The gut microbiome, composed of bacteria, fungi, and viruses, plays a crucial role in maintaining the delicate balance of human health. Emerging evidence suggests that microbiome disruptions can have far-reaching implications, ranging from the development of inflammatory diseases and cancer to metabolic disorders. Bacteriophages, or "phages", are viruses that specifically infect bacterial cells, and their interactions with the gut microbiome are receiving increased attention. Despite the recently revived interest in the gut phageome, it is still considered the "dark matter" of the gut, with more than 80% of viral genomes remaining uncharacterized. Today, research is focused on understanding the mechanisms by which phages influence the gut microbiota and their potential applications. Bacteriophages may regulate the relative abundance of bacterial communities, affect bacterial functions in various ways, and modulate mammalian host immunity. This review explores how phages can regulate bacterial functionality, particularly in gut commensals and pathogens, emphasizing their role in gut health and disease.

Carbapenem-resistant Enterobacteriaceae (CRE) are listed as a priority-one critical pathogen category by the WHO because of their abysmal treatment outcomes owing to antibiotic inefficiency. Among CRE, Klebsiella pneumoniae is prevalent in acquiring resistance genes and withstanding the last-resort drugs. Additionally, its ability to form robust biofilms further exacerbates the treatment challenges. The escalating resistance and recalcitrance of biofilm-residing bacteria against standard antibiotic treatments demand an alternative to antibiotics. Phages, being nature-tailored, are a never-ending arsenal against the bacteria because of their capacity to lyse bacteria rapidly and co-evolve with bacteria. In our study, we isolated K. pneumoniae from patients at Madras Medical Mission Hospital (MMMH), India, and assessed their antibiogram profiles, presence of carbapenemase genes, and biofilm-forming abilities. 100 % of the strains were extended-spectrum beta-lactamase producing, multidrug-resistant (ESBL-MDR), with 95 % harbouring carbapenemase genes. Among the isolates, 65 % were strong biofilm formers, and the rest were moderate. Further, we isolated a bacteriophage, SAKp11, from the hospital sewage, which was able to lyse 62 out of 167 clinical isolates and successfully reduced 99.99 % viable bacterial cells of the 24-h-old biofilm of strong biofilm forming MDR K. pneumoniae strains. Whole genome analysis revealed that SAKp11, with a genome size of 59,338bp, belonged to the Casjensviridae family, one of the less explored bacteriophage families. Comprehensive characterization of SAKp11 indicated its suitability for therapeutic use. Our study highlights the severity of drug-resistant K. pneumoniae in Indian healthcare and the inadequacy of current antibiotics, underscoring the potential of phages as an alternative therapeutic option.

The rapid spread of multidrug-resistant bacteria and the challenges in developing new antibiotics have brought renewed international attention to phage therapy. However, in bacteria-phage co-evolution, the rapid development of bacterial resistance to phage has limited its clinical application. This review consolidates the latest advancements in research on anti-phage mechanisms, encompassing strategies such as systems associated with reduced nicotinamide adenine dinucleotide (NAD+) to halt the propagation of the phage, symbiotic bacteria episymbiont-mediated modulation of gene expression in host bacteria to resist phage infection, and defence-related reverse transcriptase (DRT) encoded by bacteria to curb phage infections. We conduct an in-depth analysis of the underlying mechanisms by which bacteria undergo alterations in antibiotic susceptibility after developing phage resistance. We also discuss the remaining challenges and promising directions for phage-based therapy in the future.

Phages interact with many components of bacterial physiology from the surface to the cytoplasm. Although there are methods to determine the receptors and intracellular systems a specified phage interacts with retroactively, finding a phage that interacts with a chosen piece of bacterial physiology a priori is very challenging. Variation in phage plaque morphology does not to reliably distinguish distinct phages, and therefore many potentially redundant phages may need to be isolated, purified, and individually characterized to find phages of interest. Here, we present a method in which multiple bacterial strains are co-cultured on the same screening plate to add an extra dimension to plaque morphology data. In this method, phage discovery by co-culture (Phage DisCo), strains are isogenic except for fluorescent tags and one perturbation expected to impact phage infection. Differential plaquing on the strains is easily detectable by fluorescent signal and implies that the perturbation made to the surviving strain in a plaque prevents phage infection. We validate the Phage DisCo method by showing that characterized phages have the expected plaque morphology on Phage DisCo plates and demonstrate the power of Phage DisCo for multiple targeted discovery applications, from receptors to phage defense systems.IMPORTANCEIn this work, we describe a targeted phage discovery method that allows immediate isolation of phages with specific traits. Currently, to find a phage with specific properties, huge libraries of phages must be collected and screened retroactively. This assay, Phage Discovery by Co-culture (Phage DisCo), works by co-culture of host strains that are identical except for one perturbation that may interfere with phage infection and a unique fluorescent marker. These strains are co-cultured with an environmental sample of interest in traditional plaque assay format, making phage characteristics easily identifiable by fluorescent signal after imaging of the screening plate. We validate that Phage DisCo can identify phages with specific properties, even when these phages are rare in samples. This approach allows rapid exploration of the diversity within phage samples with vastly streamlined processes, and we anticipate it will be widely adopted within the phage discovery field.

The biofilm mode of growth and drug efflux are both important factors that impede the treatment of bacterial infections with antimicrobials. Decades of work have uncovered the mechanisms involved in both efflux and biofilm-mediated antimicrobial tolerance, but links between these phenomena have only recently been discovered. Novel findings show how efflux impacts global cellular physiology and antibiotic tolerance, underpinned by phenotypic heterogeneity. In addition efflux can mediate cell-to-cell interactions, relevant in biofilms, via mechanisms including efflux of signaling molecules and metabolites, signaling using pump components and the establishment of local antibiotic gradients via pumping. These recent findings suggest that biofilm antibiotic tolerance and efflux are closely coupled, with synergistic effects leading to the evolution of antimicrobial resistance in the biofilm environment.

The emergence of drug-resistant microorganisms requires implementing alternative therapy rather than antibiotics. Phage therapy is a fantastic substitute for antibiotics. Compared to antibiotics, phage therapy has many benefits, such as high specificity for the target bacteria, auto-dosing, biofilm penetration, and a decreased likelihood of resistance development. Regulatory issues, manufacturing barriers, the possibility of phage resistance, and interactions with the human immune system are only a few of the major obstacles that still exist. Various phage-derived enzymes and bioengineered phages may increase the therapeutic potential to combat antibiotic-resistant infections. This mini-review is compiled from research on phage mechanisms in mammalian immune systems, therapeutic uses, regulatory issues, and phage engineering advancements. Thus, it offers a hopeful future in phage therapy by offering a thorough overview of the therapeutic potentiality of phage and the global aspects of phage therapy. In conclusion, phages are expected to become an alternative treatment for antibiotics against multidrug-resistant (MDR) bacteria for medical purposes.

As the overuse of antibiotics has not yet been strictly limited in urban areas, drug-resistant Escherichia coli has become a fatal pressure for bacteremia treatment. Considering the outstanding performance of bacteriophages in vitro, bacteriophages may serve as an alternative to heal chronic refractory infections. In this study, a 49,890 bp double-stranded circular DNA phage, Henu8, was isolated and was able to lyse the group of E. coli strains tested in this study. Prominent biological characterization revealed that the highly adsorbed bacteriophage Henu8 could form a fully transparent plaque with a narrow translucent halo. The optimal multiplicity of infection of the bacteriophage Henu8 was 0.01, with a burst size of 275 PFU/cell. Genomic analysis revealed a G + C content of 44.17% Henu8, in which 65 open reading frames were located, which could be assigned as a new species in the genus Hanrivervirus of the subfamily Tempevirinae. The effective antibacterial ability and the obvious biofilm destruction and inhibition capability of phage Henu8 were observed. The time-killing assay demonstrated the synergetic potential of Henu8 with antibiotics in vitro for E. coli eradication. Henu8 has profound medicinal potential in a mouse bacteremia model. These studies indicate that Henu8 is a novel bacteriophage with therapeutic potential alone or in combination with antibiotics for clinical treatment.IMPORTANCEThe findings described in this study constitute concrete evidence that it is possible to significantly synergize the antimicrobial activity of bacteriophages and antibiotics. We showed that the newly isolated potent bacteriophage Henu8 lyses Escherichia coli rapidly but tends to produce resistant bacteria. The bacteriophage Henu8 has synergistic antimicrobial effects with several antibiotics and is not susceptible to developing resistance. These results provide further evidence that bacterial resistance to phages arises, possibly at an adaptive cost to sensitivity to antibiotics. Therefore, the findings of this study are important for increasing the potential of phages for clinical applications and developing new approaches to improve their therapeutic efficacy against bacterial drug resistance.

Multi-drug resistant Gram-negative pathogens are increasingly difficult-to-treat perpetrators of infections. New, innovative, and more multifaceted therapies for the treatment of multi-drug resistant strains are thus urgent to hinder further drug resistance and mitigate deadly, untreatable infections. Our study aimed to investigate the efficacy of cefiderocol against Gram-negative aerobic bacteria alone and in combination with phages. The minimum inhibitory concentration (MIC) of cefiderocol was determined using the microdilution broth method, while the minimum biofilm bactericidal concentration was assessed using isothermal microcalorimetry. The combined effect of cefiderocol and phages was evaluated using colony-forming unit counts. Results demonstrated a notable antibacterial effect of cefiderocol, with 83.4% of tested strains exhibiting susceptibility. When combined with phages, the MIC of cefiderocol was reduced by 2-64-fold, indicating a synergistic interaction between the two agents. Furthermore, the combination therapy showed enhanced efficacy against biofilm compared to monotherapy with either cefiderocol or phages alone, leading to complete biofilm elimination in certain cases. This study highlights the potential of combining cefiderocol with phages as a strategy to combat multi-drug resistant Gram-negative bacterial infections. The observed synergy suggests that this combination therapy could improve treatment outcomes and help address the challenges of antibiotic resistance and biofilm-associated infections.

Bacterial multidrug resistance poses an urgent challenge for the treatment of critically ill patients developing ventilator-associated pneumonia (VAP). Phage therapy, a potential alternative when conventional antibiotics fail, has been unsuccessful in first clinical trials when used alone. Whether combining antibiotics with phages may enhance effectiveness remains to be tested in experimental models. Here, we use a murine model of Pseudomonas-induced VAP to compare the efficacy of adjunctive phage cocktail for antibiotic therapy to either meropenem or phages alone. Combined treatment in murine VAP results in faster clinical improvement and prevents lung epithelial cell damage. Using human primary epithelial cells to dissect these synergistic effects, we find that adjunctive phage therapy reduces the minimum effective concentration of meropenem and prevents resistance development against both treatments. These findings suggest adjunctive phage therapy represents a promising treatment for MDR-induced VAP, enhancing the effectiveness of both antibiotics and phages while reducing adverse effects.

The significance of bacteriophages in the gut microbiota remains poorly understood due, in part, to an absence of an animal model that allows for comparative study of conditions with or without phages while retaining the microbial diversity attained by conventional colonization. We describe a mouse model that uses a broadly available chemical compound, acriflavine, to preferentially deplete virulent phages from the gut without significantly impacting gut bacteria. We then show that gut phage density can be reconstituted by oral gavage. Using this bacteriophage-conditional (BaCon) mouse model, we reveal that while phages have comparatively minimal impact during equilibrium conditions, they increase the potency of ampicillin against commensal gut bacteria. Collectively, our work presents an animal model that can be leveraged to conditionally study the role of phages in complex, physiologically relevant systems and further identifies virulent gut phages as potential sources of bacterial variability during major perturbations.

Acinetobacter baumannii (A. baumannii) has become a major hospital-acquired pathogen, well-known for its rapid development of resistance to multiple antibiotics. The rising incidence of antibiotic-resistant A. baumannii presents a significant global public health challenge. Gaining a deep understanding of the mechanisms behind this resistance is essential for creating effective treatment options. This comprehensive review explores the understanding of various antibiotic resistance mechanisms in A. baumannii. It covers intrinsic resistance, acquired resistance genes, efflux pumps, changes in outer membrane permeability, alterations in drug targets, biofilm formation, and horizontal gene transfer. Additionally, the review investigates the role of mobile genetic elements and the clinical implications of antibiotic resistance in A. baumannii infections. The insights provided may inform the development of new antimicrobial agents and the design of effective infection control strategies to curb the spread of multidrug-resistant (MDR) A. baumannii strains in healthcare environments. Unlike previous reviews, this study offers a more integrative perspective by also addressing the pathogen's environmental resilience, with particular emphasis on its resistance to desiccation and the formation of robust biofilms. It further evaluates both established and emerging therapeutic strategies, thereby expanding the current understanding of A. baumannii persistence and treatment.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause sinus infections and pneumonia in cystic fibrosis (CF) patients. Bacteriophage therapy is being investigated as a treatment for antibiotic-resistant P. aeruginosa infections. Although virulent bacteriophages have shown promise in treating P. aeruginosa infections, the development of bacteriophage-insensitive mutants (BIMs) in the presence of bacteriophages has been described. The aim of this study was to examine the genetic changes associated with the BIM phenotype. Biofilms of three genetically distinct P. aeruginosa strains, including PAO1 (ATCC 15692), and two clinical respiratory isolates (one CF and one non-CF) were grown for 7 days and treated with either a cocktail of four bacteriophages or a vehicle control for 7 consecutive days. BIMs isolated from the biofilms were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in two strains, and several small variants in all three strains. These variations involved a TonB-dependent outer membrane receptor in one strain, and mutations in lipopolysaccharide synthesis genes in two strains. Prophage deletion and induction were also noted in two strains, as well as mutations in several genes associated with virulence factors. Mutations in genes involved in susceptibility to conventional antibiotics were also identified in BIMs, with both decreased and increased antibiotic sensitivity to various antibiotics being observed. These findings may have implications for future applications of lytic phage therapy.IMPORTANCELytic bacteriophages are viruses that infect and kill bacteria and can be used to treat difficult-to-treat bacterial infections, including biofilm-associated infections and multidrug-resistant bacteria. Pseudomonas aeruginosa is a bacterium that can cause life-threatening infections. Lytic bacteriophage therapy has been trialed in the treatment of P. aeruginosa infections; however, sometimes bacteria develop resistance to the bacteriophages. This study sheds light on the genetic mechanisms of such resistance, and how this might be harnessed to restore the sensitivity of multidrug-resistant P. aeruginosa to conventional antibiotics.

Phages (bacteriophages) are viruses that specifically infect and kill bacteria. They are very abundant in nature, playing a highly relevant role in microbial ecosystems. In medicine, they are investigated as a potential alternative or supplement to antibiotics and can be used to treat wound, urinary tract and lung infections, for example. Single phages or so-called "phage cocktails" are applied.This overview article on basic knowledge of phages sheds light on well-known keywords from classical knowledge of phage biology and on state-of-the-art research focuses. Mechanisms of phage activity are presented as a basis for therapeutic application. Particularly, the phage-host interaction, lysis mechanisms, phage morphologies and specific methods for visualisation are discussed. Being part of the human microbiome, phages contribute to immune defence, especially in the mucosa. Temperate phages that are able to reside in bacterial genomes as prophages and therefore not suitable for therapy use as well as the CrAss phages (Crassvirales) and Lak megaphages discovered in recent years are also introduced. Further topics are bacterial phage defence, phage resistance and phage-antibiotic synergies. An outlook on future research is given, emphasising the importance of a coordinated collection of scientific results.Phages should not replace antibiotics, but they can even improve their efficiency. Currently, the licensing processes for phage therapy are still challenging. However, trust in phage preparations must be based on quality, which has to be guaranteed by harmonised standards.

Septic arthritis (SA) is an infection of one or more joints caused mainly by Staphylococcus aureus, to a lesser extent by streptococci and Gram-negative bacilli. It poses a huge medical problem due to its high mortality rate of 2-15%. Disease symptoms are often vague, resulting in a risk that SA may be diagnosed too late. This shows the urgency of finding a rapid diagnostic method for SA and an effective therapy. Basic treatment of SA including joint drain or empirical antimicrobial therapy does not always provide the desired results. Hence, new therapies are being sought, including the use of antimicrobial peptide or phage therapy.

Bacteriophage (phage) therapy, which uses lytic viruses as antimicrobials, is a potential strategy to address the antimicrobial resistance crisis. Cystic fibrosis, a disease complicated by recurrent Pseudomonas aeruginosa pulmonary infections, is an example of the clinical impact of antimicrobial resistance. Here, using a personalized phage therapy strategy that selects phages for a predicted evolutionary trade-off, nine adults with cystic fibrosis (eight women and one man) of median age 32 (range 22-46) years were treated with phages on a compassionate basis because their clinical course was complicated by multidrug-resistant or pan-drug-resistant Pseudomonas that was refractory to prior courses of standard antibiotics. The individuals received a nebulized cocktail or single-phage therapy without adverse events. Five to 18 days after phage therapy, sputum Pseudomonas decreased by a median of 104 CFU ml-1, or a mean difference of 102 CFU ml-1 (P = 0.006, two-way analysis of variance with Dunnett's multiple-comparisons test), without altering sputum microbiome, and an analysis of sputum Pseudomonas showed evidence of trade-offs that decreased antibiotic resistance or bacterial virulence. In addition, an improvement of 6% (median) and 8% (mean) predicted FEV1 was observed 21-35 days after phage therapy (P = 0.004, Wilcoxon signed-rank t-test), which may reflect the combined effects of decreased bacterial sputum density and phage-driven trade-offs. These results show that a personalized, nebulized phage therapy trade-off strategy may affect clinical and microbiologic endpoints, which must be evaluated in larger clinical trials.

Bacteria in polymicrobial habitats are constantly exposed to biotic threats from bacteriophages (or "phages"), antagonistic bacteria, and predatory eukaryotes. These antagonistic interactions play crucial roles in shaping the evolution and physiology of bacteria. To survive, bacteria have evolved mechanisms to protect themselves from such attacks, but the fitness costs of resisting one threat and rendering bacteria susceptible to others remain unappreciated. Here, we examined the fitness consequences of phage resistance in Salmonella enterica, revealing that phage-resistant variants exhibited significant fitness loss upon co-culture with competitor bacteria. These phage-resistant strains display varying degrees of lipopolysaccharide (LPS) deficiency and increased susceptibility to contact-dependent interbacterial antagonism, such as the type VI secretion system (T6SS). Utilizing mutational analyses and atomic force microscopy, we show that the long-modal length O-antigen of LPS serves as a protective barrier against T6SS-mediated intoxication. Notably, this competitive disadvantage can also be triggered independently by phages possessing LPS-targeting endoglycosidase in their tail spike proteins, which actively cleave the O-antigen upon infection. Our findings reveal two distinct mechanisms of phage-mediated LPS modifications that modulate interbacterial competition, shedding light on the dynamic microbial interplay within mixed populations.

Escherichia coli (E. coli), a prevalent Gram-negative bacterium, is a frequent cause of illness. The extensive use of antibiotics has led to the emergence of resistant strains, complicating antimicrobial therapy and emphasizing the need for natural alternatives such as phages.

In this study, a novel Escherichia coli phage, AUBRB02, was isolated from sewage and characterized through whole-genome sequencing, host range assays, and biofilm elimination assays. The phage's stability and infectivity were assessed under various pH and temperature conditions, and different E. coli strains.

Phage AUBRB02 has an incubation period of 45 min, a lysis period of 10 min, and a burst size of 30 phages/infected cell. It is stable across pH 5.0-9.0 and temperatures from 4 °C to 60 °C. Treatment with AUBRB02 significantly reduced post-formation E. coli biofilms, as indicated by lower OD values compared with the positive control. The whole genome sequencing revealed a genome size of 166,871 base pairs with a G + C (Guanine and Cytosine content) content of 35.47%. AUBRB02 belongs to the Tequatrovirus genus, sharing 93% intergenomic similarity with its closest RefSeq relative, and encodes 262 coding sequences, including 10 tRNAs.

AUBRB02 demonstrates high infectivity and stability under diverse conditions. Its genomic features and similarity to related phages highlight its potential for phage therapy, offering promising prospects for the targeted treatment of E. coli infections.

Pseudomonas aeruginosa, a multidrug-resistant pathogen, significantly impacts patients with chronic respiratory conditions like cystic fibrosis (CF) and non-CF chronic suppurative lung disease (CSLD), contributing to progressive lung damage and poor clinical outcomes. This bacterium thrives in the airway environments of individuals with impaired mucociliary clearance, leading to persistent infections and increased morbidity and mortality. Despite advancements in management of these conditions, treatment failure remains common, emphasising the need for alternative or adjunctive treatment strategies. Bacteriophage therapy, an emerging approach utilising viruses that specifically target bacteria, offers a potential solution to combat P. aeruginosa infections resistant to conventional antibiotics. This review examines the prevalence and disease burden of P. aeruginosa in CF and CSLD, explores the mechanisms behind antibiotic resistance, the promising role of bacteriophage therapy and clinical trials in this sphere.

Jumbo bacteriophages of the ϕKZ-like family assemble a lipid-based early phage infection (EPI) vesicle and a proteinaceous nucleus-like structure during infection. These structures protect the phage from nucleases and may create selective pressure for immunity mechanisms targeting this specific phage family. Here, we identify "jumbo phage killer" (Juk), a two-component immune system that terminates infection of ϕKZ-like phages, suppressing the expression of early phage genes and preventing phage DNA replication and phage nucleus assembly while saving the cell. JukA (formerly YaaW) rapidly senses the EPI vesicle by binding to an early-expressed phage protein, gp241, and then directly recruits JukB. The JukB effector structurally resembles a pore-forming toxin and destabilizes the EPI vesicle. Functional anti-ϕKZ JukA homologs are found across bacterial phyla, associated with diverse effectors. These findings reveal a widespread defense system that specifically targets early events executed by ϕKZ-like jumbo phages prior to phage nucleus assembly.

Antibiotic-resistant Pseudomonas aeruginosa is a pathogen notorious for its resilience in clinical settings due to biofilm formation, efflux pumps, and the rapid acquisition of resistance genes. With traditional antibiotic therapy rendered ineffective against Pseudomonas aeruginosa infections, we explore alternative therapies that have shown promise, including antimicrobial peptides, nanoparticles and quorum sensing inhibitors. While these approaches offer potential, they each face challenges, such as specificity, stability, and delivery, which require careful consideration and further study. We also delve into emerging alternative strategies, such as bacteriophage therapy and CRISPR-Cas gene editing that could enhance targeted treatment for personalized medicine. As most of them are currently in experimental stages, we highlight the need for clinical trials and additional research to confirm their feasibility. Hence, we offer insights into new therapeutic avenues that could help address the pressing issue of antibiotic-resistant Pseudomonas aeruginosa, with an eye toward practical applications in future healthcare.

Klebsiella pneumoniae (KP) infections present a significant clinical challenge and are frequently associated with elevated drug resistance. The use of phage therapy has resurged in response to escalating antibiotic resistance. This study aimed to address the multidrug resistance crisis in intensive care units by exploring the use of ceftazidime/avibactam (CAZ/AVI), a widely used clinical antimicrobial agent, in conjunction with phage therapy.

We screened a clinical strain of KP from ICU and successfully isolated phage N22 from hospital wastewater. We conducted an in-depth analysis of the physiological and biochemical properties of phage N22 and determined its optimal multiplicity of infection with the clinical KP strain. The inhibitory effects of phage N22 in combination with CAZ/AVI on biofilm formation were investigated. Comparative efficacies of these combinations were evaluated using a Galleria mellonella (G. mellonella) model.

Phage N22 inhibited KP biofilm formation. The impact of varying phage N22 concentrations when used alongside CAZ/AVI was examined, and the combination of phage N22 and CAZ/AVI was more effective against KP than CAZ/AVI alone.

This study provides a preliminary investigation into the effects of combining CAZ/AVI with phage therapy, highlighting its potential significance in developing novel therapeutic strategies for bacterial infections resistant to CAZ/AVI. The findings underscore the importance of advancing highly effective phage agents as alternative treatment modalities for patients with infections refractory to conventional antibiotics.

The current crisis in bacterial antibiotic resistance can be attributed to the overuse (or misuse) of these essential medicines in healthcare and agriculture, coupled with the slowed progression of new drug development. In the versatile, opportunistic pathogen Pseudomonas aeruginosa, the Resistance-Nodulation-Division (RND) efflux pump MexXY plays critical roles in both cell physiology and the acquisition of multidrug resistance. The mexXY operon is not constitutively expressed, but this process is instead controlled by a complex network of multiple interconnected regulatory mechanisms. These include induction by several of the pump's ribosome-targeting antibiotic substrates and transcriptional repression and anti-repression processes that are themselves influenced by various cellular factors, processes, or stresses. Although extensive studies of the MexXY complex are currently lacking as compared to other RND efflux pumps such as Escherichia coli AcrAB-TolC, recent studies have provided valuable insights into the MexXY architecture and substrate profiles, including its contribution to clinical resistance. Furthermore, while MexXY primarily associates with the outer membrane protein OprM, emerging evidence suggests that this transporter-periplasmic adaptor pair may also partner with other outer membrane proteins, potentially to alter the efflux substrate profile and activity under specific environmental conditions. In this minireview, we summarize current understanding of MexXY regulation, structure, and substrate selectivity within the context of clinical resistance and as a framework for future efflux pump inhibitor development.

Lytic bacteriophages ('phages') can limit bacterial densities and shape community structure, either directly through lysis or indirectly through costs to resistance. However, phages have also been reported to have no, and in some cases even positive, effects on host densities. Here, we investigate the mechanisms behind an increase in host density in Variovorax sp. populations following a fixation of resistance that was maintained after phage extinction. Our results demonstrate that the density increase was a genetic trait coinciding with resistance emergence. Growth curves showed that phage resistance shifted population growth curves such that density was higher in the death phase. This density-increasing effect of resistance had important implications for community structure with phage-resistant Variovorax decreasing the density of a conspecific. That resistance to lytic phage can increase host densities has implications for wider ecology and phage therapy, where lytic phages are presumed to have negative effects on their hosts.

The rise of deaths by resistant bacteria is a global threat to public health systems. Klebsiella pneumoniae is a virulent pathogen that causes serious nosocomial infections. The major obstacle to bacterial treatment is antibiotic resistance, which necessitates the introducing of alternative therapies. Phage therapy has been regarded as a promising avenue to fight multidrug-resistant (MDR) pathogens. In the current study, a novel phage vB_KpnP_KP17 was isolated from sewage, and its lytic potential was investigated against K. pneumoniae. The isolated phage vB_KpnP_kP17 was lytic to 17.5% of tested K. pneumoniae isolates. One step growth curve indicated a virulent phage with a short latent period (20 min) and large burst size (331 PFU/cell). Additionally, vB_KpnP_kP17 maintained its activity against planktonic cells over a wide range of pH, temperature and UV irradiation intervals. The potential of vB_KpnP_KP17 as antibiofilm agent was revealed by the biofilm inhibition assay. The isolated phage vB_KpnP_KP17 at multiplicity of infection (MOI) of 10 inhibited more than 50% of attached biofilms of tested K. pneumoniae isolates. The genome of vB_KpnP_kP17 was characterized and found to be a linear dsDNA of 39,936 bp in length and GC content of 52.85%. Additionally, the absence of toxicity, virulence and antibiotic resistance genes further confirms the safety of vB_KpnP_KP17 for clinical applications. These characteristics make vB_KpnP_KP17 of a potential therapeutic value to manage MDR K. pneumoniae infections. Additionally, the formulation of vB_KpnP_KP17 in a cocktail with other lytic phages or with antibiotics could be applied to further limit biofilm-producing K. pneumoniae infections.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections, and belongs to the group of ESKAPE pathogens that are the leading cause of healthcare-associated infections and have high level of antibiotic resistance. The treatment of infections caused by antibiotic-resistant P. aeruginosa is challenging, which makes it a common target for phage therapy. The successful utilization of phage therapy requires a collection of well characterized phages.

Phage fMGyn-Pae01 was isolated from a commercial phage therapy cocktail. The phage morphology was studied by transmission electron microscopy and the host range was analyzed with a liquid culture method. The phage genome was sequenced and characterized, and the genome was compared to closest phage genomes. Phage resistant bacterial mutants were isolated and whole genome sequencing and motility, phage adsorption and biofilm formation assays were performed to the mutants and host bacterium.

The genomic analysis revealed that fMGyn-Pae01 is a lytic, phiKZ-like jumbo phage with genome size of 277.8 kb. No genes associated with lysogeny, bacterial virulence, or antibiotic resistance were identified. Phage fMGyn-Pae01 did not reduce biofilm formation of P. aeruginosa, suggesting that it may not be an optimal phage to be used in monophage therapy in conditions where biofilm formation is expected. Host range screening revealed that fMGyn-Pae01 has a wide host range among P. aeruginosa strains and its infection was not dependent on O-serotype. Whole genome sequencing of the host bacterium and phage resistant mutants revealed that the mutations had inactivated either a flagellar or rpoN gene, thereby preventing the biosynthesis of a functional flagellum. The lack of functional flagella was confirmed in motility assays. Additionally, fMGyn-Pae01 failed to adsorb on non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor.

fMGyn-Pae01 is a phiKZ-like jumbo phage infecting P. aeruginosa. fMGyn-Pae01 uses the flagellum as its phage-binding receptor, supporting earlier suggestions that flagellum might be utilized by phiKZ but differs from some other previous findings showing that phiKZ-like phages use the type-IV pili as the phage-binding receptor.

In this manuscript, we highlight current literature on environmental hygiene techniques to combat reservoirs of antibiotic resistant organisms in the healthcare environment. We discuss several topics for each strategy, including mechanism of action, assessment of effectiveness based on studies, cost, and real-world translatability. The techniques and topics summarized here are not inclusive of all available environmental hygiene techniques but highlight some of the more popular and investigated strategies. We focus on the following: Ultraviolet radiation, hydrogen peroxide vapor, copper-coated surfaces, phages, interventions involving sinks, and educational initiatives.

Bacteriophage (phage) therapy has emerged as a promising solution to combat the growing crisis of multidrug-resistant (MDR) infections. There are several international centers actively engaged in implementation of phage therapy, and recent case series have reported encouraging success rates in patients receiving personalized, compassionate phage therapy for difficult-to-treat infections. Nonetheless, substantial hurdles remain in the way of more widespread adoption and more consistent success. This Review offers a comprehensive overview of current phage therapy technologies and therapeutic approaches. We first delineate the common steps in phage therapy development, from phage bank establishment to clinical administration, and examine the spectrum of therapeutic approaches, from personalized to fixed phage cocktails. Using the framework of a conventional drug development pipeline, we then identify critical knowledge gaps in areas such as cocktail design, formulation, pharmacology, and clinical trial design. We conclude that, while phage therapy holds promise, a structured drug development pipeline and sustained government support are crucial for widespread adoption of phage therapy for MDR infections.

The aim of this study was to evaluate the effects of the combination of bacteriophage therapy with antibiotics and bacteriophage treatment alone on relieving clinical symptoms of chronic recurrent cystitis caused by multidrug-resistant bacteria.

This clinical trial compared the treatment methods of 217 female patients with chronic recurrent cystitis caused by multidrug-resistant bacteria, who were investigated from June 2020 to May 2023. Patients were allocated into 4 groups: group I: received bacteriophage (Sextaphage) therapy alone; group II: received a combination of bacteriophages (Sextaphage) and furazidin; group III: received a combination of bacteriophage (Sextaphage) and furazidin with cefixime; and group IV: received furazidin and cefixime (without bacteriophage). The primary outcome included changes in the acute cystitis symptom scale and the pain visual analog scale, which were completed on days 7 and 14 following treatment. Secondary outcome measures included bladder diary records of urinary symptoms, median voided volumes, level of bacteriuria, and degree of leukocyturia.

Initially, 217 female patients were presented during baseline visits. Those who did not meet the criteria inclusions were excluded, and 178 female patients were included in the final analysis. Statistically significant improvements from baseline in acute cystitis symptom scale scores for differential, typical symptoms, and quality of life domains were observed after 14 days of treatment in groups II, III, and IV. The pain level measured on the 14th day with the visual analog scale significantly decreased in groups II, III, and IV compared with group I. The patients of group I had a reduction of mean level bacteriuria of Escherichia coli from 106 to 102 CFU/mL at 14 days of therapy. Significant improvement of voided volume from baseline was observed in groups II, III, and IV. Episodes of urinary frequency, both daytime and night-time, reduced significantly from baseline in all 4 groups only at 14 days of treatment.

Bacteriophage cocktail alone or with antibiotics may improve clinical symptoms in women with chronic recurrent cystitis caused by multidrug-resistant bacterial pathogens. In addition to improving clinical symptoms, the therapy with a phage cocktail may restore antibiotic sensitivity and increase the efficacy of antimicrobial agents.

Cell populations must adjust their phenotypic composition to adapt to changing environments. One adaptation strategy is to maintain distinct phenotypic subsets within the population and to modulate their relative abundances via gene regulation. Another strategy involves genetic mutations, which can be augmented by stress-response pathways. Here, we studied how a migrating bacterial population regulates its phenotypic distribution to traverse diverse environments. We generated isogenic Escherichia coli populations with varying distributions of swimming behaviors and observed their phenotype distributions during migration in liquid and porous environments. We found that the migrating populations became enriched with high-performing swimming phenotypes in each environment, allowing the populations to adapt without requiring mutations or gene regulation. This adaptation is dynamic and rapid, reversing in a few doubling times when migration ceases. By measuring the chemoreceptor abundance distributions during migration toward different attractants, we demonstrated that adaptation acts on multiple chemotaxis-related traits simultaneously. These measurements are consistent with a general mechanism in which adaptation results from a balance between cell growth generating diversity and collective migration eliminating underperforming phenotypes. Thus, collective migration enables cell populations with continuous, multidimensional phenotypes to flexibly and rapidly adapt their phenotypic composition to diverse environmental conditions.

Bacteria possess numerous defense systems against phage infections, which limit phage infectivity and pose challenges for phage therapy. This study aimed to engineer phages capable of evading these defense systems, using the Tmn defense system as a model. We identified an anti-Tmn protein in the ΦSMS22 phage from the Dhillonvirus genus that inhibits Tmn function in Escherichia coli. Introducing this gene into the Tmn-sensitive ΦKSS9 phage enabled it to evade Tmn immunity. Additionally, we found that a single mutation in the nmad5 gene, a DNA modification enzyme in Dhillonvirus, prevented Tmn from sensing phage infection. By mutating the nmad5 gene in the Tmn-sensitive Dhillonvirus, we demonstrated that engineering phages to evade bacterial sensing mechanisms is another viable strategy. These two phage engineering approaches-introducing anti-defense genes and mutating sensing-related genes-present a promising strategy for establishing effective phage therapy by neutralizing bacterial defense systems.

Microbes possess diverse genetic and metabolic traits that help them withstand adverse conditions. Microbial pathogens cause significant economic losses and around 7.7 million human deaths annually. While antibiotics have historically been a lifesaving treatment, their effectiveness is declining due to antibiotic-resistant strains, prompting the exploration of bacterial predation as an alternative. Bacteriophages (BPhs) have reemerged as antibacterial agents, offering advantages over antibiotics, such as (i) high specificity, (ii) self-replication, and (iii) strong killing capacity. This review explores BPh- and enzyme-based antibacterial strategies for infectious disease treatment, discussing phage-antibiotic synergy, the risks of BPh resistance, and the role of quorum sensing in BPh therapy.

Carbapenemase-producing Klebsiella pneumoniae (KPC) are globally emerging pathogens that cause life-threatening infections. Novel treatment alternatives are urgently needed. We therefore investigated the effectiveness of three novel bacteriophages (Spivey, Pharr, and Soft) in a neutropenic murine model of KPC gastrointestinal colonization, translocation, and disseminated infection. Bacteriophage efficacy was determined by residual bacterial burden of KPC (CFU/g) in kidneys. Parallel studies were conducted of bacteriophage pharmacokinetics and resistance. Treatment of mice with 5 × 109 PFU of phage cocktail via intraperitoneal injection was effective in significantly reducing renal KPC CFU by 100-fold (P < 0.01) when administered every 24 h and 1000-fold (P < 0.01) every 12 h. Moreover, a combination of bacteriophage and ceftazidime-avibactam produced a synergistic effect, resulting in a 105-fold reduction in bacterial burden in cecum and kidney (P < 0.001 in both tissues). Prophylactic administration of bacteriophages via oral gavage did not prevent KPC translocation to the kidneys. Bacteriophage decay determined by linear regression of the ln of mean concentrations demonstrated R2 values in plasma of 0.941, kidney 0.976, and cecum 0.918, with half-lives of t1/2 = 2.5 h. Furthermore, a phage-resistant mutant displayed increased sensitivity to serum killing in vitro, but did not show significant defects in renal infection in vivo. A combination of bacteriophages demonstrated significant efficacy alone and synergy with ceftazidime/avibactam in the treatment of experimental disseminated KPC infection in neutropenic mice.

Bacteriophages are an increasingly attractive option for the treatment of antibiotic-resistant infections, but their efficacy is difficult to discern due to the confounding effects of antibiotics. Phages are generally delivered in conjunction with antibiotics, and thus, when patients improve, it is unclear whether the phages, antibiotics, or both are responsible. This question is particularly relevant for enterococcus infections, as limited data suggest phages might restore antibiotic efficacy against resistant strains. Enterococci can develop high-level resistance to vancomycin, a primary treatment. We assessed clinical and laboratory isolates of Enterococcus faecium and Enterococcus faecalis to determine whether we could observe synergistic interactions between phages and antibiotics. We identified synergy between multiple phages and antibiotics including linezolid, ampicillin, and vancomycin. Notably, antibiotic susceptibility did not predict synergistic interactions with phages. Vancomycin-resistant isolates (n = 6) were eradicated by the vancomycin-phage combination as effectively as vancomycin-susceptible isolates (n = 2). Transcriptome analysis revealed significant gene expression changes under antibiotic-phage conditions, especially for linezolid and vancomycin, with upregulated genes involved in nucleotide and protein biosynthesis and downregulated stress response and prophage-related genes. While our results do not conclusively determine the mechanism of the observed synergistic interactions between antibiotics and phages, they do confirm and build upon previous research that observed these synergistic interactions. Our work highlights how using phages can restore the effectiveness of vancomycin against resistant isolates. This finding provides a promising, although unexpected, strategy for moving forward with phage treatments for vancomycin-resistant Enterococcus infections.

Bacteriophages (phages) are bacterial-specific viruses that can be used alone or with antibiotics to reduce bacterial load. Most phages are unsuitable for therapy because they are "temperate" and can integrate into the host genome, forming a lysogen that is protected from subsequent phage infections. However, integrated phages can be awakened by stressors such as antibiotics. Supported by this interaction, here we explore the potential use of combined temperate phage and antibiotic against the multi-drug-resistant pathogen, Pseudomonas aeruginosa. In all, thirty-nine temperate phages were isolated from clinical strains, and a subset was screened for synergy with six antibiotics (ciprofloxacin, levofloxacin, meropenem, piperacillin, tobramycin, and polymyxin B), using checkerboard assays. Interestingly, our screen identified phages that can synergize with each antibiotic, despite their widely differing targets; however, these are highly phage-antibiotic and phage-host pairing specific. Screening across multiple clinical strains reveals that temperate phages can reduce the antibiotic minimum inhibitory concentration up to 32-fold, even in a resistant isolate, functionally re-sensitizing the bacterium to the antibiotic. Meropenem and tobramycin did not reduce the frequency of lysogens, suggesting a mechanism of action independent of the temperate nature of the phages. By contrast, ciprofloxacin and piperacillin were able to reduce the frequency of lysogeny, the former by inducing phages-as previously reported in E. coli. Curiously, synergy with piperacillin reduced lysogen survivors, but not by inducing the phages, suggesting an alternative mechanism for biasing the phage lysis-lysogeny equilibrium. Overall, our findings indicate that temperate phages can act as adjuvants in clinically relevant pathogens, even in the presence of antibiotic resistance, thereby drastically expanding their therapeutic potential.

The recent discovery that otherwise therapeutically unusable temperate phages can potentiate the activity of antibiotics, resulting in a potent synergy, has only been tested in E. coli, and with a single model phage. Here, working with clinical isolates of Pseudomonas and phages from these isolates, we highlight the broad applicability of this synergy-across a variety of mechanisms but also highlight the limitations of predicting the phage, host, and antibiotic combinations that will synergize.

AbstractIn the face of ubiquitous threats from parasites, hosts can evolve strategies to resist infection or to altogether avoid parasitism, for instance by avoiding behavior that could expose them to parasites or by dispersing away from local parasite threats. At the microbial scale, bacteria frequently encounter viral parasites, bacteriophages. While bacteria are known to utilize a number of strategies to resist infection by phages and can have the capacity to avoid moving toward phage-infected cells, it is unknown whether bacteria can evolve dispersal to escape from phages. To answer this question, we combined experimental evolution and mathematical modeling. Experimental evolution of the bacterium Pseudomonas fluorescens in environments with differing spatial distributions of the phage Phi2 revealed that the host bacteria evolved resistance depending on parasite distribution but did not evolve dispersal to escape parasite infection. Simulations using parameterized mathematical models of bacterial growth and swimming motility showed that this is a general finding: while increased dispersal is adaptive in the absence of parasites, in the presence of parasites that fitness benefit disappears and resistance becomes adaptive, regardless of the spatial distribution of parasites. Together, these experiments suggest that parasites should rarely, if ever, drive the evolution of bacterial escape via dispersal.

Enterohemorrhagic Escherichia coli (EHEC) are pathogens that, only in the United States, cause more than 250,000 foodborne infections a year. Since antibiotics or other antidiarrheal agents may increase the hemolytic-uremic syndrome (HUS) development risk, currently only supportive therapy, including hydration, is used. Therefore, many methods to fight EHEC bacteria focus on their use in food processing to prevent human infection. One of the proposed anti-EHEC agents is bacteriophages, known for their bactericidal effect, host specificity, and lack of cross-resistance with antibiotics. In this review article, we provide an overview of the characteristics like source of isolation, morphology, kinetics of life cycle, and treatment potential of over 130 bacteriophages able to infect EHEC strains. Based on the reviewed literature, we conclude that bacteriophages may play a highly significant role in regulating EHEC propagation. In addition, we also point out the phage features that should be taken into account not only when using bacteriophages but also when examining their properties. This may contribute to accelerating the pace of work on the preventive use of bacteriophages, which is extremely needed in the modern world of the food industry, but also stimulate interest in phages and accelerate regulatory work that would enable the use of bacteriophages also in medicine, to fight the drug-resistant strains.

Phages use bacterial host resources to replicate, intrinsically linking phage and host survival. To understand phage dynamics, it is essential to understand phage-host ecology. A key step in this ecology is infection of bacterial hosts. Previous work has explored single and multiple, sequential infections. Here we focus on the theory of simultaneous infections, where multiple phages simultaneously attach to and infect one bacterial host cell. Simultaneous infections are a relevant infection dynamic to consider, especially at high phage densities when many phages attach to a single host cell in a short time window. For high bacterial growth rates, simultaneous infection can result in bi-stability: depending on initial conditions phages go extinct or co-exist with hosts, either at stable densities or through periodic oscillations of a stable limit cycle. This bears important consequences for phage applications such as phage therapy: phages can persist even though they cannot invade. Consequently, through spikes in phage densities it is possible to infect a bacterial population even when the phage basic reproductive number is less than one. In the regime of stable limit cycles, if timed right, only small densities of phage may be necessary.

Bacteriophages, or phages, depend on their bacterial hosts for proliferation, leading to a coevolutionary relationship characterized by on-going arms races, where bacteria evolve diverse antiphage defense systems. The development of in silico methods and high-throughput screening techniques has dramatically expanded our understanding of bacterial antiphage defense systems, enormously increasing the known repertoire of the distinct mechanisms across various bacterial species. These advances have revealed that bacterial antiphage defense systems exhibit a remarkable level of complexity, ranging from highly conserved to specialized mechanisms, underscoring the intricate nature of bacterial antiphage defense systems. In this review, we provide a concise snapshot of antiphage defense research highlighting two preponderantly commandeered approaches and classification of the known antiphage defense systems. A special focus is placed on the model bacterial pathogen, Pseudomonas aeruginosa in antiphage defense research. We explore the complexity and adaptability of these systems, which play crucial roles in genome evolution and adaptation of P. aeruginosa in response to an arsenal of diverse phage strains, emphasizing the importance of this organism as a key emerging model bacterium in recent antiphage defense research.

Phages, viruses of bacteria, play a pivotal role in Earth's biosphere and hold great promise as therapeutic and diagnostic tools in combating infectious diseases. Attachment of phages to bacterial cells is a crucial initial step of the interaction. The classic assay to quantify the dynamics of phage attachment involves coculturing and enumeration of bacteria and phages, which is laborious, lengthy, hence low-throughput, and only provides ensemble estimates of model-based adsorption rate constants. Here, we utilized fluorescence microscopy and particle tracking to obtain trajectories of individual virus particles interacting with cells. The trajectory durations quantified the heterogeneity in dwell time, the time that each phage spends interacting with a bacterium. The average dwell time strongly correlated with the classically measured adsorption rate constant. We successfully applied this technique to quantify host-attachment dynamics of several phages including those targeting key bacterial pathogens. This approach should benefit the field of phage biology by providing highly quantitative, model-free readouts at single-virus resolution, helping to uncover single-virus phenomena missed by traditional measurements. Owing to significant reduction in manual effort, our method should enable rapid, high-throughput screening of a phage library against a target bacterial strain for applications such as therapy or diagnosis.