A comparison of phage susceptibility testing with two liquid high-throughput methods.

Phage therapy is a promising antibacterial strategy, especially given that drug-resistant bacterial infections are escalating worldwide. Because phages are not active against all strains of a given species, phages being considered for therapeutic use would ideally be tested against bacterial isolates from individual patients prior to administration. Standardized, clinically validated phage susceptibility testing (PST) methods are needed for assessing in vitro phage activity. This study compared two high-throughput liquid-based PST assays. The first, using the Biolog Omnilog™, assessed changes in microbial respiration leading to color changes based on a tetrazolium dye. The second, Agilent BioTek Cytation 7, assessed changes in optical density. Both used 96-well microtiter plate formats. A total of 55 diverse phages with activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, or Enterococcus faecalis were studied against their respective susceptible bacterial hosts and non-susceptible controls, with susceptibility defined based on plaque assay. PST was performed by both assays in replicates, with results compared in terms of hold times (time through which bacterial growth is inhibited by phage compared to controls). Coefficients of variance and interclass correlation coefficients were used to assess inter- and intra-assay reproducibility. Based on a ≤50% coefficient of variance cutpoint, 87% of Biolog and 84% of Agilent assays were considered valid for susceptible bacteria, with 100% considered valid for non-susceptible bacteria by both systems. Using a 8 h hold time cutpoint, 100% of the results matched between the two assays. The interclass correlation coefficient showed 26% excellent agreement, 35% good agreement, and 17% moderate agreement between the two assays for susceptible isolates and 100% excellent agreement for non-susceptible isolates. Overall, the assays compared provided good/fair statistical reproducibility for the assessment of phage susceptibility.

Feasibility of using bacteriophage therapy to treat Staphylococcal aureus fracture-related infections.

OBJECTIVE: Staphylococcus aureus fracture-related infections (FRIs) are associated with significant morbidity in part because conventional antibiotic therapies have limited ability to eradicate S. aureus in sessile states. Therefore, the objective of this study was to assess the feasibility of using Staphylococcal bacteriophages for FRI by testing the activity of a library of Staphylococcal bacteriophage therapeutics against historically preserved S. aureus FRI clinical isolates. METHODS: Current Procedural Terminology codes were used to identify patients with FRI from January 1, 2021 to December 31, 2021. Preserved S. aureus FRI isolates from the cases were then tested against a library of 51 Staphylococcal bacteriophages from an American company. This was conducted by assessing the ability of bacteriophages to reduce bacterial growth over time. Growth inhibition greater than 16 h was considered adequate for this study. RESULTS: All of the S. aureus preserved clinical isolates had at least one bacteriophage with robust lytic activity and six bacteriophages (11.8 %) had robust lytic activity to seven or more of the clinical isolates. However, 41 of the bacteriophages (80.4 %) had activity to less than three of the clinical isolates and no bacteriophage had activity to all the clinical isolates. CONCLUSION: Our findings show that Staphylococcal bacteriophage therapeutics are readily available for S. aureus FRI clinical isolates. However, when correlated with the current barriers to using bacteriophages to treat FRI, designated Staphylococcal bacteriophage cocktails with broad spectrum activity should be created.

Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing.

Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P. aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP8), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P. aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series.

BACKGROUND: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. METHODS: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. FINDINGS: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once- to twice-daily regimen. CONCLUSIONS: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials. FUNDING: The study was funded in part by The Israeli Science Foundation IPMP (ISF_1349/20), Rosetrees Trust (A2232), United States-Israel Binational Science Foundation (2017123), and the Milgrom Family Support Program.

Recurrent ESBL Escherichia coli Urosepsis in a Pediatric Renal Transplant Patient Treated With Antibiotics and Bacteriophage Therapy.

INTRODUCTION: Treating recurrent multidrug resistant (MDR) urosepsis in pediatric transplant recipients can be challenging. Particularly when antibiotics fail to prevent future occurrence and the nidus is seemingly undiscoverable. While there is an increasing amount of data on phage therapy, to our knowledge, there are no published cases involving pediatric renal transplant recipients. Therefore, we present a challenging clinical case in which phage therapy was used in a pediatric renal transplant recipient who developed recurrent MDR urosepsis with an unclear source. CASE PRESENTATION: Our patient was a 17-year-old female who initially developed urosepsis caused by extended-spectrum ß-lactamase (ESBL) Escherichia coli , while being treated with an immunosuppressant regimen because of kidney rejection secondary to poor immunosuppression therapy compliance. She was admitted to our hospital intermittently for 4 months with 4 episodes of urosepsis caused by ESBL E. coli . She received multiple courses of antibiotics (mainly ertapenem) and underwent a fecal material transplant to eradicate her ESBL E. coli colonized gastrointestinal tract. Because of recurrent development of urosepsis after antibiotic treatment, she later underwent treatment with a phage cocktail consisting of 2 isolate-specific phages. After a prolonged antibiotic course and subsequent 3-week intravenous phage treatment, she had no ESBL E. coli in her urinary cultures for 4 years post-treatment. DISCUSSION: This case highlights the challenges of treating recurrent ESBL E. coli infections in a pediatric renal transplant patient and provides evidence that phage therapy may prove useful in such cases.

Diversity of SIRV-like Viruses from a North American Population.

A small subset of acidic hot springs sampled in Yellowstone National Park yielded rod-shaped viruses which lysed liquid host cultures and formed clear plaques on lawns of host cells. Three isolates chosen for detailed analysis were found to be genetically related to previously described isolates of the Sulfolobus islandicus rod-shaped virus (SIRV), but distinct from them and from each other. Functional stability of the new isolates was assessed in a series of inactivation experiments. UV-C radiation inactivated one of the isolates somewhat faster than bacteriophage λ, suggesting that encapsidation in the SIRV-like virion did not confer unusual protection of the DNA from UV damage. With respect to high temperature, the new isolates were extremely, but not equally, stable. Several chemical treatments were found to inactivate the virions and, in some cases, to reveal apparent differences in virion stability among the isolates. Screening a larger set of isolates identified greater variation of these stability properties but found few correlations among the resulting profiles. The majority of host cells infected by the new isolates were killed, but survivors exhibited heritable resistance, which could not be attributed to CRISPR spacer acquisition or the loss of the pilus-related genes identified by earlier studies. Virus-resistant host variants arose at high frequency and most were resistant to multiple viral strains; conversely, resistant host clones generated virus-sensitive variants, also at high frequency. Virus-resistant cells lacked the ability of virus-sensitive cells to bind virions in liquid suspensions. Rapid interconversion of sensitive and resistant forms of a host strain suggests the operation of a yet-unidentified mechanism that acts to allow both the lytic virus and its host to propagate in highly localized natural populations, whereas variation of virion-stability phenotypes among the new viral isolates suggests that multiple molecular features contribute to the biological durability of these viruses.

Combination of phage therapy and cefiderocol to successfully treat Pseudomonas aeruginosa cranial osteomyelitis.

Background: Pseudomonas aeruginosa has the ability to exhibit resistance to a broad range of antibiotics, highlighting the importance of identifying alternative or adjunctive treatment options, such as phages. Patients and methods: We report the case of a 25-year-old male who experienced an accidental electrocution resulting in exposed calvarium in the left parieto-temporal region, complicated by a difficult-to-treat P. aeruginosa (DTR-P. aeruginosa) infection. Cefiderocol was the sole antibiotic with consistent activity against six bacterial isolates obtained from the infected region over a 38 day period. Results: WGS analysis identified a bla GES-1 gene as well as the MDR efflux pumps MexD and MexX in all six of the patient's ST235 DTR-P. aeruginosa isolates, when compared with the reference genome P. aeruginosa PA01 and a P. aeruginosa ST235 isolate from an unrelated patient. After debridement of infected scalp and bone, the patient received approximately 6 weeks of cefiderocol in conjunction with IV phage Pa14NPøPASA16. Some improvement was observed after the initiation of cefiderocol; however, sustained local site improvement and haemodynamic stability were not achieved until phage was administered. No medication-related toxicities were observed. The patient remains infection free more than 12 months after completion of therapy. Conclusions: This report adds to the growing literature that phage therapy may be a safe and effective approach to augment antibiotic therapy for patients infected with drug-resistant pathogens. Furthermore, it highlights the importance of the GES ß-lactamase family in contributing to inactivation of a broad range of ß-lactam antibiotics in P. aeruginosa, including ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam.

Case Report: successful use of phage therapy in refractory MRSA chronic rhinosinusitis.

We present a case of refractory methicillin-resistant Staphylococcus aureus that was successfully treated with a combination of antibiotics, systemic phage and intranasal phage therapy.

Impact of Bacterial Phenotypic Variation with Bacteriophage therapy: A Pilot Study with Prosthetic Joint Infection Isolates.

BACKGROUND: Given the specificity of bacteriophage attachment receptors, a single bacterial isolate is currently utilized to match to a bacteriophage therapeutic, thereby extrapolating activity to all bacteria in vivo. Consistently, the main bacteriophage attachment receptor for Staphylococcus aureus is teichoic acid, and it is known that this receptor has phenotypic variations in different in vivo environments. Consequently, the aim of this study was to determine whether bacteriophage activity is similar across all in vivo prosthetic joint infection environments. METHODS: Three patients with prosthetic joint infections who had S. aureus grow from arthrocentesis cultures and at least three deep tissue cultures were analyzed for growth inhibition with a library of 56 bacteriophages. RESULTS: Discordant bacteriophage activity was seen across the different in vivo environments. Furthermore, bacteriophages with the most robust lytic potential to the arthrocentesis isolates usually did not demonstrate activity corresponding to all the deep tissue clinical isolates. CONCLUSION: Variations of bacteriophage activity can occur between the different in vivo clinical environments, which is likely secondary to different glycosylation patterns of teichoic acid. Consequently, if discordant activity is present, retreating with bacteriophages that have activity is likely needed for effective, reproducible outcomes.

Critically Ill Patient with Multidrug-Resistant Acinetobacter baumannii Respiratory Infection Successfully Treated with Intravenous and Nebulized Bacteriophage Therapy.

Hospitalized patients are at risk of developing serious multidrug resistant bacterial infections. This risk is heightened in patients who are on mechanical ventilation, are immunocompromised, and/or have chronic comorbidities. We report the case of a 52-year-old critically ill patient with a multidrug resistant Acinetobacter baumannii (MDR-A) respiratory infection who was successfully treated with antibiotics and intravenous and nebulized bacteriophage therapy.

Successful Treatment of Staphylococcus aureus Prosthetic Joint Infection with Bacteriophage Therapy.

Successful joint replacement is a life-enhancing procedure with significant growth in the past decade. Prosthetic joint infection occurs rarely; it is a biofilm-based infection that is poorly responsive to antibiotic alone. Recent interest in bacteriophage therapy has made it possible to treat some biofilm-based infections, as well as those caused by multidrug-resistant pathogens, successfully when conventional antibiotic therapy has failed. Here, we describe the case of a 61-year-old woman who was successfully treated after a second cycle of bacteriophage therapy administered at the time of a two-stage exchange procedure for a persistent methicillin-sensitive Staphylococcus aureus (MSSA) prosthetic knee-joint infection. We highlight the safety and efficacy of both intravenous and intra-articular infusions of bacteriophage therapy, a successful outcome with a single lytic phage, and the development of serum neutralization with prolonged treatment.

Phage Therapy for Limb-threatening Prosthetic Knee Klebsiella pneumoniae Infection: Case Report and In Vitro Characterization of Anti-biofilm Activity.

BACKGROUND: Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention. METHODS: The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient's isolate and determined by safranin staining. RESULTS: Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections.

Combining bacteriophages with cefiderocol and meropenem/vaborbactam to treat a pan-drug resistant Achromobacter species infection in a pediatric cystic fibrosis patient.

Cystic fibrosis is associated with significant morbidity and early mortality due to recurrent acute and chronic lung infections. The chronic use of multiple antibiotics without pathogen eradication increases the possibility of extensive drug resistance or even pan-drug resistance (PDR). It is imperative that new or alternative treatment options be explored. We present a clinical case of a 10-year-old female cystic fibrosis patient, infected with a PDR Achromobacter spp. She was treated with cefiderocol, meropenem/vaborbactam, and bacteriophage therapy (Ax2CJ45ϕ2) during two separate admissions in an attempt to clear her infection and restore baseline pulmonary function. The Centers for Disease Control and Prevention confirmed antibiotic susceptibilities, which showed resistance to both cefiderocol and meropenem/vaborbactam. However, after using all three agents concomitantly during the second treatment course, our patient's pulmonary function improved dramatically, and the Achromobacter spp. could not be isolated from sputum samples obtained 8 and 16 weeks after completion of therapy. Overall, the treatment regimen consisting of cefiderocol, meropenem/vaborbactam, and bacteriophage was safe and well-tolerated in our patient.

Salvage Bacteriophage Therapy for a Chronic MRSA Prosthetic Joint Infection.

This is a case of a 72 year old male with a chronic methicillin-resistant Staphylococcus aureus prosthetic joint infection. After the third intravenous dose of bacteriophage therapy, an unusual, reversible transaminitis prompted stoppage of bacteriophage therapy. Nevertheless, treatment was successful and the patient's severe chronic infection was eradicated.

Early clinical experience of bacteriophage therapy in 3 lung transplant recipients.

Bacteriophage therapy (BT) uses bacteriophages to treat pathogenic bacteria and is an emerging strategy against multidrug-resistant (MDR) infections. Experience in solid organ transplant is limited. We describe BT in 3 lung transplant recipients (LTR) with life-threatening MDR infections caused by Pseudomonas aeruginosa (n = 2) and Burkholderia dolosa (n = 1). For each patient, lytic bacteriophages were selected against their bacterial isolates. BT was administered for variable durations under emergency Investigational New Drug applications and with patient informed consent. Safety was assessed using clinical/laboratory parameters and observed clinical improvements described, as appropriate. All patients received concurrent antibiotics. Two ventilator-dependent LTR with large airway complications and refractory MDR P. aeruginosa pneumonia received BT. Both responded clinically and were discharged from the hospital off ventilator support. A third patient had recurrent B. dolosa infection following transplant. Following BT initiation, consolidative opacities improved and ventilator weaning was begun. However, infection relapsed on BT and the patient died. No BT-related adverse events were identified in the 3 cases. BT was well tolerated and associated with clinical improvement in LTRs with MDR bacterial infection not responsive to antibiotics alone. BT may be a viable adjunct to antibiotics for patients with MDR infections.

Successful Treatment of Antibiotic-resistant, Poly-microbial Bone Infection With Bacteriophages and Antibiotics Combination.

A patient with a trauma-related left tibial infection associated with extensively drug-resistant Acinetobacter baumannii and multidrug-resistant Klebsiella pneumoniae was treated with bacteriophages and antibiotics. There was rapid tissue healing and positive culture eradication. As a result, the patient's leg did not have to be amputated and he is undergoing rehabilitation.