Tiffany Luong

All Publications

• Breaking down barriers to improve phage therapy delivery. Cell host & microbe Luong, T., Rybicka, I., Blankenberg, P., Dąbrowska, K., Bollyky, P. L. 2026; 34 (7): 1285-1301

  Abstract

  There is growing interest in the development of bacteriophages as therapies for antimicrobial-resistant infections, but effective delivery of phages remains a barrier. This review examines the opportunities and challenges involved in the development of phages as drugs, focusing on phage delivery. We first review current practices and success rates for clinical phage therapy and recent advances in phage selection and design. Next, we frame ongoing delivery challenges in the context of what is known about phage biology and phage pharmacokinetics. We then explore barriers to effective phage delivery alongside dosing and administration strategies used to overcome them, followed by an examination of recent innovations in phage formulations and biomaterials technologies. Finally, we highlight outstanding questions and challenges in the field. We conclude that optimizing delivery is a key determinant of the success of phage therapy and that the integration of microbiology, materials science, and pharmacology will enable more consistent success.

  View details for DOI 10.1016/j.chom.2026.06.013

  View details for PubMedID 42419273

• Ecological partitioning enables phage-antibiotic cooperation in a human <i>Pseudomonas</i> infection NATURE COMMUNICATIONS Luong, T., Kharrat, L., Champagne-Jorgensen, K., Melendez, J. A., Pride, D., Conrad, D. J., Roach, D. R. 2026; 17 (1)

  Abstract

  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.

  View details for DOI 10.1038/s41467-026-69247-w

  View details for Web of Science ID 001718932500002

  View details for PubMedID 41667461

  View details for PubMedCentralID PMC13003088

• Pharmacodynamic individualization of phage therapy against a KPC-5-producing Pseudomonas aeruginosa JAC-ANTIMICROBIAL RESISTANCE Nguyen, T. D., Sanborn, J. T., Ho, B. M., Luong, T., Wood, T. D., Chen, L., Roach, D. R., Smith, N. M. 2026; 8 (1): dlaf257

  Abstract

  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.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.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.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.

  View details for DOI 10.1093/jacamr/dlaf257

  View details for Web of Science ID 001655438900001

  View details for PubMedID 41510198

  View details for PubMedCentralID PMC12776353

• Large-Scale Genomic Analysis of CpG-Mediated Immunogenicity in Bacteriophages and a Novel Predictive Risk Index. bioRxiv : the preprint server for biology Kharrat, L., Garcia-Botero, C. A., Ingersoll, W., Luong, T., Reyes, A., Roach, D. R. 2025

  Abstract

  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.

  View details for DOI 10.1101/2025.05.15.652987

  View details for PubMedID 40463234

  View details for PubMedCentralID PMC12132194

• Rapid Bench to Bedside Therapeutic Bacteriophage Production. Methods in molecular biology (Clifton, N.J.) Luong, T., Sue, A. D., Roach, D. R. 2024; 2734: 67-88

  Abstract

  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.

  View details for DOI 10.1007/978-1-0716-3523-0_5

  View details for PubMedID 38066363

  View details for PubMedCentralID 5923482

• Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases. Antimicrobial agents and chemotherapy Aslam, S., Roach, D., Nikolich, M. P., Biswas, B., Schooley, R. T., Lilly-Bishop, K. A., Rice, G. K., Cer, R. Z., Hamilton, T., Henry, M., Luong, T., Salabarria, A. -., Sisk-Hackworth, L., Filippov, A. A., Lebreton, F., Hall, L., Nir-Paz, R., Onallah, H., Livni, G., Shostak, E., Wieder-Finesod, A., Yahav, D., Yerushalmy, O., Alkalay-Oren, S., Braunstein, R., Khalifa, L., Rimon, A., Gelman, D., Hazan, R. 2024; 68 (4): e0172823

  Abstract

  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.

  View details for DOI 10.1128/aac.01728-23

  View details for PubMedID 38470133

  View details for PubMedCentralID PMC10989018

• A mechanism-based pathway toward administering highly active N-phage cocktails. Frontiers in microbiology Smith, N. M., Nguyen, T. D., Chin, W. H., Sanborn, J. T., de Souza, H., Ho, B. M., Luong, T., Roach, D. R. 2023; 14: 1292618

  Abstract

  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.

  View details for DOI 10.3389/fmicb.2023.1292618

  View details for PubMedID 38045026

  View details for PubMedCentralID PMC10690594

• Immunogenicity of bacteriophages. Trends in microbiology Champagne-Jorgensen, K., Luong, T., Darby, T., Roach, D. R. 2023; 31 (10): 1058-1071

  Abstract

  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.

  View details for DOI 10.1016/j.tim.2023.04.008

  View details for PubMedID 37198061

• Draft Genome Sequence of the Multidrug-Resistant Strain Pseudomonas aeruginosa PA291, Isolated from Cystic Fibrosis Sputum. Microbiology resource announcements Luong, T., Schumann, A., Conrad, D., Roach, D. 2021; 10 (36): e0057221

  Abstract

  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.

  View details for DOI 10.1128/MRA.00572-21

  View details for PubMedID 34498926

  View details for PubMedCentralID PMC8428245

• Phage Therapy in the Resistance Era: Where Do We Stand and Where Are We Going? Clinical therapeutics Luong, T., Salabarria, A. C., Roach, D. R. 2020; 42 (9): 1659-1680

  Abstract

  Widespread antibiotic-resistant bacteria are threatening the arsenal of existing antibiotics. Not only are antibiotics less likely to be effective today, but their extensive use continues to drive the emergence of multidrug-resistant pathogens. A new-old antibacterial strategy with bacteriophages (phages) is under development, namely, phage therapy. Phages are targeted bacterial viruses with multiple antibacterial effector functions, which can reduce multidrug-resistant infections within the human body. This review summarizes recent phage therapy clinical trials and patient cases and outlines the fundamentals behind phage treatment strategies under development, mainly through bench-to-bedside approaches. We discuss the challenges that remain in phage therapy and the role of phages when combined with antibiotic therapy.This narrative review presents the current knowledge and latest findings regarding phage therapy. Relevant case reports and research articles available through the Scopus and PubMed databases are discussed.Although recent clinical data suggest the tolerability and, in some cases, efficacy of phage therapy, the clinical functionality still requires careful definition. The lack of well-controlled clinical trial data and complex regulatory frameworks have driven the most recent human data generation on a single-patient compassionate use basis. These cases often include the concomitant use of antibiotics, which makes it difficult to draw conclusions regarding the effectiveness of phages alone. However, human data support using antibiotics as phage potentiators and resistance breakers; thus, phage adjuvants are a promising avenue for near-term clinical development. Current knowledge gaps exist on the appropriate routes of administration, phage selection, frequency of administration, dosage, phage resistance, and pharmacokinetic and pharmacodynamic properties of the phages. In addition, we highlight that some phage therapies have mild adverse effects in patients.Although more translational research is needed before the clinical implementation is feasible, phage therapy may well be pivotal in safeguarding humans against antibiotic-resistant infections.

  View details for DOI 10.1016/j.clinthera.2020.07.014

  View details for PubMedID 32883528

• Standardized bacteriophage purification for personalized phage therapy. Nature protocols Luong, T., Salabarria, A. C., Edwards, R. A., Roach, D. R. 2020; 15 (9): 2867-2890

  Abstract

  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 1011 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 109 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 106-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.

  View details for DOI 10.1038/s41596-020-0346-0

  View details for PubMedID 32709990

  View details for PubMedCentralID 5610518

• Isolation and Characterization of Bacteriophages That Infect Citrobacter rodentium, a Model Pathogen for Intestinal Diseases. Viruses Mizuno, C. M., Luong, T., Cederstrom, R., Krupovic, M., Debarbieux, L., Roach, D. R. 2020; 12 (7)

  Abstract

  Enteropathogenic Escherichia 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 pathogen Citrobacter rodentium in animal models. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus, and CrRp10 is a new strain within the species Escherichia virus Ime09, in the genus Tequatrovirus. Both phages appear to have independently evolved from E. coli phages, 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 of C. rodentium for 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 on C. rodentium. Together, CrRp10 is likely to be the better candidate for future phage therapy investigations.

  View details for DOI 10.3390/v12070737

  View details for PubMedID 32650458

  View details for PubMedCentralID PMC7412075

• HCV Broadly Neutralizing Antibodies Use a CDRH3 Disulfide Motif to Recognize an E2 Glycoprotein Site that Can Be Targeted for Vaccine Design. Cell host & microbe Flyak, A. I., Ruiz, S., Colbert, M. D., Luong, T., Crowe, J. E., Bailey, J. R., Bjorkman, P. J. 2018; 24 (5): 703-716.e3

  Abstract

  Hepatitis C virus (HCV) vaccine efforts are hampered by the extensive genetic diversity of HCV envelope glycoproteins E1 and E2. Structures of broadly neutralizing antibodies (bNAbs) (e.g., HEPC3, HEPC74) isolated from individuals who spontaneously cleared HCV infection facilitate immunogen design to elicit antibodies against multiple HCV variants. However, challenges in expressing HCV glycoproteins previously limited bNAb-HCV structures to complexes with truncated E2 cores. Here we describe crystal structures of full-length E2 ectodomain complexes with HEPC3 and HEPC74, revealing lock-and-key antibody-antigen interactions, E2 regions (including a target of immunogen design) that were truncated or disordered in E2 cores, and an antibody CDRH3 disulfide motif that exhibits common interactions with a conserved epitope despite different bNAb-E2 binding orientations. The structures display unusual features relevant to common genetic signatures of HCV bNAbs and demonstrate extraordinary plasticity in antibody-antigen interactions. In addition, E2 variants that bind HEPC3/HEPC74-like germline precursors may represent candidate vaccine immunogens.

  View details for DOI 10.1016/j.chom.2018.10.009

  View details for PubMedID 30439340

  View details for PubMedCentralID PMC6258177