Phage therapy: From biological mechanisms to future directions.

Increasing antimicrobial resistance rates have revitalized bacteriophage (phage) research, the natural predators of bacteria discovered over 100 years ago. In order to use phages therapeutically, they should (1) preferably be lytic, (2) kill the bacterial host efficiently, and (3) be fully characterized to exclude side effects. Developing therapeutic phages takes a coordinated effort of multiple stakeholders. Herein, we review the state of the art in phage therapy, covering biological mechanisms, clinical applications, remaining challenges, and future directions involving naturally occurring and genetically modified or synthetic phages.

HIV post-treatment controllers have distinct immunological and virological features.

HIV post-treatment controllers (PTCs) are rare individuals who maintain low levels of viremia after stopping antiretroviral therapy (ART). Understanding the mechanisms of HIV post-treatment control will inform development of strategies aiming at achieving HIV functional cure. In this study, we evaluated 22 PTCs from 8 AIDS Clinical Trials Group (ACTG) analytical treatment interruption (ATI) studies who maintained viral loads ≤400 copies/mL for ≥24 wk. There were no significant differences in demographics or frequency of protective and susceptible human leukocyte antigen (HLA) alleles between PTCs and post-treatment noncontrollers (NCs, n = 37). Unlike NCs, PTCs demonstrated a stable HIV reservoir measured by cell-associated RNA (CA-RNA) and intact proviral DNA assay (IPDA) during analytical treatment interruption (ATI). Immunologically, PTCs demonstrated significantly lower CD4+ and CD8+ T cell activation, lower CD4+ T cell exhaustion, and more robust Gag-specific CD4+ T cell responses and natural killer (NK) cell responses. Sparse partial least squares discriminant analysis (sPLS-DA) identified a set of features enriched in PTCs, including a higher CD4+ T cell% and CD4+/CD8+ ratio, more functional NK cells, and a lower CD4+ T cell exhaustion level. These results provide insights into the key viral reservoir features and immunological profiles for HIV PTCs and have implications for future studies evaluating interventions to achieve an HIV functional cure.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

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.

Phage Therapy for Antibiotic-Resistant Bacterial Infections.

Antibiotic resistance in bacterial pathogens presents a substantial threat to the control of infectious diseases. Development of new classes of antibiotics has slowed in recent years due to pressures of cost and market profitability, and there is a strong need for new antimicrobial therapies. The therapeutic use of bacteriophages has long been considered, with numerous anecdotal reports of success. Interest in phage therapy has been renewed by recent clinical successes in case studies with personalized phage cocktails, and several clinical trials are in progress. We discuss recent progress in the therapeutic use of phages and contemplate the key factors influencing the opportunities and challenges. With strong safety profiles, the main challenges of phage therapeutics involve strain variation among clinical isolates of many pathogens, battling phage resistance, and the potential limitations of host immune responses. However, the opportunities are considerable, with the potential to enhance current antibiotic efficacy, protect newly developed antibiotics, and provide a last resort in response to complete antibiotic failure.

Phylogenetic assessment within a species complex of a subterranean rodent (Geomys bursarius) with conservation implications for isolated subspecies.

Range contraction and expansion from glaciation has led to genetic divergence that may be particularly pronounced in fossorial species with low dispersal. The plains pocket gopher (Geomys bursarius) is a fossorial species that ranges widely across North America but has a poorly understood phylogeny. We used mitogenomes (14,996 base pairs) from 56 individuals across seven subspecies, plus two outgroup species, to assess genetic divergence from minimum spanning trees, measure genetic distances, and infer phylogenetic trees using BEAST. We found G. b. wisconsinensis was monophyletic with recent divergence. Further assessment is needed for G. b. major because it was paraphyletic and exhibited inconsistent groupings with other clades. Importantly, we identified G. b. illinoensis as being genetically distinct and monophyletic likely due to a unique colonization event eastward across the Mississippi River. Because G. b. illinoensis faces continued pressures from niche reduction and habitat loss, we recommend that G. b. illinoensis be considered an evolutionary significant unit warranting conservation actions to promote connectivity and restore suitable habitat. Such conservation efforts should benefit other grassland species including those originating from clades west of the Mississippi River that may also be evolutionary significant units.

Development of Host Immune Response to Bacteriophage in a Lung Transplant Recipient on Adjunctive Phage Therapy for a Multidrug-Resistant Pneumonia.

Bacteriophage therapy is the use of viruses to kill bacteria for the treatment of antibiotic-resistant infections. Little is known about the human immune response following phage therapy. We report the development of phage-specific CD4 T cells alongside rising phage-specific immunoglobulin G and neutralizing antibodies in response to adjunctive bacteriophage therapy used to treat a multidrug-resistant Pseudomonas aeruginosa pneumonia in a lung transplant recipient. Clinically, treatment was considered a success despite the development phage-specific immune responses.

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

BACKGROUND: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. METHODS: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. RESULTS: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. CONCLUSIONS: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

Jumbo phages are active against extensively drug-resistant eyedrop-associated Pseudomonas aeruginosa infections.

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

Susceptibility antibiotic screening reveals high rates of multidrug resistance of Salmonella, Shigella and Campylobacter in HIV infected and uninfected patients from Mozambique.

BACKGROUND: Antibacterial resistance is a growing concern worldwide, including in Mozambique. Diarrhea is an important cause of mortality in Mozambique, yet few local studies have reported on the resistance of bacterial pathogens in this context. Therefore, this study aims to characterize antibiotic susceptibility patterns of Salmonella, Shigella and Campylobacter spp. among patients with diarrhea, including those who are HIV-infected and-uninfected. METHODS: We conducted antibiotic susceptibility testing on 157 stool isolates recovered from 129 patients aged between 0 and 80 years with diarrhea, including HIV infected (n = 68) and-uninfected individuals (n = 61), assisted at two health centers in Maputo city. The isolates comprised of 99 Salmonella, 45 Shigella and 13 Campylobacter strains. The Kirby-Bauer disk diffusion method was used on Mueller-Hinton II agar for Salmonella and Shigella spp., while Mueller-Hinton II agar with 5% defibrinated sheep blood was used for Campylobacter spp. We tested six antibiotics listed on the national essential medicines list, including ciprofloxacin, erythromycin, azithromycin, trimethoprim-sulfamethoxazole, gentamicin, and tetracycline. RESULTS: All isolates were resistant to at least one antibiotic. A high percentage of Salmonella spp. isolates were found to be resistant to trimethoprim-sulfamethoxazole (89.9%, n = 89), erythromycin (88.9%, n = 88) and tetracycline (76.8%, n = 76). In addition, 86.6% (n = 39) and 68.9% (n = 31) of Shigella isolates were resistant to trimethoprim-sulfamethoxazole and tetracycline, respectively. The majority of Campylobacter isolates (92.3%, n = 12) were resistant to erythromycin, azithromycin and tetracycline. Multidrug resistance (MDR) was observed in 79.8% of Salmonella spp., 76.9% of Campylobacter spp., and 57.8% of Shigella spp. Drug susceptibility profiles for Salmonella spp. and Campylobacter were similar in both HIV-1 infected and uninfected patients. However, Shigella spp. isolates obtained from patients without HIV infection were significantly more likely to be resistant to erythromycin, azithromycin or to exhibit multidrug resistance than those obtained from patients with HIV-1 infection (p < 0.05). All Shigella spp. and Campylobacter spp. isolates were susceptible to gentamicin. CONCLUSION: Our study highlights concerning rates of antibiotic resistance and MDR among diarrheal bacterial pathogens in Mozambique. Further research is needed to understand the impact of HIV, ART therapy and immunosuppression on antibiotic resistance. Urgent interventions are essential to prevent the spread of resistant strains.

Evaluation of Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Mitigation Strategies on a University Campus Using an Agent-Based Network Model.

Universities are faced with decisions on how to resume campus activities while mitigating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) risk. To provide guidance for these decisions, we developed an agent-based network model of SARS-CoV-2 transmission to assess the potential impact of strategies to reduce outbreaks. The model incorporates important features related to risk at the University of California San Diego. We found that structural interventions for housing (singles only) and instructional changes (from in-person to hybrid with class size caps) can substantially reduce the basic reproduction number, but masking and social distancing are required to reduce this to at or below 1. Within a risk mitigation scenario, increased frequency of asymptomatic testing from monthly to twice weekly has minimal impact on average outbreak size (1.1-1.9), but substantially reduces the maximum outbreak size and cumulative number of cases. We conclude that an interdependent approach incorporating risk mitigation, viral detection, and public health intervention is required to mitigate risk.

Strategies and Progress in CXCR4-Targeted Anti-Human Immunodeficiency Virus (HIV) Therapeutic Development.

The acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV), has been a global public health challenge for several decades. The majority of HIV infection is caused by the human immunodeficiency virus type 1 (HIV-1), which enters and infects a host cell via the cell surface proteins of CD4 as the primary receptor, and chemokine receptors CXCR4 or CCR5 as the coreceptor-then undergoing replication using the cell's intracellular machinery. Whereas many drugs targeting CCR5-mediated entry or HIV-1 replication via reverse transcriptase or proteases have long been used clinically, agents targeting CXCR4 are yet to be advanced to clinical application. Here in this review we highlight some of the strategies for and progress made in the discovery of novel small molecules, peptides, and larger molecules that target CXCR4, and their future prospects for translation into the clinic as a new class of anti-HIV therapeutics.

COVID-19-Lessons Learned and Questions Remaining.

In this article, the editors of Clinical Infectious Diseases review some of the most important lessons they have learned about the epidemiology, clinical features, diagnosis, treatment and prevention of SARS-CoV-2 infection and identify essential questions about COVID-19 that remain to be answered.

Rethinking Remdesivir: Synthesis, Antiviral Activity, and Pharmacokinetics of Oral Lipid Prodrugs.

Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

Declines in rodent abundance and diversity track regional climate variability in North American drylands.

Regional long-term monitoring can enhance the detection of biodiversity declines associated with climate change, improving future projections by reducing reliance on space-for-time substitution and increasing scalability. Rodents are diverse and important consumers in drylands, regions defined by the scarcity of water that cover 45% of Earth's land surface and face increasingly drier and more variable climates. We analyzed abundance data for 22 rodent species across grassland, shrubland, ecotone, and woodland ecosystems in the southwestern USA. Two time series (1995-2006 and 2004-2013) coincided with phases of the Pacific Decadal Oscillation (PDO), which influences drought in southwestern North America. Regionally, rodent species diversity declined 20%-35%, with greater losses during the later time period. Abundance also declined regionally, but only during 2004-2013, with losses of 5% of animals captured. During the first time series (wetter climate), plant productivity outranked climate variables as the best regional predictor of rodent abundance for 70% of taxa, whereas during the second period (drier climate), climate best explained variation in abundance for 60% of taxa. Temporal dynamics in diversity and abundance differed spatially among ecosystems, with the largest declines in woodlands and shrublands of central New Mexico and Colorado. Which species were winners or losers under increasing drought and amplified interannual variability in drought depended on ecosystem type and the phase of the PDO. Fewer taxa were significant winners (18%) than losers (30%) under drought, but the identities of winners and losers differed among ecosystems for 70% of taxa. Our results suggest that the sensitivities of rodent species to climate contributed to regional declines in diversity and abundance during 1995-2013. Whether these changes portend future declines in drought-sensitive consumers in the southwestern USA will depend on the climate during the next major PDO cycle.

Herd Immunity Likely Protected the Men Who Have Sex With Men in the Recent Hepatitis A Outbreak in San Diego, California.

A high seroprevalence of hepatitis A virus (81%) among human immunodeficiency virus-negative high-risk men who have sex with men is likely why this community was largely spared from a recent hepatitis A virus outbreak in San Diego, California.

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.

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.

What's Old Is New Again: Bacteriophage Therapy in the 21st Century.

We highlight features associated with bacteriophage therapy that make it an attractive treatment option for multidrug-resistant infections and also discuss some of the challenges that need to be considered in the design and execution of clinical trials directed at evaluating the efficacy of bacteriophage therapy in humans.