Cell Shape and Matrix Stiffness Impact Schwann Cell Plasticity via YAP/TAZ and Rho GTPases.

Schwann cells (SCs) are a highly plastic cell type capable of undergoing phenotypic changes following injury or disease. SCs are able to upregulate genes associated with nerve regeneration and ultimately achieve functional recovery. During the regeneration process, the extracellular matrix (ECM) and cell morphology play a cooperative, critical role in regulating SCs, and therefore highly impact nerve regeneration outcomes. However, the roles of the ECM and mechanotransduction relating to SC phenotype are largely unknown. Here, we describe the role that matrix stiffness and cell morphology play in SC phenotype specification via known mechanotransducers YAP/TAZ and RhoA. Using engineered microenvironments to precisely control ECM stiffness, cell shape, and cell spreading, we show that ECM stiffness and SC spreading downregulated SC regenerative associated proteins by the activation of RhoA and YAP/TAZ. Additionally, cell elongation promoted a distinct SC regenerative capacity by the upregulation of Rac1/MKK7/JNK, both necessary for the ECM and morphology changes found during nerve regeneration. These results confirm the role of ECM signaling in peripheral nerve regeneration as well as provide insight to the design of future biomaterials and cellular therapies for peripheral nerve regeneration.

Potential Mechanisms of Mucin-Enhanced Acinetobacter baumannii Virulence in the Mouse Model of Intraperitoneal Infection.

Porcine mucin has been commonly used to enhance the infectivity of bacterial pathogens, including Acinetobacter baumannii, in animal models, but the mechanisms for enhancement by mucin remain relatively unknown. In this study, using the mouse model of intraperitoneal (i.p.) mucin-enhanced A. baumannii infection, we characterized the kinetics of bacterial replication and dissemination and the host innate immune responses, as well as their potential contribution to mucin-enhanced bacterial virulence. We found that mucin, either admixed with or separately injected with the challenge bacterial inoculum, was able to enhance the tissue and blood burdens of A. baumannii strains of different virulence. Intraperitoneal injection of A. baumannii-mucin or mucin alone induced a significant but comparable reduction of peritoneal macrophages and lymphocytes, accompanied by a significant neutrophil recruitment and early interleukin-10 (IL-10) responses, suggesting that the resulting inflammatory cellular and cytokine responses were largely induced by the mucin. Depletion of peritoneal macrophages or neutralization of endogenous IL-10 activities showed no effect on the mucin-enhanced infectivity. However, pretreatment of mucin with iron chelator DIBI, but not deferoxamine, partially abolished its virulence enhancement ability, and replacement of mucin with iron significantly enhanced the bacterial burdens in the peritoneal cavity and lung. Taken together, our results favor the hypothesis that iron at least partially contributes to the mucin-enhanced infectivity of A. baumannii in this model.

Contribution of Active Iron Uptake to Acinetobacter baumannii Pathogenicity.

Acinetobacter baumannii is an important nosocomial pathogen. Mechanisms that allow A. baumannii to cause human infection are still poorly understood. Iron is an essential nutrient for bacterial growth in vivo, and the multiplicity of iron uptake systems in A. baumannii suggests that iron acquisition contributes to the ability of A. baumannii to cause infection. In Gram-negative bacteria, active transport of ferrisiderophores and heme relies on the conserved TonB-ExbB-ExbD energy-transducing complex, while active uptake of ferrous iron is mediated by the Feo system. The A. baumannii genome invariably contains three tonB genes (tonB1, tonB2, and tonB3), whose role in iron uptake is poorly understood. Here, we generated A. baumannii mutants with knockout mutations in the feo and/or tonB gene. We report that tonB3 is essential for A. baumannii growth under iron-limiting conditions, whereas tonB1, tonB2, and feoB appear to be dispensable for ferric iron uptake. tonB3 deletion resulted in reduced intracellular iron content despite siderophore overproduction, supporting a key role of TonB3 in iron uptake. In contrast to the case for tonB1 and tonB2, the promoters of tonB3 and feo contain functional Fur boxes and are upregulated in iron-poor media. Both TonB3 and Feo systems are required for growth in complement-free human serum and contribute to resistance to the bactericidal activity of normal human serum, but only TonB3 appears to be essential for virulence in insect and mouse models of infection. Our findings highlight a central role of the TonB3 system for A. baumannii pathogenicity. Hence, TonB3 represents a promising target for novel antibacterial therapies and for the generation of attenuated vaccine strains.

Effects of moderate, voluntary ethanol consumption on the rat and human gut microbiome.

Many alcohol-induced health complications are directly attributable to the toxicity of alcohol or its metabolites, but another potential health impact of alcohol may be on the microbial communities of the human gut. Clear distinctions between healthy and diseased-state gut microbiota have been observed in subjects with metabolic diseases, and recent studies suggest that chronic alcoholism is linked to gut microbiome dysbiosis. Here, we investigated the effects of moderate levels of alcohol consumption on the gut microbiome in both rats and humans. The gut microbiota of rats voluntarily consuming a 20 percent ethanol solution, on alternate days, were compared with a non-exposed control group to identify differential taxonomic and functional profiles. Gut microbial diversity profiles were determined using culture-independent amplification, next-generation sequencing and bioinformatic analysis of bacterial 16S ribosomal RNA gene sequence libraries. Our results showed that, compared with controls, ethanol-consuming rats experienced a significant decline in the biodiversity of their gut microbiomes, a state generally associated with dysbiosis. We also observed significant shifts in the overall diversity of the gut microbial communities and a dramatic change in the relative abundance of particular microbes, such as the Lactobacilli. We also compared our results to human fecal microbiome data collected as part of the citizen science American Gut Project. In contrast to the rat data, human drinkers had significantly higher gut microbial biodiversity than non-drinkers. However, we also observed that microbes that differed among the human subjects displayed similar trends in the rat model, including bacteria implicated in metabolic disease.

Aging and Intermittent Fasting Impact on Transcriptional Regulation and Physiological Responses of Adult Drosophila Neuronal and Muscle Tissues.

The progressive decline of the nervous system, including protein aggregate formation, reflects the subtle dysregulation of multiple functional pathways. Our previous work has shown intermittent fasting (IF) enhances longevity, maintains adult behaviors and reduces aggregates, in part, by promoting autophagic function in the aging Drosophila brain. To clarify the impact that IF-treatment has upon aging, we used high throughput RNA-sequencing technology to examine the changing transcriptome in adult Drosophila tissues. Principle component analysis (PCA) and other analyses showed ~1200 age-related transcriptional differences in head and muscle tissues, with few genes having matching expression patterns. Pathway components showing age-dependent expression differences were involved with stress response, metabolic, neural and chromatin remodeling functions. Middle-aged tissues also showed a significant increase in transcriptional drift-variance (TD), which in the CNS included multiple proteolytic pathway components. Overall, IF-treatment had a demonstrably positive impact on aged transcriptomes, partly ameliorating both fold and variance changes. Consistent with these findings, aged IF-treated flies displayed more youthful metabolic, behavioral and basal proteolytic profiles that closely correlated with transcriptional alterations to key components. These results indicate that even modest dietary changes can have therapeutic consequences, slowing the progressive decline of multiple cellular systems, including proteostasis in the aging nervous system.

Identification of sphingolipid metabolites that induce obesity via misregulation of appetite, caloric intake and fat storage in Drosophila.

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C14:0) and ceramide diene (C14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.

Survival response to increased ceramide involves metabolic adaptation through novel regulators of glycolysis and lipolysis.

The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart.

Predicting Marathon Time Using Exhaustive Graded Exercise Test in Marathon Runners.

The study aimed to investigate the correlation between time on a treadmill test and exhaustion 2 weeks before a road marathon and the subsequent road marathon performance time (MPT). The study recruited 59 runners entered in the Melbourne 2012 marathon, Canberra 2013 marathon, and Gold Coast 2013 marathon. Forty runners completed both the graded exercise treadmill test to exhaustion and the 42.2 km marathon. Nineteen participants dropped out of the study due to illness, injury, or did not begin the treadmill test. A statistically significant correlation was found between treadmill time and MPT (adjusted R(2) = 0.447). Sex, weekly running duration (t = -1.58, p = 0.12), years of running (t = 1.10, p = 0.28), and age (t = 0.94, p = 0.36) did not statistically correlate with MPT. The relationship between the graded exercise test and MPT can be used to predict MPT using y = -3.85x + 351.57, where y is MPT and x is treadmill time. This is a simple, accessible, and cost-effective method to aid athletes in predicting their race time over 42.2 km. Prediction of marathon time in a simple and accessible manner was believed to be useful to the growing population of marathon runners around the world.

Impact of the Baltimore Experience Corps Trial on cortical and hippocampal volumes.

INTRODUCTION: There is a substantial interest in identifying interventions that can protect and buffer older adults from atrophy in the cortex and particularly, the hippocampus, a region important to memory. We report the 2-year effects of a randomized controlled trial of an intergenerational social health promotion program on older men's and women's brain volumes. METHODS: The Brain Health Study simultaneously enrolled, evaluated, and randomized 111 men and women (58 interventions; 53 controls) within the Baltimore Experience Corps Trial to evaluate the intervention impact on biomarkers of brain health at baseline and annual follow-ups during the 2-year trial exposure. RESULTS: Intention-to-treat analyses on cortical and hippocampal volumes for full and sex-stratified samples revealed program-specific increases in volumes that reached significance in men only (P's ≤ .04). Although men in the control arm exhibited age-related declines for 2 years, men in the Experience Corps arm showed a 0.7% to 1.6% increase in brain volumes. Women also exhibited modest intervention-specific gains of 0.3% to 0.54% by the second year of exposure that contrasted with declines of about 1% among women in the control group. DISCUSSION: These findings showed that purposeful activity embedded within a social health promotion program halted and, in men, reversed declines in brain volume in regions vulnerable to dementia. CLINICAL TRIAL REGISTRATION: NCT0038.

Deconstructing the Effects of Matrix Elasticity and Geometry in Mesenchymal Stem Cell Lineage Commitment.

A wide variety of environmental factors including physical and biochemical signals are responsible for stem cell behavior and function. In particular, matrix elasticity and cell shape have been shown to determine stem cell function, yet little is known about the interplay between how these physical cues control cell differentiation. For the first time, by using ultraviolet (UV) lithography to pattern poly(ethylene) glycol (PEG) hydrogels we are able to manufacture microenvironments capable of parsing the effects of matrix elasticity, cell shape, and cell size in order to explore the relationship between matrix elasticity and cell shape in mesenchymal stem cell (MSC) lineage commitment. Our data shows that cells cultured on 1,000 µm2 circles, squares, and rectangles were primarily adipogenic lineage regardless of matrix elasticity, while cells cultured on 2,500 and 5,000 µm2 shapes more heavily depended on shape and elasticity for lineage specification. We further went on to characterize how modifying the cell cytoskeleton through pharmacological inhibitors can modify cell behavior. By showing MSC lineage commitment relationships due to physical signals, this study highlights the importance of cell shape and matrix elasticity in further understanding stem cell behavior for future tissue engineering strategies.

Serum resistance, gallium nitrate tolerance and extrapulmonary dissemination are linked to heme consumption in a bacteremic strain of Acinetobacter baumannii.

Bacteremia caused by Acinetobacter baumannii is a highly lethal complication of hospital-acquired pneumonia. In the present study, we investigated the serum resistance, gallium nitrate tolerance and heme consumption of A. baumannii strain LAC-4 which was recently reported to display high virulence in a mouse pneumonia model with extrapulmonary dissemination leading to fatal bacteremia. This strain showed enhanced growth in mouse and fetal bovine serum that was independent of complement and was not observed with regular growth media. The LAC-4 strain was found to possess a high tolerance to gallium nitrate (GaN), whereas serum synergized with GaN in inhibiting A. baumannii strain ATCC 17978. We found that LAC-4 contains a heme oxygenase gene and expresses a highly efficient heme consumption system. This system can be fully blocked in vitro and in vivo by gallium protoporphyrin IX (GaPPIX). Inhibition of heme consumption by GaPPIX completely abrogated the growth advantage of LAC-4 in serum as well as its tolerance to GaN. More importantly, GaPPIX treatment of mice intranasally infected with LAC-4 prevented extrapulmonary dissemination and death. Thus, we propose that heme provides an additional source of iron for LAC-4 to bypass iron restriction caused by serum transferrin, lactoferrin or free gallium salts. Heme consumption systems in A. baumannii may constitute major virulence factors for lethal bacteremic isolates.

A mouse model of Acinetobacter baumannii-associated pneumonia using a clinically isolated hypervirulent strain.

Acinetobacter baumannii is an important emerging pathogen in health care-acquired infections and is responsible for severe nosocomial and community-acquired pneumonia. Currently available mouse models of A. baumannii pneumonia show poor colonization with little to no extrapulmonary dissemination. Here, we describe a mouse model of A. baumannii pneumonia using a clinical isolate (LAC-4 strain) that reliably reproduces the most relevant features of human pulmonary A. baumannii infection and pathology. Using this model, we have shown that LAC-4 infection induced rapid bacterial replication in the lungs, significant extrapulmonary dissemination, and severe bacteremia by 24 h postintranasal inoculation. Infected mice showed severe bronchopneumonia and dilatation and inflammatory cell infiltration in the perivascular space. More significantly, 100% of C57BL/6 and BALB/c mice succumbed to 10(8) CFU of LAC-4 inoculation within 48 h. When this model was used to assess the efficacy of antimicrobials, all mice treated with imipenem and tigecycline survived a lethal intranasal challenge, with minimal clinical signs and body weight loss. Moreover, intranasal immunization of mice with formalin-fixed LAC-4 protected 40% of mice from a lethal (100× 100% lethal dose) intraperitoneal challenge. Thus, this model offers a reproducible acute course of A. baumannii pneumonia without requiring additional manipulation of host immune status, which will facilitate the development of therapeutic agents and vaccines against A. baumannii pneumonia in humans.

Ultrasound-Activated Piezoelectric Polyvinylidene Fluoride-Trifluoroethylene Scaffolds for Tissue Engineering Applications.

Severe peripheral nervous system (PNS) injuries have limited options for therapeutic solutions to regain functional recovery. This can be attributed in part to the lack of regeneration pathways promoted by recapitulating chemical, physical, and electrical cues to direct nerve guidance. To address this, we examined ultrasonic stimulation of a piezoelectric polyvinylidene fluoride-triflouroethylene (PVDF-TrFE) scaffold as a potentially clinically relevant therapy for PNS regeneration. Owing to the piezoelectric modality of PVDF-TrFE, we hypothesize that ultrasound stimulation will activate the scaffold to electrically stimulate cells in response to the mechanical deformation mediated by sound waves. Biocompatible PVDF-TrFE scaffolds were fabricated to be used as an ultrasound-activated, piezoelectric biomaterial to enhance cellular activity for PNS applications. NIH-3T3 fibroblasts were cultured on PVDF-TrFE nanofibers and stimulated with low-, medium-, or high-powered ultrasound. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays were performed on fibroblasts to measure the metabolic activity of the cells following stimulation. MTT assays showed that ultrasound-stimulated fibroblasts on PVDF-TrFE scaffolds had increased metabolic activity as power was increased, whereas on plain polystyrene, an opposite trend was observed where cells had a decreased metabolic activity with ascending levels of ultrasound power. Ultrasound-stimulated PVDF-TrFE nanofibers hold exciting potential as a therapy for PNS injuries by promoting increased metabolic activity and proliferation. The ability to noninvasively stimulate implantable piezoelectric nanofibers to promote mechanical and electrical stimulation for nerve repair offers a promising benefit to severe trauma patients.

Innervation of an Ultrasound-Mediated PVDF-TrFE Scaffold for Skin-Tissue Engineering.

In this work, electrospun polyvinylidene-trifluoroethylene (PVDF-TrFE) was utilized for its biocompatibility, mechanics, and piezoelectric properties to promote Schwann cell (SC) elongation and sensory neuron (SN) extension. PVDF-TrFE electrospun scaffolds were characterized over a variety of electrospinning parameters (1, 2, and 3 h aligned and unaligned electrospun fibers) to determine ideal thickness, porosity, and tensile strength for use as an engineered skin tissue. PVDF-TrFE was electrically activated through mechanical deformation using low-intensity pulsed ultrasound (LIPUS) waves as a non-invasive means to trigger piezoelectric properties of the scaffold and deliver electric potential to cells. Using this therapeutic modality, neurite integration in tissue-engineered skin substitutes (TESSs) was quantified including neurite alignment, elongation, and vertical perforation into PVDF-TrFE scaffolds. Results show LIPUS stimulation promoted cell alignment on aligned scaffolds. Further, stimulation significantly increased SC elongation and SN extension separately and in coculture on aligned scaffolds but significantly decreased elongation and extension on unaligned scaffolds. This was also seen in cell perforation depth analysis into scaffolds which indicated LIPUS enhanced perforation of SCs, SNs, and cocultures on scaffolds. Taken together, this work demonstrates the immense potential for non-invasive electric stimulation of an in vitro tissue-engineered-skin model.

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

In vitro and in vivo biological activities of iron chelators and gallium nitrate against Acinetobacter baumannii.

We investigated the ability of compounds interfering with iron metabolism to inhibit the growth of Acinetobacter baumannii. Iron restriction with transferrin or 2,2-bipyridyl significantly inhibited A. baumannii growth in vitro. Gallium nitrate alone was moderately effective at reducing A. baumannii growth but became bacteriostatic in the presence of serum or transferrin. More importantly, gallium nitrate treatment reduced lung bacterial burdens in mice. The use of gallium-based therapies shows promise for the control of multidrug-resistant A. baumannii.

The use of intra-articular platelet rich plasma for the symptomatic management of osteoarthritis of the knee: a pilot study.

BACKGROUND: Osteoarthritis of the knee is a chronic inflammatory condition resulting in significant patient disability, with intra-articular platelet rich plasma (PRP) injections having shown potential to improve symptomatic outcomes. This retrospective cohort pilot study aimed to observe whether PRP injections were beneficial in the symptomatic management of knee osteoarthritis in an Australian population, based on patient reported outcomes. An additional aim was to observe for an association between the number of injections and patient characteristics, such as body mass index, age, sex and radiologically determined severity of the disease. METHODS: The cohort was drawn from those who attended Ballarat Orthopaedic and Sports Medicine for PRP injections and who had completed the appropriate pre- and post-injection Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire. WOMAC scores were analysed to observe for any difference following a course of PRP injections. RESULTS: The data suggest that the use of PRP improved patient reported WOMAC scores. Additionally, it was shown that two injections had a greater effect than one injection, with a third injection providing no further benefit. Finally, there was an association with lower WOMAC scores post PRP therapy amongst male participants compared to female participants. CONCLUSION: These results suggest two PRP injections are optimal for the symptomatic management of knee osteoarthritis, identifying a need for further prospective research in this Australian population.

The impact of physical, biochemical, and electrical signaling on Schwann cell plasticity.

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.

Bacteriophage Isolation, Purification, and Characterization Techniques Against Ubiquitous Opportunistic Pathogens.

Healthcare-associated infection with "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) is a global health crisis due to their extensive intrinsic antibiotic resistance and the ability to quickly acquire resistance determinants. Alternative treatment options are required to combat this crisis, and one possibility is the use of bacteriophages, or viruses that strictly infect the pathogenic bacteria. Currently, there is a renaissance in research and development into the use of phages to target multi-, extensively, and pan-resistant bacterial infections in humans, known as phage therapy. Using A. baumannii as an example, this article describes the isolation and purification of bacteriophages from sewage and soil samples, as well as general methods used in phage research such as precipitation of phages using polyethylene glycol, host range analysis, single-cell burst size determination, DNA extraction, and restriction fragment length polymorphism analysis. © 2022 National Research Council Canada. Current Protocols © 2022 Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Industry. Basic Protocol 1: Isolation of bacteriophages against A. baumannii from sewage samples Alternate Protocol 1: Isolation of bacteriophages against A. baumannii from soil samples Support Protocol 1: Titering a bacteriophage stock Basic Protocol 2: Purification of phage to an axenic working stock Support Protocol 2: Liquid propagation of bacteriophage Basic Protocol 3: Host range analysis using the spot plate method Basic Protocol 4: Single burst size analysis Alternate Protocol 2: One-step growth curve Basic Protocol 5: Precipitation of bacteriophage using PEG 6000 Basic Protocol 6: DNA extraction from dsDNA bacteriophages Basic Protocol 7: Restriction fragment length polymorphism analysis of novel phage genomes.