Bacteriophage-antibiotic coadministration is increasingly used for refractory infections, yet the in vivo interactions among phages, bacteria, antibiotics, and host immunity remain poorly defined. We report a longitudinal, multiomic case analysis of a male in his seventies with cystic fibrosis (CF) experiencing an acute-on-chronic pulmonary exacerbation caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. After colistin discontinuation due to nephrotoxicity, ciprofloxacin was initiated, with an intravenous two-phage cocktail introduced days later. Distinct mucoid and nonmucoid bacterial subpopulations associated differentially with antibiotic versus phage exposure, consistent with nonoverlapping selective pressures. Phage activity was temporally constrained, with one phage dominating early bacterial and genomic signals before attenuating after approximately seven days, despite continued genomic detectability. In contrast, the second phage showed no evidence of productive activity. This asymmetry coincided with phage-reactive humoral immunity: pre-existing IgM was associated with lack of recoverability of one phage, while treatment-associated IgM emergence temporally tracked attenuation of the dominant phage. Although phage-resistant variants arose during therapy, they showed limited expansion relative to susceptible populations. These findings define a mechanistic framework-chemobiotherapy-in which chemical and biological antimicrobials coordinate through ecological and immunologic complementarity rather than direct pharmacologic synergy.

Accurate quantitation of therapeutic bacteriophages (phages) remains a challenge for clinical development. Plaque-based enumeration is the current standard but is laborious, host-dependent, and variable, particularly when distinguishing individual phages in cocktails. Targeted mass spectrometry of virion structural proteins offers an orthogonal, structure-based approach amenable to reproducible and scalable phage quantitation. Here, we describe a targeted proteomic liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for host-independent quantitation of the Pseudomonas aeruginosa podovirus LUZ19. Proteomic characterization was performed on an LTQ Orbitrap XL to assess sequence coverage and select surrogate peptide candidates based on specificity and sensitivity. High-resolution peptide mapping identified multiple structural proteins of LUZ19 and provided 55% sequence coverage for the major head protein (YP_001671977.1). Fifteen peptides were detected and evaluated, from which the tryptic peptide EVAELDGQELAR was selected based on abundance, stability, and chromatographic performance. Quantitative analysis was conducted on a QTRAP 7500+ using optimized multiple reaction monitoring transitions for targeted peptide detection. Back-calculated concentrations met accuracy criteria across a validated range of 0.008 to 80 pg/mL, with bias spanning -8.2 to 8.2%, intra-day precision ranging from 0.5 to 9.8%, and inter-day precision ranging from 6.3 to 9.7%. Peptide concentrations from digested lysate samples were related to phage concentrations determined by double layer agar assay, yielding an estimated three copies of the major head protein per virion.

Background:There is a resurgence of interest in bacteriophage (phage) therapy as antimicrobials, resulting from growing antimicrobial resistance to small-molecule antibiotics. Phages are bacterial viruses long studied, but there is a need for high resolution and systematic assessment of clinical dosing strategies for phages to better inform therapy. Methods:We hypothesized that empirical in vitro assessment of clinically relevant phages facilitates pharmacodynamic-driven individualization. Three clinically relevant phage strains (LUZ19, PYO2 and E215) were evaluated as mono- or dual-phage therapy against a clinical Pseudomonas aeruginosa in 24 h static time kills and in 7-day hollow fibre infection model. Results:PYO2 single-bolus administration achieved a bacterial log reduction of 6.82 log10 cfu/mL, with eradication at 4 h. Dual-phage therapy (LUZ19 + PYO2) achieved a bacterial log reduction of 6.81 log10 cfu/mL, with delayed eradication at 12 h. Conclusions:This highlights the potential of reverse translational pharmacokinetic/pharmacodynamic-driven approaches to guide rational phage selection strategies against individual clinical isolates while identifying potential antagonistic phage-phage interactions.

Nosocomial Pseudomonas aeruginosa infections are among the most challenging infections to treat, and resistance to last-line agents, including polymyxins and aztreonam-based therapies, risks a future with limited or no clinical treatment options. Bacteriophages (phages) have emerged as a promising therapeutic option, both in cocktails and when given in combination with antibiotics. In this study, we show that the synergy between a lipopolysaccharide (LPS)-specific phage and antibiotics is driven by clinically relevant increases in beta-lactam permeability due to the selection of phage-resistant subpopulations. First, in the Hollow Fiber Infection Model (HFIM), we show that the combination of phage LUZ19 (pili-targeted), phage E215 (LPS-specific), and aztreonam (ATM) eradicated the laboratory host PAO1, whereas monotherapy with neither phage nor ATM could eradicate PAO1. Static time-kill studies (STKS) evaluated the LPS-specific phage PYO2 in combination with either aztreonam/avibactam (ATM/AVI) or polymyxin B (PMB) against an extensively drug-resistant clinical isolate of P. aeruginosa (AR-0231). PYO2 combined with ATM/AVI was determined to be synergistic, with a mean excess-over-bliss (EOB) of 0.350 (P < 0.05), while PYO2 with PMB was also synergistic, with a mean EOB of 0.361 (P < 0.05). Linear regression of STKS using sequential administration showed that, compared to antibiotic-first treatment, phage-first treatment reduced bacterial concentrations at 24 h by -6.19 log10 colony-forming unit (CFU)/mL (P < 0.05), with statistically significant interactions estimated for each antibiotic. These results indicated that bacterial pre-selection by PYO2 improved antibiotic activity. Mass spectrometry studies showed that the PYO2-resistant AR-0231 strain exhibited a >25% increase in the outer membrane permeability surface area coefficient for all major anti-Pseudomonal beta-lactams, including aztreonam. Altogether, our study provides mechanistic insights into phage-induced collateral sensitivity of beta-lactams. These results show how specific beta-lactams can be prioritized and optimized in phage-antibiotic combinations to maximize the antibacterial activity.

Bacteriophage (phage) therapy is a promising alternative to antibiotics, yet phage-induced immune responses can affect treatment efficacy. However, current methods for assessing phage immunogenicity are limited, hindering the development of safer, more effective therapies. Here, we introduce the Bacteriophage Risk Index (BRI), a novel metric that quantifies phage immunogenic potential based on CpG dinucleotide frequency, motif spacing, and sequence context, key factors influencing Toll-like receptor 9 (TLR9) activation. Applying the BRI to 7,011 phage genomes, we classified them into five risk tiers, revealing substantial immunogenic variability, even among phages targeting the same bacterial host. BRI scores correlated with immune responses in human lung epithelial cells, validating its predictive power. Experimental testing further confirmed this, as exposure of lung epithelial cells to two phages from distinct risk tiers showed that the high-risk phage (Category 4) induced a strong pro-inflammatory response, upregulating CXCL1, CXCL8, IRF7, and TNFAIP3, while the low-risk phage (Category 2) triggered minimal immune activation with limited cytokine expression. These findings confirm that higher BRI scores predict stronger immune responses, providing a robust tool for evaluating phage immunogenicity. By enabling the selection of phages with lower immunogenic potential, the BRI enhances the safety and efficacy of phage therapy while offering a framework for regulatory agencies, clinical researchers, and biologic drug development, with applications extending beyond phage therapy to other immunogenic biologics.

The global rise in multidrug-resistant (MDR) Klebsiella pneumoniae, a critical ESKAPE pathogen, has outpaced the development of effective antibiotics. Bacteriophage therapy offers a promising alternative, but therapeutic candidates must be carefully selected for broad activity, genetic safety, synergistic cocktail performance, and clinical stability. We isolated and characterized six novel lytic phages (vB_Kpn_AM.K1 to vB_Kpn_AM.K6) targeting K. pneumoniae by assessing morphology, host range, growth kinetics, physicochemical stability, and resistance frequency. Genomes were sequenced to confirm absence of lysogeny and virulence genes. Infection dynamics was visualized via fluorescence microscopy. Phage activity was tested across 60 different MDR K. pneumoniae clinical isolates, obtained from diverse sources such as blood, sputum, occult feces, urine etc. All six isolated phages were identified as novel dsDNA phages belonging to Caudoviricetes, with genome sizes ranging from 111 to 169 Kbp, devoid of virulence and AMR genes and demonstrating strong bacteriolytic activity. Growth kinetics indicated burst sizes varying from 12-148 PFU/infected cell. The phages displayed stability between 4-50°C, pH 4 -10 and sustained complete activity after lyophilization. More significantly, the phages and their cocktail combinations could effectively kill 93

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

It has been over 100 years since bacteriophages (phages) were used as a human therapeutic. Since then, phage production has dramatically evolved. Current phage preparations have fewer adverse effects due to their low bacterial toxin content. As a result, therapeutic phages have become a predominant class of new antimicrobials and are being widely used for compassionate treatment of multidrug-resistant (MDR) infections. We describe herein a protocol for the production and ultrapurification of phages. By this technique, it is possible for a lab experienced with the process to produce >109 plaque-forming units (PFU) per mL of Gram-negative phages that meet FDA endotoxins limits for intravenous infusions in as little as 48 hours. We provide illustrations of the process and tips on how to safely remove bacterial toxins from phage lysates. Although dependent on the phage strain, the approach described can rapidly generate and purify phages for a variety of applications.

Bacteriophage (phage) cocktail therapy has been relied upon more and more to treat antibiotic-resistant infections. Understanding of the complex kinetics between phages, target bacteria, and the emergence of phage resistance remain hurdles to successful clinical outcomes. Building upon previous mathematical concepts, we develop biologically-motivated nonlinear ordinary differential equation models to explore single, cocktail, and sequential phage treatment modalities. While the optimal pairwise phage treatment strategy was the double simultaneous administration of two highly potent and asymmetrically binding phage strains, it appears unable to prevent the evolution of resistance. This treatment regimen did have a greater lysis efficiency, promoted higher phage population sizes, reduced bacterial density the most, and suppressed the evolution of resistance the longest compared to all other treatments strategies tested. Conversely, the combination of phages with polar potencies allows the more efficiently replicating phages to monopolize susceptible host cells, thereby quickly negating the intended compounding effect of cocktails. Together, we demonstrate that a biologically-motivated modeling-based framework can be leveraged to quantify the effects of each phage’s properties to more precisely predict treatment responses.

Bacteriophage (phage) therapy is being explored as a possible response to the antimicrobial resistance public health emergency. Administering a mixture of different phage types as a cocktail is one proposed strategy for therapeutic applications, but the optimal method for formulating phage cocktails remains a major challenge. Each phage strain has complex pharmacokinetic/pharmacodynamic (PK/PD) properties which depend on the nano-scale size, target-mediated, self-dosing nature of each phage strain, and rapid selection of resistant subpopulations. The objective of this study was to explore the pharmacodynamics (PD) of three unique and clinically relevant anti-Pseudomonas phages after simulation of dynamic dosing strategies. The Hollow Fiber Infection Model (HFIM) is an in vitro system that mimics in vivo pharmacokinetics (PK) with high fidelity, providing an opportunity to quantify phage and bacteria concentration profiles over clinical time scales with rich sampling. Exogenous monotherapy-bolus (producing max concentrations of Cmax = 7 log10 PFU/mL) regimens of phages LUZ19, PYO2, and E215 produced Pseudomonas aeruginosa nadirs of 0, 2.14, or 2.99 log10 CFU/mL after 6 h of treatment, respectively. Exogenous combination therapy bolus regimens (LUZ19 + PYO2 or LUZ19 + E215) resulted in bacterial reduction to <2 log10 CFU/mL. In contrast, monotherapy as a continuous infusion (producing a steady-state concentration of Css,avg = 2 log10PFU/mL) was less effective at reducing bacterial densities. Specifically, PYO2 failed to reduce bacterial density. Next, a mechanism-based mathematical model was developed to describe phage pharmacodynamics, phage–phage competition, and phage-dependent adaptive phage resistance. Monte Carlo simulations supported bolus dose regimens, predicting lower bacterial counts with bolus dosing as compared to prolonged phage infusions. Together, in vitro and in silico evaluation of the time course of phage pharmacodynamics will better guide optimal patterns of administration of individual phages as a cocktail.

Developing optimized regimens for combination antibiotic therapy is challenging and often performed empirically over many clinical studies. Novel implementation of a hybrid machine-learning pharmacokinetic/pharmacodynamic/toxicodynamic (ML-PK/PD/TD) approach optimizes combination therapy using human PK/TD data along with in vitro PD data. This study utilized human population PK (PopPK) of aztreonam, ceftazidime/avibactam, and polymyxin B along with in vitro PDs from the Hollow Fiber Infection Model (HFIM) to derive optimal multi-drug regimens de novo through implementation of a genetic algorithm (GA). The mechanism-based PD model was constructed based on 7-day HFIM experiments across 4 clinical, extensively drug resistant Klebsiella pneumoniae isolates. GA-led optimization was performed using 13 different fitness functions to compare the effects of different efficacy (60%, 70%, 80%, or 90% of simulated subjects achieving bacterial counts of 102  CFU/mL) and toxicity (66% of simulated subjects having a target polymyxin B area under the concentration-time curve [AUC] of 100 mg·h/L and aztreonam AUC of 1,332 mg·h/L) on the optimized regimen. All regimens, except those most heavily weighted for toxicity prevention, were able to achieve the target efficacy threshold (102  CFU/mL). Overall, GA-based regimen optimization using preclinical data from animal-sparing in vitro studies and human PopPK produced clinically relevant dosage regimens similar to those developed empirically over many years for all three antibiotics. Taken together, these data provide significant insight into new therapeutic approaches incorporating ML to regimen design and treatment of resistant bacterial infections.

The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses.

Hundreds of trillions of diverse bacteriophages (phages) peacefully thrive within and on the human body. However, whether and how phages influence their mammalian hosts is poorly understood. In this review, we explore current knowledge and present growing evidence that direct interactions between phages and mammalian cells often induce host inflammatory and antiviral immune responses. We show evidence that, like viruses of the eukaryotic host, phages are actively internalized by host cells and activate conserved viral detection receptors. This interaction often generates proinflammatory cytokine secretion and recruitment of adaptive immune programs. However, significant variability exists in phage-immune interactions, suggesting an important role for structural phage characteristics. The factors leading to the differential immunogenicity of phages remain largely unknown but are highly influenced by their human and bacterial hosts.

Here, we report the complete genome sequence of the virulent podovirus PaCe, which was isolated from wastewater in San Diego, California, using the host Pseudomonas aeruginosa. Its complete genome is 45,365 bp in length, with a GC content of 52.5%. PaCe belongs to the genus Bruynoghevirus in the class Caudoviricetes.

Viruses that infect bacteria (i.e., phages) are abundant and widespread in the human body, and new anti-infective approaches such as phage therapy are essential for the future of effective medicine. Our understanding of microenvironmental factors such as tissue oxygen availability at the site of phage–bacteria interaction remains limited, and it is unknown whether evolved resistance is sculpted differentially under normoxia vs. hypoxia. We, therefore, analyzed the phage–bacteria interaction landscape via adsorption, one-step, time-kill dynamics, and genetic evolution under both normoxia and hypoxia. This revealed that adsorption of phages to Pseudomonas aeruginosa decreased under 14% environmental oxygen (i.e., hypoxia), but phage time-kill and one-step growth kinetics were not further influenced. Tracking the adaptation of P. aeruginosa to phages uncovered a higher frequency of phage resistance and constrained types of spontaneous mutation under hypoxia. Given the interest in developing phage therapies, developing our understanding of the phage–pathogen interaction under microenvironmental conditions resembling those in the body offers insight into possible strategies to overcome multidrug-resistant (MDR) bacteria.

Pseudomonas aeruginosa is a frequent cause of life-threatening opportunistic infections in the critically ill and immunocompromised. Its treatment is challenging due to the increasing prevalence of resistance to most conventional antibiotics. Although numerous alternative therapies are currently under investigation, bacteriophage (phage) cocktail therapy appears poised for long-term success. Here, we investigate potency and longevity of individual Pseudomonas phages in cocktail to determine viral co-factors that promote optimal treatment efficacy. We combined in vitro and in silico models to predict sixty-eight treatment permutations with three phages that adsorb symmetrically and asymmetrically when administered singly, double simultaneously, or double sequentially. We showed that simultaneously administering two asymmetrically binding phages with high cell lysis efficiencies improved cocktail potency. Use of a higher-potency cocktail, along with a reduction in the net probability of independent gene mutations was associated with prolonged bacterial suppression. Nevertheless, in vitro we almost always observed evolution of multiphage resistance. Simulations also predict that when combining phages with polar potencies, susceptible host cells are monopolized by the more efficiently replicating phage. Thus, further perpetuating the growth demise of the weaker phage in cocktail. Our mathematical model was used to explore and predict changes in phage and bacterial populations that were difficult to measure experimentally. This framework has many inferential and exploratory uses for clinical investigation such as identifying the most sensitive parameters for phage selection and exploring different treatment regimens. Collectively, our findings attempt to dissect the mechanisms of phage cocktails combating P. aeruginosa infections and highlight the viral co-factors necessary for treatment efficacy.

Here, we report the genome sequence of PA291, a nonmucoid, multidrug-resistant strain of Pseudomonas aeruginosa isolated from cystic fibrosis sputum. Short reads were de novo assembled into 190 contigs and scaffold assembled to a length of 6.26 Mbp. PhiSpy predicts that PA291 is free of prophages.

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward—by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 10 11 plaque-forming units (PFUs) per milliliter for Pseudomonas , Klebsiella , and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 10 9 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 10 6 -fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.

EnteropathogenicEscherichia coli(EPEC) is a major pathogen for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse have led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy as an alternative antibacterial therapy. Because EPEC in vivo models have shortcomings, a surrogate is used to study the mouse pathogenCitrobacter rodentiumin animal models. In this study, two new phages CrRp3 and CrRp10, which infectC. rodentium,were isolated and characterized. CrRp3 was found to be a new species within the genusVectrevirus,and CrRp10 is a new strain within the speciesEscherichia virus Ime09, in the genusTequatrovirus.Both phages appear to have independently evolved fromE. coliphages, rather than other Citrobacter spp. phages. Neither phage strain carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 is more potent, having a 24-fold faster adsorption rate and shorter lytic cycle when compared to the same properties of CrRp10. However, a lysis curve analysis revealed that CrRp10 prevented growth ofC. rodentiumfor 18 h, whereas resistance developed against CrRp3 within 9 h. We also show that hypoxic (5% oxygen) conditions decreased CrRp3 ability to control bacterial densities in culture. In contrast, low oxygen conditions did not affect CrRp10 ability to replicate onC. rodentium. Together, CrRp10 is likely to be the better candidate for future phage therapy investigations.