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ABSTRACT Bacteriophages, known for their ability to kill bacteria, are hampered in their effectiveness because bacteria are able to rapidly develop resistance, thereby posing a significant challenge for the efficacy of phage therapy. The impact of evolutionary trajectories on the long-term success of phage therapy remains largely unclear. Herein, we conducted evolutionary experiments, genomic analysis, and CRISPR-mediated gene editing, to illustrate the evolutionary trajectory occurring between phages and their hosts. Our results illustrate the ongoing “arms race” between a lytic phage and its host, a carbapenem-resistant Klebsiella pneumoniae clinical strain Kp2092, suggesting their respective evolutionary adaptations that shape the efficacy of phage therapy. Specifically, Kp2092 rapidly developed resistance to phages through mutations in a key phage receptor (galU) and bacterial membrane defenses such as LPS synthesis, however, this evolution coincides with unexpected benefits. Evolved bacterial clones not only exhibited increased sensitivity to clinically important antibiotics but also displayed a loss of virulence in an in-vivo model. In contrast, phages evolved under the selection pressure against Kp2092 mutants and exhibited enhanced bacterial killing potency, targeting mutations in phage tail proteins gp12 and gp17. These parallel evolutionary trajectories suggest a common genetic mechanism driving adaptation, ultimately favoring the efficacy of phage therapy. Overall, our findings highlight the potential of phages not only as agents for combating bacterial resistance, but also a driver of evolution outcomes that could lead to more favorable clinical outcomes in the treatment of multidrug resistance pathogens.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae represents one of the leading pathogens for infectious diseases. With traditional antibiotics often being ineffective, phage therapy has emerged as a promising alternative. However, phage predation imposes a strong evolutionary pressure on the rapid evolution of bacteria, challenging treatment efficacy. Our findings illustrate how co-evolution enhances phage lytic capabilities through accumulated mutations in the tail proteins gp12 and gp17, while simultaneously reducing bacterial virulence and antibiotic resistance. These insights advance our understanding of phage-host interactions in clinical settings, potentially inspiring new approaches akin to an “arms race” model to combat multidrug-resistant crises effectively.

ABSTRACT Daptomycin (DAP) is a cyclic lipopeptide antibiotic that targets the cell membrane and is commonly used for the treatment of multidrug-resistant Staphylococcus aureus infections, especially methicillin-resistant strains (MRSA). Emergence of DAP resistance during DAP therapy is increasingly reported; however, the mechanisms by which resistance occurs are diverse. Using phages in combination with antibiotics is of high interest to circumvent antibiotic resistance, due to their minimal side effects, accessibility, and synergistic effects with antibiotics. Here, we have investigated the relationship between DAP resistance and sensitivity to phage Sb-1 in a cohort of 14 clinical MRSA strains. This cohort consists of four isogenic pairs (a clinical DAP-susceptible parent and its isogenic DAP-resistant variant isolated during DAP treatment), and three DAP-susceptible/DAP-resistant strain pairs are created by traditional serial passaging in the presence of increasing DAP concentrations. Efficiency of plating (EOP) and bacteriophage plaque sizes were recorded, and synergy between the antibacterial agents was tested using the phage-antibiotic combination (PAC) checkerboard method. Plaque sizes and EOP significantly increased (P = 0.019 and P = 0.008, respectively) as strains developed resistance to DAP. The PAC checkerboards conveyed less antagonistic patterns in DAP-resistant mutant strains compared with their DAP-susceptible counterparts. Antagonism was displayed in 71.4% (5/7) of the DAP-susceptible strains, while it was only present in 14.3% (1/7) of the DAP-resistant mutants. Transmission electron microscopy images showed significantly thicker cell walls in DAP-susceptible strains exposed to DAP monotherapy compared with either growth control or DAP-phage (P = 0.0002 and 0.0007, respectively). These data indicate increased sensitivity to phage Sb-1 concurrent with the emergence of DAP resistance.IMPORTANCEMultidrug-resistant Staphylococcus aureus is a threat to the health care system, especially cross-resistance between daptomycin (DAP) and glycopeptides through various mutations such as mprF (which is involved in the modification of membrane phospholipids in some bacteria) and yycG (part of a two-component regulatory system in bacteria that is important for regulating cell wall biosynthesis and other cellular processes) has been reported previously. Our current study shows adjunctive treatment with phage in DAP-resistant strains will lead to synergistic activity and larger phage plaque sizes, translating to elevated lytic performance. The addition of bacteriophage to standard-of-care antibiotic therapies for multidrug-resistant S. aureus infections has the potential to hinder, and possibly revert, resistance to antibiotics. Applying this strategy can potentially lead to the preservation of the current antibiotics. Verification of this salutary outcome in relevant ex vivo and in vivo models of endovascular infections is required to validate translatability.

ABSTRACT Acute lung injury caused by Pseudomonas aeruginosa is attributed to the translocation of cytotoxin into pulmonary epithelial cells via the P. aeruginosa type III secretion system. This virulence can be blocked with a specific antibody against PcrV in this secretion system. However, because anti-PcrV antibodies do not have bactericidal activity, the treatment of bacteria depends on the phagocytic system of the host. In this study, we investigated the therapeutic effect of combination therapy with an anti-PcrV antibody and bactericidal bacteriophages on acute lung injury and subsequent death in mice compared with a single treatment. After the mice intratracheally received a lethal dose of the cytotoxic P. aeruginosa strain, a second instillation was performed with saline, anti-PcrV IgG, bacteriophages, or a mixture of anti-PcrV and bacteriophages. The survival rates 24 h after infection were as follows: 7.1% in the saline group, 26.7% in the anti-PcrV group, 41.2% in the phage group, and 66.7% in the anti-PcrV + phage group (P < 0.001 vs saline-treated group). The activity of surviving mice in the anti-PcrV + phage group was significantly greater than that in the saline group. The lung weight in the anti-PcrV + phage group was significantly lower than that in the anti-PcrV group. In conclusion, combination therapy with an anti-PcrV antibody and a bacteriophage reduces acute lung injury and suggests improved survival compared with each treatment alone. This combination therapy, which does not rely on conventional antibiotics, could constitute a new strategy for treating multidrug-resistant P. aeruginosa infections.IMPORTANCECombination therapy with either bacteriophages alone or in combination with anti-PcrV antibodies in a mouse model of Pseudomonas aeruginosa pneumonia may reduce the acute lung injury and improve survival. This combination therapy, which does not rely on conventional antibiotics, may be a new strategy to treat multidrug-resistant Pseudomonas aeruginosa infections.

Klebsiella spp. are causative agents of healthcare-associated infections in patients who are immunocompromised and use medical devices. The antibiotic resistance crisis has led to an increase in infections caused by these bacteria, which can develop into potentially life-threatening illnesses if not treated swiftly and effectively. Thus, new treatment options for Klebsiella are urgently required. Phage therapy can offer an alternative to ineffective antibiotic treatments for antibiotic-resistant bacteria infections. The aim of the present study was to produce a safe and effective phage cocktail treatment against Klebsiella pneumoniae and Klebsiella oxytoca, both in liquid in vitro culture and an in vivo Galleria mellonella infection model. The phage cocktail was significantly more effective at killing K. pneumoniae and K. oxytoca strains compared with monophage treatments. Preliminary phage cocktail safety was demonstrated through application in the in vivo G. mellonella model: where the phage cocktail induced no toxic side effects in G. mellonella. In addition, the phage cocktail significantly improved the survival of G. mellonella when administered as a prophylactic treatment, compared with controls. In conclusion, our phage cocktail was demonstrated to be safe and effective against Klebsiella spp. in the G. mellonella infection model. This provides a strong case for future treatment for Klebsiella infections, either as an alternative or adjunct to antibiotics. IMPORTANCE Klebsiella infections are a concern in individuals who are immunocompromised and are becoming increasingly difficult to treat with antibiotics due to their drug-resistant properties. Bacteriophage is one potential alternative therapy that could be used to tackle these infections. The present study describes the design of a non-toxic phage cocktail that improved the survival of Galleria mellonella infected with Klebsiella. This phage cocktail demonstrates potential for the safe and effective treatment of Klebsiella infections, as an adjunct or alternative to antibiotics. [ABSTRACT FROM AUTHOR]

ABSTRACT Infections caused by multidrug resistant (MDR) pathogenic bacteria are a global health threat. Bacteriophages (“phage”) are increasingly used as alternative or last-resort therapeutics to treat patients infected by MDR bacteria. However, the therapeutic outcomes of phage therapy may be limited by the emergence of phage resistance during treatment and/or by physical constraints that impede phage–bacteria interactions in vivo. In this work, we evaluate the role of lung spatial structure on the efficacy of phage therapy for Pseudomonas aeruginosa infections. To do so, we developed a spatially structured metapopulation network model based on the geometry of the bronchial tree, including host innate immune responses and the emergence of phage-resistant bacterial mutants. We model the ecological interactions between bacteria, phage, and the host innate immune system at the airway (node) level. The model predicts the synergistic elimination of a P. aeruginosa infection due to the combined effects of phage and neutrophils, given the sufficient innate immune activity and efficient phage-induced lysis. The metapopulation model simulations also predict that MDR bacteria are cleared faster at distal nodes of the bronchial tree. Notably, image analysis of lung tissue time series from wild-type and lymphocyte-depleted mice revealed a concordant, statistically significant pattern: infection intensity cleared in the bottom before the top of the lungs. Overall, the combined use of simulations and image analysis of in vivo experiments further supports the use of phage therapy for treating acute lung infections caused by P. aeruginosa, while highlighting potential limits to therapy in a spatially structured environment given impaired innate immune responses and/or inefficient phage-induced lysis.IMPORTANCEPhage therapy is increasingly employed as a compassionate treatment for severe infections caused by multidrug-resistant (MDR) bacteria. However, the mixed outcomes observed in larger clinical studies highlight a gap in understanding when phage therapy succeeds or fails. Previous research from our team, using in vivo experiments and single-compartment mathematical models, demonstrated the synergistic clearance of acute P. aeruginosa pneumonia by phage and neutrophils despite the emergence of phage-resistant bacteria. In fact, the lung environment is highly structured, prompting the question of whether immunophage synergy explains the curative treatment of P. aeruginosa when incorporating realistic physical connectivity. To address this, we developed a metapopulation network model mimicking the lung branching structure to assess phage therapy efficacy for MDR P. aeruginosa pneumonia. The model predicts the synergistic elimination of P. aeruginosa by phage and neutrophils but emphasizes potential challenges in spatially structured environments, suggesting that higher innate immune levels may be required for successful bacterial clearance. Model simulations reveal a spatial pattern in pathogen clearance where P. aeruginosa are cleared faster at distal nodes of the bronchial tree than in primary nodes. Interestingly, image analysis of infected mice reveals a concordant and statistically significant pattern: infection intensity clears in the bottom before the top of the lungs. The combined use of modeling and image analysis supports the application of phage therapy for acute P. aeruginosa pneumonia while emphasizing potential challenges to curative success in spatially structured in vivo environments, including impaired innate immune responses and reduced phage efficacy.

ABSTRACT Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

ABSTRACTClostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%–92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry.IMPORTANCEClostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

ABSTRACT Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient’s gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient’s antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections.IMPORTANCEPhage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

ABSTRACT In recent years, bacteriophages (or phages) have reentered the spotlight as alternative or adjuvant therapeutic agents to antibiotics. Their efficacy in more recalcitrant forms of infections like biofilms, frequently encountered in the orthopedic setting, remains less characterized. The present study aimed at evaluating the activity of phage-antibiotic combinations against Pseudomonas aeruginosa biofilms. A large collection of phages was de novo isolated from a wide variety of environmental sources. Three phages with a large host range against a.o. clinical orthopedic isolates, PSP2 (Yuavirus), PSP3 (Pbunavirus), and PSP30 (Bruynoghevirus), were selected for phage-antibiotic synergy assays on mature P. aeruginosa PAO1 biofilms. Phages were combined with ciprofloxacin, meropenem, and ceftazidime, all used as standard of care in bone-related infections. Significant reductions in both cell counts and biomass were observed for all phage-antibiotic combinations. The highest reduction in viable cell counts was observed for PSP3 and PSP2 in combination with ceftazidime [4.58 and 4.30 log10 Colony Forming Units (CFU)/well], followed by all phage combinations with ciprofloxacin (up to 3.56 log10 CFU/well). The highest reduction in biomass was obtained by combining PSP3 with ciprofloxacin (29.8%). Metabolic assays confirmed these findings with reductions in biofilm respiratory rate of up to 65%. Scanning electron microscopy imaging of PAO1 biofilms grown on titanium coupons and treated by ciprofloxacin and PSP30 confirmed the efficacy of the combined treatment with PSP30 and ciprofloxacin. This study highlights the synergetic effects of phage-antibiotic combinations on P. aeruginosa biofilms, thereby offering a promising approach to combat biofilm-associated infections. IMPORTANCE Biofilm-related infections are among the most difficult-to-treat infections in all fields of medicine due to their antibiotic tolerance and persistent character. In the field of orthopedics, these biofilms often lead to therapeutic failure of medical implantable devices and urgently need novel treatment strategies. This forthcoming article aims to explore the dynamic interplay between newly isolated bacteriophages and routinely used antibiotics and clearly indicates synergetic patterns when used as a dual treatment modality. Biofilms were drastically more reduced when both active agents were combined, thereby providing additional evidence that phage-antibiotic combinations lead to synergism and could potentially improve clinical outcome for affected patients.

ABSTRACTMycobacterium abscessus is a nontuberculous mycobacteria that contributes to the decline and death of patients with lung diseases such as cystic fibrosis and other muco-obstructive airway diseases. M. abscessus is challenging to treat due to its extensive antibiotic resistance and ability to survive inside mammalian cells. An alternative to antibiotics is the therapeutic use of bacteriophages (phages). There are recent cases of phage therapy being used to treat M. abscessus infections in people under compassionate-use conditions. However, little is known about the ability of phages to kill bacteria, such as M. abscessus, which reside in an intracellular environment. Here, we used M. abscessus strains and phages from recent phage therapy cases to determine if phages can enter mammalian cells and if they can infect and kill intracellular M. abscessus. Using fluorescence microscopy, we demonstrate phage uptake by macrophages and lung epithelial cells, and we further demonstrate phage infection of intracellular M. abscessus with fluorescent reporter phages. Transmission electron microscopy was additionally used to image phage infection of intracellular M. abscessus. Together, these findings provide the first visualizations of phage-M. abscessus interactions in an intracellular environment. Finally, we show that phage treatment can significantly reduce the intracellular burden of M. abscessus in a manner that depends on both the specific phage and mammalian cell type involved. These results demonstrate the potential to use phage therapy to treat intracellular bacteria, specifically M. abscessus, while also highlighting the importance of prescreening phage therapy candidates for activity in an intracellular environment.IMPORTANCEAs we rapidly approach a post-antibiotic era, bacteriophage (phage) therapy may offer a solution for treating drug-resistant bacteria. Mycobacterium abscessus is an emerging, multidrug-resistant pathogen that causes disease in people with cystic fibrosis, chronic obstructive pulmonary disease, and other underlying lung diseases. M. abscessus can survive inside host cells, a niche that can limit access to antibiotics. As current treatment options for M. abscessus infections often fail, there is an urgent need for alternative therapies. Phage therapy is being used to treat M. abscessus infections as an option of last resort. However, little is known about the ability of phages to kill bacteria in the host environment and specifically in an intracellular environment. Here, we demonstrate the ability of phages to enter mammalian cells and to infect and kill intracellular M. abscessus. These findings support the use of phages to treat intracellular bacterial pathogens.

ABSTRACT Phage therapy represents a promising strategy for curing bacterial infections refractory to antibiotics. However, the success rate of phage therapy may be lowered by the emergence of bacterial resistance to the phages used for therapy. In this work, we studied the resistance to the CK4 cocktail, which is a mixture composed of four phages able to cure Pseudomonas aeruginosa infections in animal models. CK4-resistant mutants were easily isolated from cultures grown in either a standard laboratory medium or an artificial sputum medium mimicking the composition of the airway fluid of cystic fibrosis (CF) patients, who are highly susceptible to P. aeruginosa chronic lung infections. In both cases, CK4-resistant mutants resulted in being defective in lipopolysaccharide (LPS) biosynthesis. Accordingly, all CK4 phages were unable to infect wzy mutants lacking the O-antigen polymerase. A survey of the other 15 P. aeruginosa phages isolated from different environmental sources showed that they all needed either wzy or the Type IV-pilus (T4P) biosynthetic gene pilQ for the infection. Overall, our data suggest that 16 out of the 19 analyzed Pseudomonas phages may use either the LPS or the T4P as a receptor. Interestingly, CK4-resistant mutants devoid of the O-antigen had strongly attenuated virulence in a zebrafish embryo infection model, and the lack of T4P also decreased virulence in zebrafish. With respect to isolates from patients with CF, phages not reproducing in the Δwzy mutant had a wider host range than those requiring pilQ, suggesting that phages dependent on PAO1-type T4P may have limited therapeutic value for treating CF-related infections. IMPORTANCE In this work, we identified the putative receptors of 16 Pseudomonas phages and evaluated how resistance to phages recognizing different bacterial receptors may affect the virulence. Our findings are relevant for the implementation of phage therapy of Pseudomonas aeruginosa infections, which are difficult to treat with antibiotics. Overall, our results highlight the need to modify natural phages to enlarge the repertoire of receptors exploited by therapeutic phages and suggest that phages using the PAO1-type T4P as receptor may have limited value for the therapy of the cystic fibrosis infection.

The Burkholderia cepacia complex (Bcc) causes life-threatening respiratory tract infections in persons with cystic fibrosis (CF). In CF patients, end-stage pulmonary disease often requires lung transplantation, and pre-transplant colonization with antibiotic-resistant Burkholderia is predictive of poor post-transplant outcomes. To address this issue, phage therapy has been proposed as a treatment for these infections. However, the majority of characterized Bcc phages are temperate and are therefore difficult to use as therapeutics, and the few obligately lytic phages that have been isolated have limited host ranges. To overcome these limitations, we have produced a virulent, broad-host range derivative of the temperate Burkholderia cenocepacia phage Milagro. Phage Milagro is a 39.1-kb temperate myophage related to phage KL3 and the paradigm coliphage P2. This phage showed a phenotype of spontaneous autoplaquing on lawns of Milagro lysogens, and these autoplaques were found to be produced by virulent mutants of the parental phage Milagro. Mutations associated with virulence were identified as single base changes, insertions or deletions in the phage lysogeny control region that define potential operator sites required for lysogen maintenance. To improve phage host range, the C-terminal domain of the Milagro tail fiber was replaced with the receptor-binding domain of the broad-host range tailocin (high molecular weight bacteriocin) BceTMilo. A spontaneous virulent mutant of this engineered phage, designated Milagro vir gp20:Milo, exhibited an expanded host range over the parental phage and is able to infect multiple Bcc species including B. cenocepacia, Burkholderia multivorans, Burkholderia gladioli, Burkholderia dolosa, and Burkholderia vietnamensis. [ABSTRACT FROM AUTHOR]

ABSTRACT Antimicrobial resistance is a danger to global public health and threatens many aspects of modern medicine. Bacterial species such as those of the Burkholderia cepacia complex (Bcc) cause life-threatening respiratory infections and are highly resistant to antibiotics. One promising alternative being explored to combat Bcc infections is phage therapy (PT): the use of phages to treat bacterial infections. Unfortunately, the utility of PT against many pathogenic species is limited by its prevailing paradigm: that only obligately lytic phages should be used therapeutically. It is thought that ‘lysogenic’ phages do not lyse all bacteria and can transfer antimicrobial resistance or virulence factors to their hosts. We argue that the tendency of a lysogenization-capable (LC) phage to form stable lysogens is not predicated exclusively on its ability to do so, and that the therapeutic suitability of a phage must be evaluated on a case-by-case basis. Concordantly, we developed several novel metrics—Efficiency of Phage Activity, Growth Reduction Coefficient, and Stable Lysogenization Frequency—and used them to evaluate eight Bcc-specific phages. Although these parameters vary considerably among Bcc phages, a strong inverse correlation (R2 = 0.67; P < 0.0001) exists between lysogen formation and antibacterial activity, indicating that certain LC phages with low frequency of stable lysogenization may be therapeutically efficacious. Moreover, we show that many LC Bcc phages interact synergistically with other phages in the first reported instance of mathematically defined polyphage synergy, and that these interactions result in the eradication of in vitro bacterial growth. Together, these findings reveal a novel therapeutic role for LC phages and challenge the current paradigm of PT. IMPORTANCE The spread of antimicrobial resistance is an imminent threat to public health around the world. Particularly concerning are species of the Burkholderia cepacia complex (Bcc), which cause life-threatening respiratory infections and are notoriously resistant to antibiotics. Phage therapy is a promising alternative being explored to combat Bcc infections and antimicrobial resistance in general, but its utility against many pathogenic species, including the Bcc, is restricted by the currently prevailing paradigm of exclusively using rare obligately lytic phages due to the perception that ‘lysogenic’ phages are therapeutically unsuitable. Our findings show that many lysogenization-capable phages exhibit powerful in vitro antibacterial activity both alone and through mathematically defined synergistic interactions with other phages, demonstrating a novel therapeutic role for LC phages and therefore challenging the currently prevailing paradigm of PT.

ABSTRACT The increase in antibiotic-resistant avian-pathogenic Escherichia coli (APEC), the causative agent of colibacillosis in poultry, warrants urgent research and the development of alternative therapies. This study describes the isolation and characterization of 19 genetically diverse, lytic coliphages, 8 of which were tested in combination for their efficacy in controlling in ovo APEC infections. Genome homology analysis revealed that the phages belong to nine different genera, one of them being a novel genus (Nouzillyvirus). One phage, REC, was derived from a recombination event between two Phapecoctavirus phages (ESCO5 and ESCO37) isolated in this study. Twenty-six of the 30 APEC strains tested were lysed by at least one phage. Phages exhibited varying infectious capacities, with narrow to broad host ranges. The broad host range of some phages could be partially explained by the presence of receptor-binding protein carrying a polysaccharidase domain. To demonstrate their therapeutic potential, a phage cocktail consisting of eight phages belonging to eight different genera was tested against BEN4358, an APEC O2 strain. In vitro, this phage cocktail fully inhibited the growth of BEN4358. In a chicken lethality embryo assay, the phage cocktail enabled 90% of phage-treated embryos to survive infection with BEN4358, compared with 0% of nontreated embryos, indicating that these novel phages are good candidates to successfully treat colibacillosis in poultry. IMPORTANCE Colibacillosis, the most common bacterial disease affecting poultry, is mainly treated by antibiotics. Due to the increased prevalence of multidrug-resistant avian-pathogenic Escherichia coli, there is an urgent need to assess the efficacy of alternatives to antibiotherapy, such as phage therapy. Here, we have isolated and characterized 19 coliphages that belong to nine phage genera. We showed that a combination of 8 of these phages was efficacious in vitro to control the growth of a clinical isolate of E. coli. Used in ovo, this phage combination allowed embryos to survive APEC infection. Thus, this phage combination represents a promising treatment for avian colibacillosis.

ABSTRACT The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis-infecting phage, Lacachita, and compare its genome with those of five other phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but Lacachita is capable of transducing antibiotic resistance and conferring phage resistance to transduced cells. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that Lacachita may be temperate and members of this novel genus are not suitable for phage therapy. IMPORTANCE This project describes the discovery of a culturable bacteriophage infecting Staphylococcus epidermidis that is a member of a rapidly growing novel siphovirus genus. A member of this genus was recently characterized and proposed for phage therapy, as there are few phages currently available to treat S. epidermidis infections. Our data contradict this, as we show Lacachita is capable of moving DNA from one bacterium to another, and it may be capable of maintaining itself in a plasmid-like state in infected cells. These phages’ putative plasmid-like extrachromosomal state appears to be due to a simplified maintenance mechanism found in true plasmids of Staphylococcus and related hosts. We suggest Lacachita and other identified members of this novel genus are not suitable for phage therapy.

Lysogenic bacteriophages can integrate their genome into the bacterial chromosome in the form of a prophage and can promote genetic transfer between bacterial strains in vitro . However, the contribution of lysogenic bacteriophages to the incidence of antimicrobial resistance (AMR) in clinical settings is poorly understood. Here, in a set of 186 clinical isolates of Pseudomonas aeruginosa collected from respiratory cultures from 82 patients with cystic fibrosis, we evaluate the links between prophage counts and both genomic and phenotypic resistance to six anti-pseudomonal antibiotics: tobramycin, colistin, ciprofloxacin, meropenem, aztreonam, and piperacillin-tazobactam. We identified 239 different prophages in total. We find that P. aeruginosa isolates contain on average 3.06 ± 1.84 (SD) predicted prophages. We find no significant association between the number of prophages per isolate and the minimum inhibitory concentration for any of these antibiotics. We then investigate the relationship between particular prophages and AMR. We identify a single lysogenic phage associated with phenotypic resistance to the antibiotic tobramycin and, consistent with this association, we observe that AMR genes associated with resistance to tobramycin are more likely to be found when this prophage is present. However, we find that they are not encoded directly on prophage sequences. Additionally, we identify a single prophage statistically associated with ciprofloxacin resistance but do not identify any genes associated with ciprofloxacin phenotypic resistance. These findings suggest that prophages are only infrequently associated with the AMR genes in clinical isolates of P. aeruginosa .IMPORTANCEAntibiotic-resistant infections of Pseudomonas aeruginosa ( Pa ), a leading pathogen in patients with cystic fibrosis (CF), are a global health threat. While lysogenic bacteriophages are known to facilitate horizontal gene transfer, their role in promoting antibiotic resistance in clinical settings remains poorly understood. In our analysis of 186 clinical isolates of P. aeruginosa from CF patients, we find that prophage abundance does not predict phenotypic resistance to key antibiotics but that specific prophages are infrequently associated with tobramycin resistance genes. In addition, we do not find antimicrobial resistance (AMR) genes encoded directly on prophages. These results highlight that while phages can be associated with AMR, phage-mediated AMR transfer may be rare in clinical isolates and difficult to identify. This work is important for future efforts on mitigating AMR in CFCF and other vulnerable populations affected by Pa infections and advances our understanding of bacterial-phage dynamics in clinical infections.

Ashwin and Chanagan are double-stranded siphoviral mycobacteriophages isolated from a single soil sample from a lavender flowerpot in Johannesburg, South Africa. Both are capable of infecting and lyzing Mycobacterium smegmatis and are predicted to contain 122 and 91 open reading frames, respectively, most without known function., Competing Interests: The authors declare no conflict of interest.

ABSTRACT The mosquito microbiota has a profound impact on multiple biological processes ranging from reproduction to disease transmission. Interestingly, the adult mosquito microbiota is largely derived from the larval microbiota, which in turn is dependent on the microbiota of their water habitat. The larval microbiota not only plays a crucial role in larval development but also has a significant impact on the adult stage of the mosquito. By precisely engineering the larval microbiota, it is feasible to alter larval development and other life history traits of the mosquitoes. Bacteriophages, given their host specificity, can serve as a tool for modulating the microbiota. For this proof-of-principle study, we selected representative strains of five common Anopheles mosquito-associated bacterial genera, namely, Enterobacter, Serratia, Pseudomonas, Elizabethkingia, and Asaia. Our results with monoaxenic cultures showed that Anopheles larvae with Enterobacter and Pseudomonas displayed normal larval development with no significant mortality. However, monoaxenic Anopheles larvae with Elizabethkingia showed delayed larval development and higher mortality. Serratia and Asaia gnotobiotic larvae failed to develop past the first instar. We isolated and characterized three novel bacteriophages (EP1, SP1, and EKP1) targeting Enterobacter, Serratia, and Elizabethkingia, respectively, and utilized a previously characterized bacteriophage (GH1) targeting Pseudomonas to modulate larval water microbiota. Gnotobiotic Anopheles larvae with all five bacterial genera showed reduced survival and larval development with the addition of bacteriophages EP1 and GH1, targeting Enterobacter and Pseudomonas, respectively. The effect was synergistic when both EP1 and GH1 were added together. Our results demonstrate a novel application of bacteriophages for mosquito control. IMPORTANCE Mosquitoes are efficient vectors of multiple human and animal pathogens. The biology of mosquitoes is strongly affected by their associated microbiota. Because of the important role of the larval microbiota in mosquito biology, the microbiota can potentially serve as a target for altering mosquito life-history traits. Our study provides proof of principle that bacteriophages can be used as tools to modulate the mosquito larval habitat microbiota and can, in turn, affect larval development and survival. These results highlight the utility of bacteriophages in mosquito microbiota research and also provide a new potential mosquito control tool.

ABSTRACT Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.

ABSTRACT Nontyphoidal Salmonella spp. are a leading cause of human food poisoning and can be transmitted to humans via consuming contaminated pork. To reduce Salmonella spread to the human food chain, bacteriophage (phage) therapy could be used to reduce bacteria from animals’ preslaughter. We aimed to determine if adding a two-phage cocktail to feed reduces Salmonella colonization in piglets. This first required spray drying phages to allow them to be added as a powder to feed, and phages were spray dried in different excipients to establish maximum recovery. Although laboratory phage yields were not maintained during scale up in a commercial spray dryer (titers fell from 3 × 108 to 2.4 × 106 PFU/g respectively), the phage titers were high enough to progress. Spray dried phages survived mixing and pelleting in a commercial feed mill, and sustained no further loss in titer when stored at 4°C or barn conditions over 6 months. Salmonella-challenged piglets that were prophylactically fed the phage-feed diet had significantly reduced Salmonella colonization in different gut compartments (P

Phage-based biocontrol is an emerging method for managing the plant pathogen Erwinia amylovora. Control of E. amylovora in North America is achieved chiefly through the application of streptomycin and has led to the development of streptomycin resistance. Resistant E. amylovora can be tracked through the analysis of CRISPR spacer sequences. An alternative to antibiotics are bacterial viruses, known as phages, which lyse their hosts during replication to control the bacterial population. Endogenous CRISPR-Cas systems act as phage resistance mechanisms however, preliminary genomic analysis suggests this activity is limited in E. amylovora. This leaves the functionality of the CRISPR-Cas system, any clade-based differences, and the impact which this system may have on phage-based biocontrol in question. In this study, the CRISPR arrays from 127 newly available genomic sequences of E. amylovora were analyzed through a novel bioinformatic pipeline. Through this, the Eastern and Western North American clades were shown to be incompatible with the current PCR-based approaches for tracking E. amylovora given the size and composition of their CRISPR arrays. Two artificial CRISPR arrays were designed to investigate the functionality of the CRISPR-Cas system in E. amylovora. This system was capable of curing a targeted plasmid and providing phage resistance but was not the source of phage resistance observed within the controls. This suggests that while the CRISPR-Cas system is an important defense mechanism for invasive plasmids, an as yet unidentified mechanism is the primary source of phage resistance in E. amylovora. [ABSTRACT FROM AUTHOR]

Extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli have disseminated in food-producing animals globally, attributed to horizontal transmission of blaCTX-M variants, as seen in the InCI1-blaCTX-M-1 plasmid. This ease of transmission, coupled with its demonstrated long-term persistence, presents a significant One Health antimicrobial resistance (AMR) risk. Bacteriophage (phage) therapy is a potential strategy in eliminating ESC-R E. coli in food-producing animals; however, it is hindered by the development of phage-resistant bacteria and phage biosafety concerns. Another alternative to antimicrobials is probiotics, with this study demonstrating that AMR-free commensal E. coli, termed competitive exclusion clones (CECs), can be used to competitively exclude ESC-R E. coli. This study isolated and characterized phages that lysed E. coli clones harboring the InCI1-blaCTX-M-1 plasmid, before investigation of the effect and synergy of phage therapy and competitive exclusion as a novel strategy for decolonizing ESC-resistant E. coli. In vitro testing demonstrated superiority in the combined therapy, reducing and possibly eliminating ESC-R E. coli through phage-mediated lysis coupled with simultaneous prevention of regrowth of phage-resistant mutants due to competitive exclusion with the CEC. Further investigation into this combined therapy in vivo is warranted, with on-farm application possibly reducing ESC-R prevalence, while constricting newly emergent ESC-R E. coli outbreaks prior to their dissemination throughout food-producing animals or humans. [ABSTRACT FROM AUTHOR]

The genomes of three Pseudomonas aeruginosa Phikzvirus bacteriophages isolated in Kenya are described. The genomes of phages vB&#95;PaePAO1-KEN19, vB&#95;Pae3705-KEN49, and vB&#95;Pae10145-KEN51, respectively, had lengths of 278,921, 280,231, and 280,173 bp, with 36.93%, 36.84%, and 36.86% GC content, containing 419, 417, and 417 coding sequences (including seven tRNAs in each genome)., Competing Interests: The authors declare no conflict of interest.

We report the genome sequences of 12 Enterococcus faecalis phages isolated in Kenya, belonging to the genus Copernicusvirus , Efquatrovirus , Saphexavirus , and Kochikohdavirus . They have double-stranded DNA with lengths varying from 17,979 to 147,374 bp and G+C content from 33.14% to 40.05%. The genomes contain 28-250 coding sequences., Competing Interests: The authors declare no conflict of interest.

SUMMARYDespite the early recognition of their therapeutic potential and the current escalation of multidrug-resistant (MDR) pathogens, the adoption of bacteriophages into mainstream clinical practice is hindered by unfamiliarity with their basic pharmacokinetic (PK) and pharmacodynamic (PD) properties, among others. Given the self-replicative nature of bacteriophages in the presence of host bacteria, the adsorption rate, and the clearance by the host's immunity, their PK/PD characteristics cannot be estimated by conventional approaches, and thus, the introduction of new considerations is required. Furthermore, the multitude of different bacteriophage types, preparations, and treatment schedules impedes drawing general conclusions on their in vivo PK/PD features. Additionally, the drawback of acquired bacteriophage resistance of MDR pathogens with clinical and environmental implications should be taken into consideration. Here, we provide an overview of the current state of the field of PK and PD of bacteriophage therapy with a focus on its application against MDR Gram-negative infections, highlighting the potential knowledge gaps and the challenges in translation from the bench to the bedside. After reviewing the in vitro PKs and PDs of bacteriophages against the four major MDR Gram-negative pathogens, Klebsiella pneumoniae , Acinetobacter baumannii complex, Pseudomonas aeruginosa , and Escherichia coli , specific data on in vivo PKs (tissue distribution, route of administration, and basic PK parameters in animals and humans) and PDs (survival and reduction of bacterial burden in relation to the route of administration, timing of therapy, dosing regimens, and resistance) are summarized. Currently available data merit close scrutiny, and optimization of bacteriophage therapy in the context of a better understanding of the underlying PK/PD principles is urgent to improve its therapeutic effect and to minimize the occurrence of bacteriophage resistance., Competing Interests: The authors declare no conflict of interest.

Burn wounds are a major burden, with high mortality rates due to infections. Staphylococcus aureus is a major causative agent of burn wound infections, which can be difficult to treat because of antibiotic resistance and biofilm formation. An alternative to antibiotics is the use of bacteriophages, viruses that infect and kill bacteria. We investigated the efficacy of bacteriophage therapy for burn wound infections, in both a porcine and a newly developed human ex vivo skin model. In both models, the efficacy of a reference antibiotic treatment (fusidic acid) and bacteriophage treatment was determined for a single treatment, successive treatment, and prophylaxis. Both models showed a reduction in bacterial load after a single bacteriophage treatment. Increasing the frequency of bacteriophage treatments increased bacteriophage efficacy in the human ex vivo skin model, but not in the porcine model. In both models, prophylaxis with bacteriophages increased treatment efficacy. In all cases, bacteriophage treatment outperformed fusidic acid treatment. Both models allowed investigation of bacteriophage-bacteria dynamics in burn wounds. Overall, bacteriophage treatment outperformed antibiotic control underlining the potential of bacteriophage therapy for the treatment of burn wound infections, especially when used prophylactically., Competing Interests: The authors declare no conflict of interest.

ABSTRACT In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffer from batch-to-batch variations and require manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans, which feeds on bacteria, a fast growing and easy to handle organism that can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae, and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae, and EHEC/EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages. IMPORTANCE Validating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism C. elegans as a quick, reliable, and simple alternative for testing phages. The data show that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from 90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

ABSTRACT Rising antimicrobial resistance severely limits efforts to treat infections and is a cause for critical concern. Renewed interest in bacteriophage therapy has advanced understanding of the breadth of species capable of targeting bacterial antimicrobial resistance mechanisms, but many questions concerning ideal application remain unanswered. The following minireview examines bacterial resistance mechanisms, the current state of bacteriophage therapy, and how bacteriophage therapy can augment strategies to combat resistance with a focus on the clinically relevant bacterium Pseudomonas aeruginosa, as well as the role of efflux pumps in antimicrobial resistance. Methods to prevent antimicrobial efflux using efflux pump inhibitors and phage steering, a type of bacteriophage therapy, are also covered. The evolutionary context underlying antimicrobial resistance and the need to include theory in the ongoing development of bacteriophage therapy are also discussed.

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a comprehensive view of lysins' structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G2) (higher net charge and amphipathic helices) are responsible for improved interaction with the G2 envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecule design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G2 surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages' host bacteria, and sequence-predicted physicochemical properties. Based on such classifiers, an investigation of the differential appearance of certain features was conducted. This analysis revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. In particular, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment, and aliphatic index) were associated with G2 phage lysins, appearing specifically at their C-terminal end. Information on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts is provided, specifically supporting the nowadayscommon hypothesis that lysins from G2 usually contain AMP-like regions. IMPORTANCE Phage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The potential of lysins as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysin diversity landscape by examining a set of phage lysin genes. We have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures and, thus, the reach of their specialization regarding cell wall structures. These results provide clarity and evidence to sustain some of the common hypotheses in current literature, as well as making available an updated and characterized database of lysins sequences for further developments. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K-12 strain, but not of the O157 strain, for adsorption, even though OmpCs from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in loop 4 of the K-12 OmpC as essential for T4 phage adsorption in the copresence of loops 1 and 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the distal tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K-12 strain. These results shed light on the mechanism underlying the phage host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential health care applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (chi) infects serovars of the pathogenic enterobacterium Salmonella enterica. However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multigene deletion library of S. enterica serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multigene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. In this way, we identified motile mutants with various degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multidrug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an acrABtolC triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene tig, encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and inform research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections. [ABSTRACT FROM AUTHOR]

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae. In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus. Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio. A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by .90% (P,0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries. IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade. [ABSTRACT FROM AUTHOR]

Delivery of therapeutic compounds to the site of action is crucial. While many chemical substances such as beta-lactam antibiotics can reach therapeutic levels in most parts throughout the human body after administration, substances of higher molecular weight such as therapeutic proteins may not be able to reach the site of action (e.g., an infection) and are therefore ineffective. In the case of therapeutic phages, i.e., viruses that infect microbes, to treat bacterial infections, this problem is exacerbated; not only are phages unable to penetrate tissues, but phage particles can be cleared by the immune system, and phage proteins are rapidly degraded by enzymes or inactivated by the low pH in the stomach. Yet, the use of therapeutic phages is a highly promising strategy, in particular for infections caused by bacteria that exhibit multidrug resistance. Clinicians increasingly encounter situations where no treatment options remain available for such infections where antibiotic compounds are ineffective. While the number of drug-resistant pathogens continues to rise due to the overuse and misuse of antibiotics, no new compounds are becoming available, as many pharmaceutical companies discontinue their search for chemical antimicrobials. In recent years, phage therapy has undergone massive innovation for the treatment of infections caused by pathogens resistant to conventional antibiotics. While most therapeutic applications of phages are well described in the literature, other aspects of phage therapy are less well-documented. In this review, we focus on the issues that are critical for phage therapy to become a reliable standard therapy and describe methods for efficient and targeted delivery of phages, including their encapsulation. [ABSTRACT FROM AUTHOR]

ABSTRACT The spread of multidrug-resistant (MDR) bacteria is a global public health crisis. Bacteriophage therapy (or “phage therapy”) constitutes a potential alternative approach to treat MDR infections. However, the effective use of phage therapy may be limited when phage-resistant bacterial mutants evolve and proliferate during treatment. Here, we develop a nonlinear population dynamics model of combination therapy that accounts for the system-level interactions between bacteria, phage, and antibiotics for in vivo application given an immune response against bacteria. We simulate the combination therapy model for two strains of Pseudomonas aeruginosa, one which is phage sensitive (and antibiotic resistant) and one which is antibiotic sensitive (and phage resistant). We find that combination therapy outperforms either phage or antibiotic alone and that therapeutic effectiveness is enhanced given interaction with innate immune responses. Notably, therapeutic success can be achieved even at subinhibitory concentrations of antibiotics, e.g., ciprofloxacin. These in silico findings provide further support to the nascent application of combination therapy to treat MDR bacterial infections, while highlighting the role of innate immunity in shaping therapeutic outcomes. IMPORTANCE This work develops and analyzes a novel model of phage-antibiotic combination therapy, specifically adapted to an in vivo context. The objective is to explore the underlying basis for clinical application of combination therapy utilizing bacteriophage that target antibiotic efflux pumps in Pseudomonas aeruginosa. In doing so, the paper addresses three key questions. How robust is combination therapy to variation in the resistance profiles of pathogens? What is the role of immune responses in shaping therapeutic outcomes? What levels of phage and antibiotics are necessary for curative success? As we show, combination therapy outperforms either phage or antibiotic alone, and therapeutic effectiveness is enhanced given interaction with innate immune responses. Notably, therapeutic success can be achieved even at subinhibitory concentrations of antibiotic. These in silico findings provide further support to the nascent application of combination therapy to treat MDR bacterial infections, while highlighting the role of system-level feedbacks in shaping therapeutic outcomes.

ABSTRACT The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro. Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design. IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

ABSTRACT Metazoans were proposed to host bacteriophages on their mucosal surfaces in a symbiotic relationship, where phages provide an external immunity against bacterial infections and the metazoans provide phages a medium for interacting with bacteria. However, scarce empirical evidence and model systems have left the phage-mucus interaction poorly understood. Here, we show that phages bind both to porcine mucus and to rainbow trout (Oncorhynchus mykiss) primary mucus, persist up to 7 days in the mucosa, and provide protection against Flavobacterium columnare. Also, exposure to mucus changes the bacterial phenotype by increasing bacterial virulence and susceptibility to phage infections. This trade-off in bacterial virulence reveals ecological benefit of maintaining phages in the metazoan mucosal surfaces. Tests using other phage-bacterium pairs suggest that phage binding to mucus may be widespread in the biosphere, indicating its importance for disease, ecology, and evolution. This phenomenon may have significant potential to be exploited in preventive phage therapy. IMPORTANCE The mucosal surfaces of animals are habitat for microbes, including viruses. Bacteriophages—viruses that infect bacteria—were shown to be able to bind to mucus. This may result in a symbiotic relationship in which phages find bacterial hosts to infect, protecting the mucus-producing animal from bacterial infections in the process. Here, we studied phage binding on mucus and the effect of mucin on phage-bacterium interactions. The significance of our research is in showing that phage adhesion to mucus results in preventive protection against bacterial infections, which will serve as basis for the development of prophylactic phage therapy approaches. Besides, we also reveal that exposure to mucus upregulates bacterial virulence and that this is exploited by phages for infection, adding one additional layer to the metazoan-bacterium-phage biological interactions and ecology. This phenomenon might be widespread in the biosphere and thus crucial for understanding mucosal diseases, their outcome and treatment.

ABSTRACT The competition between bacteria often involves both nutrients and phage predators and may give rise to abrupt regime shifts between the alternative stable states characterized by different species compositions. While such transitions have been previously studied in the context of competition for nutrients, the case of phage-induced bistability between competing bacterial species has not been considered yet. Here we demonstrate a possibility of regime shifts in well-mixed phage-bacterium ecosystems. In one of the bistable states, the fast-growing bacteria competitively exclude the slow-growing ones by depleting their common nutrient. Conversely, in the second state, the slow-growing bacteria with a large burst size generate such a large phage population that the other species cannot survive. This type of bistability can be realized as the competition between a strain of bacteria protected from phage by abortive infection and another strain with partial resistance to phage. It is often desirable to reliably control the state of microbial ecosystems, yet bistability significantly complicates this task. We discuss successes and limitations of one control strategy in which one adds short pulses to populations of individual species. Our study proposes a new type of phage therapy, where introduction of the phage is supplemented by the addition of a partially resistant host bacteria. IMPORTANCE Phage-microbe communities play an important role in human health as well as natural and industrial environments. Here we show that these communities can assume several alternative species compositions separated by abrupt regime shifts. Our model predicts these regime shifts in the competition between bacterial strains protected by two different phage defense mechanisms: abortive infection/CRISPR and partial resistance. The history dependence caused by regime shifts greatly complicates the task of manipulation and control of a community. We propose and study a successful control strategy via short population pulses aimed at inducing the desired regime shifts. In particular, we predict that a fast-growing pathogen could be eliminated by a combination of its phage and a slower-growing susceptible host.

ABSTRACT Phage therapy is a promising alternative to chemotherapeutic antibiotics for the treatment of bacterial infections. However, despite recent clinical uses of combinations of phages to treat multidrug-resistant infections, a mechanistic understanding of how bacteria evolve resistance against multiple phages is lacking, limiting our ability to deploy phage combinations optimally. Here, we show, using Pseudomonas aeruginosa and pairs of phages targeting shared or distinct surface receptors, that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance. Whereas sequential exposure allowed bacteria to acquire multiple resistance mutations effective against both phages, this evolutionary trajectory was prevented by simultaneous exposure, resulting in quantitatively weaker resistance. The order of phage exposure determined the fitness costs of sequential resistance, such that certain sequential orders imposed much higher fitness costs than the same phage pair in the reverse order. Together, these data suggest that phage combinations can be optimized to limit the strength of evolved resistances while maximizing their associated fitness costs to promote the long-term efficacy of phage therapy. IMPORTANCE Globally rising rates of antibiotic resistance have renewed interest in phage therapy where combinations of phages have been successfully used to treat multidrug-resistant infections. To optimize phage therapy, we first need to understand how bacteria evolve resistance against combinations of multiple phages. Here, we use simple laboratory experiments and genome sequencing to show that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance evolution in the opportunistic pathogen Pseudomonas aeruginosa. These findings suggest that phage combinations can be optimized to limit the emergence and persistence of resistance, thereby promoting the long-term usefulness of phage therapy.

Two bacteriophages (phages) of Klebsiella pneumoniae were isolated from sewage water collected from Dakar, Senegal. Phage vKpIN17 belongs to the Przondovirus genus within the Autographiviridae family, with double-stranded DNA genomes, whereas vKpIN18 belongs to the Webervirus genus of the Drexlerviridae family., Competing Interests: The authors declare no conflict of interest.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening microorganisms for global human health. The current strategies to reduce the impact of S. aureus include a restrictive control of worldwide antibiotic use, prophylactic measures to hinder contamination, and the search for novel antimicrobials to treat human and animal infections caused by this bacterium. The last strategy is currently the focus of considerable research. In this regard, phage lytic proteins (endolysins and virion-associated peptidoglycan hydrolases [VAPGHs]) have been proposed as suitable candidates. Indeed, these proteins display narrow-spectrum antimicrobial activity and a virtual lack of bacterial-resistance development. Additionally, the therapeutic use of phage lytic proteins in S. aureus animal infection models is yielding promising results, showing good efficacy without apparent side effects. Nonetheless, human clinical trials are still in progress, and data are not available yet. This minireview also analyzes the main obstacles for introducing phage lytic proteins as human therapeutics against S. aureus infections. Besides the common technological problems derived from large-scale production of therapeutic proteins, a major setback is the lack of a proper legal framework regulating their use. In that sense, the relevant health authorities should urgently have a timely discussion about these new antimicrobials. On the other hand, the research community should provide data to dispel any doubts regarding their efficacy and safety. Overall, the appropriate scientific data and regulatory framework will encourage pharmaceutical companies to invest in these promising antimicrobials.

ABSTRACT Drug-resistant staphylococci, particularly Staphylococcus aureus and Staphylococcus epidermidis, are leading causes of hospital-acquired infections. Bacteriophages and their peptidoglycan hydrolytic enzymes (lysins) are currently being explored as alternatives to conventional antibiotics; however, only a limited diversity of staphylococcal phages and their lysins has yet been characterized. Here, we describe a novel staphylococcal phage and its lysins. Bacteriophage Andhra is the first reported S. epidermidis phage belonging to the family Podoviridae. Andhra possesses an 18,546-nucleotide genome with 20 open reading frames. BLASTp searches revealed that gene product 10 (gp10) and gp14 harbor putative catalytic domains with predicted peptidase and amidase activities, characteristic functions of phage lysins. We purified these proteins and show that both Andhra_gp10 and Andhra_gp14 inhibit growth and degrade cell walls of diverse staphylococci, with Andhra_gp10 exhibiting more robust activity against the panel of cell wall substrates tested. Site-directed mutagenesis of its predicted catalytic residues abrogated the activity of Andhra_gp10, consistent with the presence of a catalytic CHAP domain on its C terminus. The active site location combined with the absence of an SH3b cell wall binding domain distinguishes Andhra_gp10 from the majority of staphylococcal lysins characterized to date. Importantly, close homologs of Andhra_gp10 are present in related staphylococcal podophages, and we propose that these constitute a new class of phage-encoded lysins. Altogether, our results reveal insights into the biology of a rare family of staphylococcal phages while adding to the arsenal of antimicrobials with potential for therapeutic use. IMPORTANCE The spread of antibiotic resistance among bacterial pathogens is inciting a global public health crisis. Drug-resistant Staphylococcus species, especially S. aureus and S. epidermidis, have emerged in both hospital and community settings, underscoring the urgent need for new strategies to combat staphylococcal infections. Bacterial viruses (phages) and the enzymes that they use to degrade bacterial cell walls (lysins) show promise as alternative antimicrobials; however, only a limited variety of staphylococcal phages and their lysins have yet been identified. Here, we report the discovery and characterization of a novel staphylococcal phage, Andhra. We show that Andhra encodes two lysins (Andhra_gp10 and Andhra_gp14) that inhibit growth and degrade the cell walls of diverse staphylococci, including S. aureus and S. epidermidis strains. Andhra and its unique lysins add to the arsenal of antimicrobials with potential for therapeutic use.

The ubiquitous bacterial pathogen Pseudomonas aeruginosa is responsible for severe infections in patients with burns, cystic fibrosis, and neutropenia. Biofilm formation gives physical refuge and a protected microenvironment for sessile cells, rendering cure by antibiotics a challenge. Bacteriophages have evolved to prey on these biofilms over millions of years, using hydrolases and depolymerases to penetrate biofilms and reach cellular targets. Here, we assessed how a newly discovered KMV-like phage (ΦJB10) interacts with antibiotics to treat P. aeruginosa more effectively in both planktonic and biofilm forms. By testing representatives of four classes of antibiotics (cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems), we demonstrated class-dependent interactions between ΦJB10 and antibiotics in both biofilm clearance and P. aeruginosa killing. Despite identifying antagonism between some antibiotic classes and ΦJB10 at early time points, all classes showed neutral to favorable interactions with the phage at later time points. In one notable example where the antibiotic alone had poor activity against both biofilm and high-density planktonic cells, we found that addition of ΦJB10 demonstrated synergy and resulted in effective treatment of both. Further, ΦJB10 seemed to act as an adjuvant to several antibiotics, reducing the concentration of antibiotics required to ablate the biofilm. This report shows that phages such as ΦJB10 may be valuable additions to the armamentarium against difficult-to-treat biofilm-based infections., Competing Interests: The authors declare no conflict of interest.

Recurrent urinary tract infections (UTIs) are common in kidney transplant recipients, and novel prevention approaches are needed. The case presented by Le et al. (Antimicrob Agents Chemother, in press) describes a patient with recurrent UTIs due to extended-spectrum β-lactamase-producing Klebsiella pneumoniae who was successfully treated with bacteriophage therapy. This commentary highlights the potential for bacteriophage therapy to prevent recurrent UTIs, as well as outstanding questions that require further investigation., Competing Interests: The authors declare a conflict of interest. J.R.L. holds patent US-2020-0048713-A1 titled “Methods of Detecting Cell-Free DNA in Biological Samples” licensed to Eurofins Viracor, received research support under an investigator-initiated research grant from BioFire Diagnostics, LLC, and receives speakers' fees from Astellas. M.J.S. has received fees for consulting for Shionogi, for participating in a Data Safety Monitoring Board for Abbvie, and has received research funding from SNIPRBiome, Merck, and bioMerieux.