A Retrospective, Observational Study of 12 Cases of Expanded-Access Customized Phage Therapy: Production, Characteristics, and Clinical Outcomes.

BACKGROUND: Antimicrobial resistance (AMR) is undermining modern medicine, a problem compounded by bacterial adaptation to antibiotic pressures. Phages are viruses that infect bacteria. Their diversity and evolvability offer the prospect of their use as a therapeutic solution. Reported are outcomes of customized phage therapy for patients with difficult-to-treat antimicrobial resistant infections. METHODS: We retrospectively assessed 12 cases of customized phage therapy from a phage production center. Phages were screened, purified, sequenced, characterized, and Food and Drug Administration-approved via the IND (investigational new drug) compassionate-care route. Outcomes were assessed as favorable or unfavorable by microbiologic and clinical standards. Infections were device-related or systemic. Other experiences such as time to treatment, antibiotic synergy, and immune responses were recorded. RESULTS: Fifty requests for phage therapy were received. Customized phages were generated for 12 patients. After treatment, 42% (5/12) of cases showed bacterial eradication and 58% (7/12) showed clinical improvement, with two-thirds of all cases (66%) showing favorable responses. No major adverse reactions were observed. Antibiotic-phage synergy in vitro was observed in most cases. Immunological neutralization of phages was reported in 5 cases. Several cases were complicated by secondary infections. Complete characterization of the phages (morphology, genomics, and activity) and their production (methods, sterility, and endotoxin tests) are reported. CONCLUSIONS: Customized phage production and therapy was safe and yielded favorable clinical or microbiological outcomes in two-thirds of cases. A center or pipeline dedicated to tailoring the phages against a patient's specific AMR bacterial infection may be a viable option where standard treatment has failed.

Phage therapy for hidradenitis suppurativa: a unique challenge and possible opportunity for personalized treatment of a complex, inflammatory disease.

Phage therapy is an emerging antimicrobial treatment for critical multidrug-resistant pathogens. In this review, the specific potential and challenges of phage therapy for patients with hidradenitis suppurativa (HS) are discussed. This represents a unique challenge as HS is a chronic inflammatory disease, but presenting with acute exacerbations, which have an enormous negative impact on patient's quality of life. The therapeutic arsenal for HS has expanded in the past decade, for example, with adalimumab and several other biologicals that are currently under investigation. However, treatment of HS remains challenging for dermatologists because there are individuals who do not respond to any classes of the current treatment options when used for a first or second time. Furthermore, after several courses of treatment, a patient may lose their response to therapy, meaning long-term use is not always an option. Culturing studies and 16S ribosomal RNA profiling highlight the complex polymicrobial nature of HS lesions. Despite the detection of various bacterial species in lesion samples, several key pathogens, including Staphylococcus, Corynebacterium and Streptococcus, may be potential targets for phage therapy. Using phage therapy for the treatment of a chronic inflammatory disease could potentially provide new insights into the role of bacteria and the immune system in HS development. In addition, it is possible more details on the immunomodulatory effects of phages may come to light.

Corrigendum: Development of phage cocktails to treat E. coli catheter-associated urinary tract infection and associated biofilms.

[This corrects the article DOI: 10.3389/fmicb.2022.796132.].

Development of Phage Cocktails to Treat E. coli Catheter-Associated Urinary Tract Infection and Associated Biofilms.

High rates of antimicrobial resistance and formation of biofilms makes treatment of Escherichia coli catheter-associated urinary tract infections (CAUTI) particularly challenging. CAUTI affect 1 million patients per year in the United States and are associated with morbidity and mortality, particularly as an etiology for sepsis. Phage have been proposed as a potential therapeutic option. Here, we report the development of phage cocktails that lyse contemporary E. coli strains isolated from the urine of patients with spinal cord injury (SCI) and display strong biofilm-forming properties. We characterized E. coli phage against biofilms in two in vitro CAUTI models. Biofilm viability was measured by an MTT assay that determines cell metabolic activity and by quantification of colony forming units. Nine phage decreased cell viability by >80% when added individually to biofilms of two E. coli strains in human urine. A phage cocktail comprising six phage lyses 82% of the strains in our E. coli library and is highly effective against young and old biofilms and against biofilms on silicon catheter materials. Using antibiotics together with our phage cocktail prevented or decreased emergence of E. coli resistant to phage in human urine. We created an anti-biofilm phage cocktail with broad host range against E. coli strains isolated from urine. These phage cocktails may have therapeutic potential against CAUTI.

Antiviral Resistance and Phage Counter Adaptation to Antibiotic-Resistant Extraintestinal Pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC), often multidrug resistant (MDR), is a leading cause of urinary tract and systemic infections. The crisis of emergent MDR pathogens has led some to propose bacteriophages as a therapeutic. However, bacterial resistance to phage is a concerning issue that threatens to undermine phage therapy. Here, we demonstrate that E. coli sequence type 131, a circulating pandemic strain of ExPEC, rapidly develops resistance to a well-studied and therapeutically active phage (ϕHP3). Whole-genome sequencing of the resisters revealed truncations in genes involved in lipopolysaccharide (LPS) biosynthesis, the outer membrane transporter ompA, or both, implicating them as phage receptors. We found ExPEC resistance to phage is associated with a loss of fitness in host microenvironments and attenuation in a murine model of systemic infection. Furthermore, we constructed a novel phage-bacterium bioreactor to generate an evolved phage isolate with restored infectivity to all LPS-truncated ExPEC resisters. This study suggests that although the resistance of pandemic E. coli to phage is frequent, it is associated with attenuation of virulence and susceptibility to new phage variants that arise by directed evolution.IMPORTANCE In response to the rising crisis of antimicrobial resistance, bacteriophage (phage) therapy has gained traction. In the United States, there have been over 10 cases of largely successful compassionate-use phage therapy to date. The resilience of pathogens allowing their broad antibiotic resistance means we must also consider resistance to therapeutic phages. This work fills gaps in knowledge regarding development of phage resisters in a model of infection and finds critical fitness losses in those resisters. We also found that the phage was able to rapidly readapt to these resisters.

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract.

The human gastrointestinal mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. In the gastrointestinal tract, the interaction of bacteriophages (phages) and their prokaryotic hosts influences the health of the mammalian host, especially colonization with invasive pathobionts. Antibiotics may be used, but they also kill protective commensals. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, positions it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing mucosal targeting phage to selectively remove invasive pathobiont species from mucosal surfaces.IMPORTANCE Invasive pathobionts or microbes capable of causing disease can reside deep within the mucosal epithelium of our gastrointestinal tract. Targeted effective antibacterial therapies are needed to combat these disease-causing organisms, many of which may be multidrug resistant. Here, we isolated a lytic bacteriophage (phage) that can localize to the epithelial surface by binding heparan sulfated glycans, positioning it near its host, Escherichia coli This targeted therapy can be used to selectively remove invasive pathobionts from the gastrointestinal tract, preventing the development of disease.

Enteroaggregative E. coli Adherence to Human Heparan Sulfate Proteoglycans Drives Segment and Host Specific Responses to Infection.

Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding that was ameliorated when mucins were removed, a process dependent on AafA. Pan-screening for glycans for binding to purified AafA identified the human ligand as heparan sulfate proteoglycans (HSPGs). Removal of HSPG abrogated EAEC association with HIEs. These results may mean that the human intestine responds remarkably different to distinct pathobionts that is dependent on the both the individual and intestinal segment in question, and uncover a major role for surface heparan sulfate proteoglycans as tropism-driving factor in adherence and/or colonization.

Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry.

The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments.IMPORTANCE Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.

Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research: Personalized Infectious Disease Medicine for the Most Vulnerable At-Risk Patients.

Mutation is the most powerful driver of change for life on Earth. Pathogenic bacteria utilize mutation as a means to survive strong live-die selective pressures generated by chemical antibiotics. As such, the traditional drug-making pipeline, characterized by significant financial and time investment, is insufficient to keep pace with the rapid evolution of bacterial resistance to structurally fixed and chemically unmalleable antibacterial compounds. In contrast, the genetic diversity and adaptive mutability of the bacteriophage can be leveraged to not only overcome resistance but also used for the development of enhanced traits that increase lytic potential and therapeutic efficacy in relevant host microenvironments. This is the fundamental premise behind Baylor College of Medicine's Tailored Antibacterials and Innovative Laboratories for Phage (Φ) Research (TAILΦR) initiative. In this perspective, we outline the concept, structure, and process behind TAILΦR's attempt to generate a personalized therapeutic phage that addresses the most clinically challenging of bacterial infections.

Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections.

Phage therapy requires libraries of well-characterized phages. Here we describe the generation of phage libraries for three target species: Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae. The basic phage characteristics on the isolation host, sequence analysis, growth properties, and host range and virulence on a number of contemporary clinical isolates are presented. This information is required before phages can be added to a phage library for potential human use or sharing between laboratories for use in compassionate use protocols in humans under eIND (emergency investigational new drug). Clinical scenarios in which these phages can potentially be used are discussed. The phages presented here are currently being characterized in animal models and are available for eINDs.

Metals Enhance the Killing of Bacteria by Bacteriophage in Human Blood.

Multidrug-resistant bacterial pathogens are a major medical concern. E. coli, particularly the pathotype extraintestinal pathogenic E. coli (ExPEC), is a leading cause of bloodstream infections. As natural parasites of bacteria, bacteriophages are considered a possible solution to treat patients infected with antibiotic resistant strains of bacteria. However, the development of phage as an anti-infective therapeutic is hampered by limited knowledge of the physiologic factors that influence their properties in complex mammalian environments such as blood. To address this barrier, we tested the ability of phage to kill ExPEC in human blood. Phages are effective at killing ExPEC in conventional media but are substantially restricted in this ability in blood. This phage killing effect is dependent on the levels of free metals and is inhibited by the anticoagulant EDTA. The EDTA-dependent inhibition of ExPEC killing is overcome by exogenous iron, magnesium, and calcium. Metal-enhanced killing of ExPEC by phage was observed for several strains of ExPEC, suggesting a common mechanism. The addition of metals to a murine host infected with ExPEC stimulated a phage-dependent reduction in ExPEC levels. This work defines a role for circulating metals as a major factor that is essential for the phage-based killing of bacteria in blood.

Role for FimH in Extraintestinal Pathogenic Escherichia coli Invasion and Translocation through the Intestinal Epithelium.

The translocation of bacteria across the intestinal epithelium of immunocompromised patients can lead to bacteremia and life-threatening sepsis. Extraintestinal pathogenic Escherichia coli (ExPEC), so named because this pathotype infects tissues distal to the intestinal tract, is a frequent cause of such infections, is often multidrug resistant, and chronically colonizes a sizable portion of the healthy population. Although several virulence factors and their roles in pathogenesis are well described for ExPEC strains that cause urinary tract infections and meningitis, they have not been linked to translocation through intestinal barriers, a fundamentally distant yet important clinical phenomenon. Using untransformed ex situ human intestinal enteroids and transformed Caco-2 cells, we report that ExPEC strain CP9 binds to and invades the intestinal epithelium. ExPEC harboring a deletion of the gene encoding the mannose-binding type 1 pilus tip protein FimH demonstrated reduced binding and invasion compared to strains lacking known E. coli virulence factors. Furthermore, in a murine model of chemotherapy-induced translocation, ExPEC lacking fimH colonized at levels comparable to that of the wild type but demonstrated a statistically significant reduction in translocation to the kidneys, spleen, and lungs. Collectively, this study indicates that FimH is important for ExPEC translocation, suggesting that the type 1 pilus is a therapeutic target for the prevention of this process. Our study also highlights the use of human intestinal enteroids in the study of enteric diseases.

Bacteriophages from ExPEC Reservoirs Kill Pandemic Multidrug-Resistant Strains of Clonal Group ST131 in Animal Models of Bacteremia.

Multi-drug resistant (MDR) enteric bacteria are of increasing global concern. A clonal group, Escherichia coli sequence type (ST) 131, harbors both MDR and a deadly complement of virulence factors. Patients with an immunocompromised system are at high risk of infections with these E. coli and there is strong epidemiologic evidence that the human intestinal tract, as well as household pets, may be a reservoir. Here, we examine if phages are an effective treatment strategy against this clonal group in murine models of bacteremia that recapitulate clinical infections. Bacteriophages isolated from known E. coli reservoirs lyse a diverse array of MDR ST131 clinical isolates. Phage HP3 reduced E. coli levels and improved health scores for mice infected with two distinct ST131 strains. Efficacy was correlated to in vitro lysis ability by the infecting phage and the level of virulence of the E. coli strain. Importantly, it is also demonstrated that E. coli bacteremia initiated from translocation across the intestinal tract in an immunocompromised host is substantially reduced after phage treatment. This study demonstrates that phage, isolated from the environment and with little experimental manipulation, can be effective in combating even the most serious of infections by E. coli "superbugs".

A dual component heme biosensor that integrates heme transport and synthesis in bacteria.

Bacterial pathogens acquire host iron to power cellular processes and replication. Heme, an iron-containing cofactor bound to hemoglobin, is scavenged by bacterial proteins to attain iron. Methods to measure intracellular heme are laborious, involve complex chemistry, or require radioactivity. Such drawbacks limit the study of the mechanistic steps of heme transport and breakdown. Hypothesizing heme homeostasis could be measured with fluorescent methods, we coupled the conversion of heme to biliverdin IXα (a product of heme catabolism) by heme oxygenase 1 (HO1) with the production of near-infrared light upon binding this verdin by infrared fluorescent protein (IFP1.4). The resultant heme sensor, IFP-HO1, was fluorescent in pathogenic E. coli exposed to heme but not in the absence of the heme transporter ChuA and membrane coupling protein TonB, thereby validating their long-standing proposed role in heme uptake. Fluorescence was abolished in a strain lacking hemE, the central gene in the heme biosynthetic pathway, but stimulated by iron, signifying the sensor reports on intracellular heme production. Finally, an invasive strain of E. coli harboring the sensor was fluorescent during an active infection. This work will allow researchers to expand the molecular toolbox used to study heme and iron acquisition in culture and during infection.

Murine model of chemotherapy-induced extraintestinal pathogenic Escherichia coli translocation.

Escherichia coli is a major cause of life-threatening infections in patients with neutropenia, particularly those receiving chemotherapy for the treatment of cancer. In most cases, these infections originate from opportunistic strains living within the patient's gastrointestinal tract which then translocate to major organ systems. There are no animal models that faithfully recapitulate these infections, and, as such, the host or bacterial factors that govern this process remain unidentified. We present here a novel model of chemotherapy-induced bacterial translocation of E. coli. Oral gavage of BALB/c mice with a clinical isolate of extraintestinal pathogenic E. coli (ExPEC) leads to stable and long-term colonization of the murine intestine. Following the induction of neutropenia with the chemotherapeutic drug cyclophosphamide, ExPEC translocates from the intestine to the lungs, liver, spleen, and kidneys with concomitant morbidity in infected animals. Translocation can also occur in mice bearing mammary tumors, even in the absence of chemotherapy. Translocation of ExPEC is also associated with an increase of the diversity of bacterial DNA detected in the blood. This is the first report of a chemotherapy-based animal model of ExPEC translocation in cancerous mice, a system that can be readily used to identify important virulence factors for this process.

A product of heme catabolism modulates bacterial function and survival.

Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC), a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.