly do not infect nor replicate within mammalian cells, they do interact with and gain unfettered access to the eukaryotic cell structure. With the growing appreciation for the human virome, coupled with our increased application of phages to patients within clinical settings, the potential impact of phage-mammalian interactions is progressively recognized. In this review, we provide a detailed mechanistic overview of how phages interact with the mammalian cell surface, the processes through which said phages are internalized by the cell, and the intracellular processing and fate of the phages. We then summarize the current state-of-the-field with respect to phage-mammalian interactions and their associations with health and disease states.

The gut virome and the relevance of temperate phages in human health.

Alterations in the gut virome impact human health. Bacteriophages, viruses that infect bacteria, dominate the gut virome and are mainly composed by virulent and temperate phages. While virulent phages exclusively replicate within and lyse their bacterial host's cell, temperate phages switch from an integrated state residing within their bacterial host's chromosome to an induced free virion state via an induction event. How often do these induction events occur and what are their implications on gut homeostasis? Here, we summarize the current knowledge of the gut virome based on metagenomics and present how the proportion of induced temperate phages varies amongst individuals, age, and disease states. Finally, we highlight the importance of building upon classical culture-dependent techniques and sequencing approaches to improve our understanding of temperate phages to enable their potential therapeutic use.

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. Here, 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-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

Single-arm, open-labelled, safety and tolerability of intrabronchial and nebulised bacteriophage treatment in children with cystic fibrosis and Pseudomonas aeruginosa.

INTRODUCTION: Cystic fibrosis (CF) is a multisystem condition that is complicated by recurrent pulmonary infections requiring aggressive antibiotic treatment. This predisposes the patient to complications such as sensorineural hearing loss, renal impairment, hypersensitivity and the development of antibiotic resistance. Pseudomonas aeruginosa is one of the more common organisms which cause recurrent infections and result in greater morbidity and mortality in people living with CF. Bacteriophages have been identified as a potential alternative or adjunct to antibiotics. We hypothesise that bacteriophage therapy is a safe and well-tolerated treatment in children with CF infected with P. aeruginosa infection in their airways. METHODS: This single-arm, open-labelled, non-randomised trial will run for a maximum period of 36 months with up to 10 participants. Adolescents (≥12 years and <18 years of age) who continue to shed P.aeruginosa (within 3 months of enrolment) despite undergoing eradication therapy previously, will be considered for this trial. Non-genetically modified bacteriophages that have demonstrated obligate lytic activity against each of the study participants' P. aeruginosa strains will be selected and prepared according to a combination of established protocols (isolation, purification, sterility testing and packaging) to achieve close to good manufacturing practice recommendations. The selected bacteriophage will be administered endo-bronchially first under direct vision, followed by two times a day nebulisation for 7 days in addition to standard CF treatment (intravenous antibiotics, physiotherapy to be completed as inpatient for 10-14 days). Safety and tolerability will be defined as the absence of (1) fever above 38.5°C occurring within 1 hour of the administration of the nebulised bacteriophage, (2) a 10% decline in spirometry (forced expiratory volume in 1 s %) measured preadministration and postadministration of the first dose of nebulised bacteriophage. Clinical reviews including repeat sputum cultures and spirometry will be performed at 3, 6, 9 and 12 months following bacteriophage treatment. ETHICS AND DISSEMINATION: Our clinical trial is conducted in accordance with (1) good clinical practice, (2) Australian legislation, (3) National Health and Medical Research Council guidelines for the ethical conduct of research. TRIAL REGISTRATION NUMBER: Australia and New Zealand Clinical Trial Registry (ACTRN12622000767707).

Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

Fit-Tested N95 Masks Combined With Portable High-Efficiency Particulate Air Filtration Can Protect Against High Aerosolized Viral Loads Over Prolonged Periods at Close Range.

BACKGROUND: Healthcare workers (HCWs) are at risk from aerosol transmission of severe acute respiratory syndrome coronavirus 2. The aims of this study were to (1) quantify the protection provided by masks (surgical, fit-testFAILED N95, fit-testPASSED N95) and personal protective equipment (PPE), and (2) determine if a portable high-efficiency particulate air (HEPA) filter can enhance the benefit of PPE. METHODS: Virus aerosol exposure experiments using bacteriophage PhiX174 were performed. An HCW wearing PPE (mask, gloves, gown, face shield) was exposed to nebulized viruses (108 copies/mL) for 40 minutes in a sealed clinical room. Virus exposure was quantified via skin swabs applied to the face, nostrils, forearms, neck, and forehead. Experiments were repeated with a HEPA filter (13.4 volume-filtrations/hour). RESULTS: Significant virus counts were detected on the face while the participants were wearing either surgical or N95 masks. Only the fit-testPASSED N95 resulted in lower virus counts compared to control (P = .007). Nasal swabs demonstrated high virus exposure, which was not mitigated by the surgical/fit-testFAILED N95 masks, although there was a trend for the fit-testPASSED N95 mask to reduce virus counts (P = .058). HEPA filtration reduced virus to near-zero levels when combined with fit-testPASSED N95 mask, gloves, gown, and face shield. CONCLUSIONS: N95 masks that have passed a quantitative fit-test combined with HEPA filtration protects against high virus aerosol loads at close range and for prolonged periods of time.

Phage Cocktail Targeting STEC O157:H7 Has Comparable Efficacy and Superior Recovery Compared with Enrofloxacin in an Enteric Murine Model.

O157:H7 is the most important Shiga toxin-producing Escherichia coli (STEC) serotype in relation to public health. Given that antibiotics may contribute to the exacerbation of STEC-related disease and an increased frequency of antibiotic-resistant strains, bacteriophage (phage) therapy is considered a promising alternative. However, phage therapy targeting enteric pathogens is still underdeveloped with many confounding effects from the microbiota. Here we comprehensively compared the therapeutic efficacy of a phage cocktail with the antibiotic enrofloxacin in a mouse model of STEC O157:H7 EDL933 infection. Enrofloxacin treatment provided 100% survival and the phage cocktail treatment provided 90% survival. However, in terms of mouse recovery, the phage cocktail outperformed enrofloxacin in all measured outcomes. Compared with enrofloxacin treatment, phage treatment led to a faster elimination of enteric pathogens, decreased expression levels of inflammatory markers, increased weight gain, maintenance of a stable relative organ weight, and improved homeostasis of the gut microbiota. These results provide support for the potential of phage therapy to combat enteric pathogens and suggest that phage treatment leads to enhanced recovery of infected mice compared with antibiotics. IMPORTANCE With the increasing severity of antibiotic resistance and other adverse consequences, animal experiments and clinical trials investigating the use of phages for the control and prevention of enteric bacterial infections are growing. However, the effects of phages and antibiotics on organisms when treating intestinal infections have not been precisely studied. Here, we comprehensively compared the therapeutic efficacy of a phage cocktail to the antibiotic enrofloxacin in a mouse model of STEC O157:H7 EDL933 infection. We found that, despite a slightly lower protection rate, phage treatment contributed to a faster recovery of infected mice compared with enrofloxacin. These results highlight the potential benefits of phage therapy to combat enteric infections.

Phage-antibiotic combination is a superior treatment against Acinetobacter baumannii in a preclinical study.

BACKGROUND: Clinical phage therapy is often delivered alongside antibiotics. However, the phenomenon of phage-antibiotic synergy has been mostly studied in vitro. Here, we assessed the in vivo bactericidal effect of a phage-antibiotic combination on Acinetobacter baumannii AB900 using phage øFG02, which binds to capsular polysaccharides and leads to antimicrobial resensitisation in vitro. METHODS: We performed a two-stage preclinical study using a murine model of severe A. baumannii AB900 bacteraemia. In the first stage, with an endpoint of 11 h, mice (n = 4 per group) were treated with either PBS, ceftazidime, phage øFG02, or the combination of phage and ceftazidime. The second stage involved only the latter two groups (n = 5 per group), with a prolonged endpoint of 16 h. The primary outcome was the average bacterial burden from four body sites (blood, liver, kidney, and spleen). Bacterial colonies from phage-treated mice were retrieved and screened for phage-resistance. FINDINGS: In the first stage, the bacterial burden (CFU/g of tissue) of the combination group (median: 4.55 × 105; interquartile range [IQR]: 2.79 × 105-2.81 × 106) was significantly lower than the PBS (median: 2.42 × 109; IQR: 1.97 × 109-3.48 × 109) and ceftazidime groups (median: 3.86 × 108; IQR: 2.15 × 108-6.35 × 108), but not the phage-only group (median: 1.28 × 107; IQR: 4.71 × 106-7.13 × 107). In the second stage, the combination treatment (median: 1.72 × 106; IQR: 5.11 × 105-4.00 × 106) outperformed the phage-only treatment (median: 7.46 × 107; IQR: 1.43 × 107-1.57 × 108). Phage-resistance emerged in 96% of animals receiving phages, and all the tested isolates (n = 11) had loss-of-function mutations in genes involved in capsule biosynthesis and increased sensitivity to ceftazidime. INTERPRETATION: øFG02 reliably drives the in vivo evolution of A. baumannii AB900 towards a capsule-deficient, phage-resistant phenotype that is resensitised to ceftazidime. This mechanism highlights the clinical potential of using phage therapy to target A. baumannii and restore antibiotic activity. FUNDING: National Health and Medical Research Council (Australia).

The gut virome in two indigenous populations from Malaysia.

The human gut contains a complex microbiota dominated by bacteriophages but also containing other viruses and bacteria and fungi. There are a growing number of techniques for the extraction, sequencing, and analysis of the virome but currently no standardized protocols. This study established an effective workflow for virome analysis to investigate the virome of stool samples from two understudied ethnic groups from Malaysia: the Jakun and Jehai Orang Asli. By using the virome extraction and analysis workflow with the Oxford Nanopore Technology, long-read sequencing successfully captured close to full-length viral genomes. The virome composition of the two indigenous Malaysian communities were remarkably different from those found in other parts of the world. Additionally, plant viruses found in the viromes of these individuals were attributed to traditional food-seeking methods. This study establishes a human gut virome workflow and extends insights into the healthy human gut virome, laying the groundwork for comparative studies.

Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant Klebsiella pneumoniae by a bacteriophage-polymyxin combination.

Resistance to the last-line polymyxins is emerging in multidrug-resistant Klebsiella pneumoniae and phage therapy is a promising alternative. However, phage monotherapy often rapidly causes resistance and few studies have examined antibiotic-phage combinations against K. pneumoniae. Here, we investigated the combination of polymyxin B with a novel phage pK8 against an mcr-1-carrying polymyxin-resistant clinical isolate Kp II-503 (polymyxin B MIC, 8 mg/L). The phage genome was sequenced and bacterial metabolomes were analysed at 4 and 24 h following the treatment with polymyxin B (16 mg/L), phage pK8 (102 PFU/mL) and their combination. Minimal metabolic changes across 24 h were observed with polymyxin B alone; whereas a significant inhibition of the citrate cycle, pentose phosphate pathway, amino acid and nucleotide metabolism occurred with the phage-polymyxin combination at both 4 and 24 h, but with phage alone only at 4 h. The development of resistance to phage alone was associated with enhanced membrane lipid and decreased amino acid biosynthesis in Kp II-503. Notably, cAMP, cGMP and cCMP were significantly enriched (3.1-6.6 log2fold) by phage alone and the combination only at 4 h. This is the first systems pharmacology study to investigate the enhanced bacterial killing by polymyxin-phage combination and provides important mechanistic information on phage killing, resistance and antibiotic-phage combination in K. pneumoniae.

Protocols for studying bacteriophage interactions with in vitro epithelial cell layers.

Interactions between bacteriophages and mammalian cells are poorly understood. Establishing common methodologies investigating these interactions is important for advancing our understanding in this area. The protocols presented here provide an overview of key approaches investigating interactions between bacteriophages and eukaryotic cells using a variety of techniques, including transwells, microscopy, and whole-cell analysis. These techniques allow for the direct measurement of phage-cellular interactions and characterization of how the presence of phages affects cellular pathways, cell biology, immunology, and the microbiome. For complete details on the use and execution of this protocol, please refer to Nguyen et al. (2017) and Bichet et al. (2021).

Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages.

Lysogenic phages can integrate into their bacterial host's genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications. Lysogenic behavior is typically seen in phages that create turbid plaques or possess subpar bactericidal activity; yet, these are not definitive indicators. As a result, the presence of integrase genes is often used as a hallmark for lysogenic behavior; however, the accuracy of genetic screening for lysogeny depends on the quality of the extraction, sequencing and assembly of the phage genome, and database comparison. The present protocol describes a simple phenotypic test that can be used to screen therapeutically relevant phages for lysogenic behavior. This test relies on the identification of spontaneous phage release from their lysogenized host and can be reliably used in cases where no sequencing data are available. The protocol does not require specialized equipment, is not work-intensive, and is broadly applicable to any phage with an easily culturable bacterial host, making it particularly amenable to settings with limited resources. Graphical abstract: Screening pipeline for lysogen activity of a given phage.

Component Parts of Bacteriophage Virions Accurately Defined by a Machine-Learning Approach Built on Evolutionary Features.

Antimicrobial resistance (AMR) continues to evolve as a major threat to human health, and new strategies are required for the treatment of AMR infections. Bacteriophages (phages) that kill bacterial pathogens are being identified for use in phage therapies, with the intention to apply these bactericidal viruses directly into the infection sites in bespoke phage cocktails. Despite the great unsampled phage diversity for this purpose, an issue hampering the roll out of phage therapy is the poor quality annotation of many of the phage genomes, particularly for those from infrequently sampled environmental sources. We developed a computational tool called STEP3 to use the "evolutionary features" that can be recognized in genome sequences of diverse phages. These features, when integrated into an ensemble framework, achieved a stable and robust prediction performance when benchmarked against other prediction tools using phages from diverse sources. Validation of the prediction accuracy of STEP3 was conducted with high-resolution mass spectrometry analysis of two novel phages, isolated from a watercourse in the Southern Hemisphere. STEP3 provides a robust computational approach to distinguish specific and universal features in phages to improve the quality of phage cocktails and is available for use at http://step3.erc.monash.edu/. IMPORTANCE In response to the global problem of antimicrobial resistance, there are moves to use bacteriophages (phages) as therapeutic agents. Selecting which phages will be effective therapeutics relies on interpreting features contributing to shelf-life and applicability to diagnosed infections. However, the protein components of the phage virions that dictate these properties vary so much in sequence that best estimates suggest failure to recognize up to 90% of them. We have utilized this diversity in evolutionary features as an advantage, to apply machine learning for prediction accuracy for diverse components in phage virions. We benchmark this new tool showing the accurate recognition and evaluation of phage component parts using genome sequence data of phages from undersampled environments, where the richest diversity of phage still lies.