Broad protective vaccination against systemic Escherichia coli with autotransporter antigens.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

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.

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.

Identifying Causative Microorganisms in Left Ventricular Assist Device Infections as a Guide for Developing Bacteriophage Therapy.

BACKGROUND: As more left ventricular-assist devices (LVADs) are implanted, multidrug-resistant LVAD infections are becoming increasingly common, partly due to bacterial biofilm production. To aid in developing bacteriophage therapy for LVAD infections, we have identified the most common bacterial pathogens that cause LVAD driveline infections (DLIs) in our heart transplant referral center. MATERIALS AND METHODS: We studied a retrospective cohort of patients who received LVADs from November 2003 to August 2017 to identify the common causative organisms of LVAD infection. We also studied a prospective cohort of patients diagnosed with DLIs from October 2018 to May 2019 to collect bacterial strains from DLIs for developing bacteriophages to lyse causative pathogens. LVAD infections were classified as DLI, bacteremia, and pump/device infections in the retrospective cohort. RESULTS: In the retrospective cohort of 582 patients, 186 (32.0%) developed an LVAD infection, with 372 microbial isolates identified. In the prospective cohort, 96 bacterial strains were isolated from 54 DLIs. The microorganisms causing DLIs were similar in the two cohorts; the most common isolate was Staphylococcus aureus. We identified 6 prospective S. aureus strains capable of biofilm formation. We developed 3 bacteriophages that were able to lyse 5 of 6 of the biofilm-forming S. aureus strains. CONCLUSIONS: Similar pathogens caused LVAD DLIs in our retrospective and prospective cohorts, indicating our bacterial strain bank will be representative of future DLIs. Our banked bacterial strains will be useful in developing phage cocktails that can lyse ≥80% of the bacteria causing LVAD infections at our institution.

Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model.

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund's Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

Heme catabolism in the causative agent of anthrax.

A challenge common to all bacterial pathogens is to acquire nutrients from hostile host environments. Iron is an important cofactor required for essential cellular processes such as DNA repair, energy production and redox balance. Within a mammalian host, most iron is sequestered within heme, which in turn is predominantly bound by hemoglobin. While little is understood about the mechanisms by which bacterial hemophores attain heme from host-hemoglobin, even less is known about intracellular heme processing. Bacillus anthracis, the causative agent of anthrax, displays a remarkable ability to grow in mammalian hosts. Hypothesizing this pathogen harbors robust ways to catabolize heme, we characterize two new intracellular heme-binding proteins that are distinct from the previously described IsdG heme monooxygenase. The first of these, HmoA, binds and degrades heme, is necessary for heme detoxification and facilitates growth on heme iron sources. The second protein, HmoB, binds and degrades heme too, but is not necessary for heme utilization or virulence. The loss of both HmoA and IsdG renders B. anthracis incapable of causing anthrax disease. The additional loss of HmoB in this background increases clearance of bacilli in lungs, which is consistent with this protein being important for survival in alveolar macrophages.

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.

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.

Use of the Naturally Occurring Bacteriophage Grouping Model for the Design of Potent Therapeutic Cocktails.

The specificity of phages and their ability to evolve and overcome bacterial resistance make them potentially useful as adjuncts in the treatment of antibiotic-resistant bacterial infections. The goal of this study was to mimic a natural grouping of phages of interest and to evaluate the nature of their proliferation dynamics with bacteria. We have, for the first time, transferred naturally occurring phage groups directly from their sources of isolation to in vitro and identified 13 P. aeruginosa and 11 K. pneumoniae phages of 18 different genera, whose host range was grouped as 1.2-17%, 28-48% and 60-87%, using a large collection of P. aeruginosa (n = 102) and K. pneumoniae (n = 155) strains carrying different virulence factors and phage binding receptors. We introduced the interpretation model curve for phage liquid culturing, which allows easy and quick analysis of bacterial and phage co-proliferation and growth of phage-resistant mutants (PRM) based on qualitative and partially quantitative evaluations. We assayed phage lytic activities both individually and in 14 different cocktails on planktonic bacterial cultures, including three resistotypes of P. aeruginosa (PAO1, PA14 and PA7) and seven K. pneumoniae strains of different capsular serotypes. Based on the results, the natural phage cocktails designed and tested in this study largely performed well and inhibited PRM growth either synergistically or in proto-cooperation. This study contributes to the knowledge of phage behavior in cocktails and the formulation of therapeutic phage preparations. The paper also provides a detailed description of the methods of working with phages.

Personalized bacteriophage therapy outcomes for 100 consecutive cases: a multicentre, multinational, retrospective observational study.

In contrast to the many reports of successful real-world cases of personalized bacteriophage therapy (BT), randomized controlled trials of non-personalized bacteriophage products have not produced the expected results. Here we present the outcomes of a retrospective observational analysis of the first 100 consecutive cases of personalized BT of difficult-to-treat infections facilitated by a Belgian consortium in 35 hospitals, 29 cities and 12 countries during the period from 1 January 2008 to 30 April 2022. We assessed how often personalized BT produced a positive clinical outcome (general efficacy) and performed a regression analysis to identify functional relationships. The most common indications were lower respiratory tract, skin and soft tissue, and bone infections, and involved combinations of 26 bacteriophages and 6 defined bacteriophage cocktails, individually selected and sometimes pre-adapted to target the causative bacterial pathogens. Clinical improvement and eradication of the targeted bacteria were reported for 77.2% and 61.3% of infections, respectively. In our dataset of 100 cases, eradication was 70% less probable when no concomitant antibiotics were used (odds ratio = 0.3; 95% confidence interval = 0.127-0.749). In vivo selection of bacteriophage resistance and in vitro bacteriophage-antibiotic synergy were documented in 43.8% (7/16 patients) and 90% (9/10) of evaluated patients, respectively. We observed a combination of antibiotic re-sensitization and reduced virulence in bacteriophage-resistant bacterial isolates that emerged during BT. Bacteriophage immune neutralization was observed in 38.5% (5/13) of screened patients. Fifteen adverse events were reported, including seven non-serious adverse drug reactions suspected to be linked to BT. While our analysis is limited by the uncontrolled nature of these data, it indicates that BT can be effective in combination with antibiotics and can inform the design of future controlled clinical trials. BT100 study, ClinicalTrials.gov registration: NCT05498363 .

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.

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.].

Phage Therapy Related Microbial Succession Associated with Successful Clinical Outcome for a Recurrent Urinary Tract Infection.

We rationally designed a bacteriophage cocktail to treat a 56-year-old male liver transplant patient with complex, recurrent prostate and urinary tract infections caused by an extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) (UCS1). We screened our library for phages that killed UCS1, with four promising candidates chosen for their virulence, mucolytic properties, and ability to reduce bacterial resistance. The patient received 2 weeks of intravenous phage cocktail with concomitant ertapenem for 6 weeks. Weekly serum and urine samples were collected to track the patient's response. The patient tolerated the phage therapy without any adverse events with symptom resolution. The neutralization of the phage activity occurred with sera collected 1 to 4 weeks after the first phage treatment. This was consistent with immunoassays that detected the upregulation of immune stimulatory analytes. The patient developed asymptomatic recurrent bacteriuria 6 and 11 weeks following the end of phage therapy-a condition that did not require antibiotic treatment. The bacteriuria was caused by a sister strain of E. coli (UCS1.1) that remained susceptible to the original phage cocktail and possessed putative mutations in the proteins involved in adhesion and invasion compared to UCS1. This study highlights the utility of rationally designed phage cocktails with antibiotics at controlling E. coli infection and suggests that microbial succession, without complete eradication, may produce desirable clinical outcomes.

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.

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.