Experimental Evolution Studies in Φ6 Cystovirus.

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

Neutralizing antibodies after nebulized phage therapy in cystic fibrosis patients.

BACKGROUND: Cystic fibrosis (CF) patients are prone to recurrent multi-drug-resistant (MDR) bacterial lung infections. Under this scenario, phage therapy has been proposed as a promising tool. However, the limited number of reported cases hampers the understanding of clinical outcomes. Anti-phage immune responses have often been overlooked and only described following invasive routes of administration. METHODS: Three monophage treatments against Staphylococcus aureus and/or Pseudomonas aeruginosa lung infections were conducted in cystic fibrosis patients. In-house phage preparations were nebulized over 10 days with standard-of-care antibiotics. Clinical indicators, bacterial counts, phage and antibiotic susceptibility, phage detection, and immune responses were monitored. FINDINGS: Bacterial load was reduced by 3-6 log in two of the treatments. No adverse events were described. Phages remained in sputum up to 33 days after completion of the treatment. In all cases, phage-neutralizing antibodies were detected in serum from 10 to 42 days post treatment, with this being the first report of anti-phage antibodies after nebulized therapy. CONCLUSIONS: Nebulized phage therapy reduced bacterial load, improving quality of life even without bacterial eradication. The emergence of antibodies emphasizes the importance of long-term monitoring to better understand clinical outcomes. These findings encourage the use of personalized monophage therapies in contrast to ready-to-use cocktails, which might induce undesirable antibody generation. FUNDING: This study was supported by the Spanish Ministry of Science, Innovation and Universities; Generalitat Valenciana; and a crowdfunding in collaboration with the Spanish Cystic Fibrosis Foundation.

Evolutionary Invasion Analysis of Modern Epidemics Highlights the Context-Dependence of Virulence Evolution.

Models are often employed to integrate knowledge about epidemics across scales and simulate disease dynamics. While these approaches have played a central role in studying the mechanics underlying epidemics, we lack ways to reliably predict how the relationship between virulence (the harm to hosts caused by an infection) and transmission will evolve in certain virus-host contexts. In this study, we invoke evolutionary invasion analysis-a method used to identify the evolution of uninvadable strategies in dynamical systems-to examine how the virulence-transmission dichotomy can evolve in models of virus infections defined by different natural histories. We reveal peculiar patterns of virulence evolution between epidemics with different disease natural histories (SARS-CoV-2 and hepatitis C virus). We discuss the findings with regards to the public health implications of predicting virus evolution, and in broader theoretical canon involving virulence evolution in host-parasite systems.

Robustness in population-structure and demographic-inference results derived from the Aedes aegypti genotyping chip and whole-genome sequencing data.

The mosquito Aedes aegypti is the primary vector of many human arboviruses such as dengue, yellow fever, chikungunya, and Zika, which affect millions of people worldwide. Population genetic studies on this mosquito have been important in understanding its invasion pathways and success as a vector of human disease. The Axiom aegypti1 SNP chip was developed from a sample of geographically diverse A. aegypti populations to facilitate genomic studies on this species. We evaluate the utility of the Axiom aegypti1 SNP chip for population genetics and compare it with a low-depth shotgun sequencing approach using mosquitoes from the native (Africa) and invasive ranges (outside Africa). These analyses indicate that results from the SNP chip are highly reproducible and have a higher sensitivity to capture alternative alleles than a low-coverage whole-genome sequencing approach. Although the SNP chip suffers from ascertainment bias, results from population structure, ancestry, demographic, and phylogenetic analyses using the SNP chip were congruent with those derived from low-coverage whole-genome sequencing, and consistent with previous reports on Africa and outside Africa populations using microsatellites. More importantly, we identified a subset of SNPs that can be reliably used to generate merged databases, opening the door to combined analyses. We conclude that the Axiom aegypti1 SNP chip is a convenient, more accurate, low-cost alternative to low-depth whole-genome sequencing for population genetic studies of A. aegypti that do not rely on full allelic frequency spectra. Whole-genome sequencing and SNP chip data can be easily merged, extending the usefulness of both approaches.

Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells.

Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

Lytic bacteriophages interact with respiratory epithelial cells and induce the secretion of antiviral and proinflammatory cytokines.

Phage therapy is a therapeutic approach to treat multidrug resistant infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. We determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

Optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa at Yale University.

Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d'Hérelle's first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.

The future of evolutionary medicine: sparking innovation in biomedicine and public health.

Evolutionary medicine - i.e. the application of insights from evolution and ecology to biomedicine - has tremendous untapped potential to spark transformational innovation in biomedical research, clinical care and public health. Fundamentally, a systematic mapping across the full diversity of life is required to identify animal model systems for disease vulnerability, resistance, and counter-resistance that could lead to novel clinical treatments. Evolutionary dynamics should guide novel therapeutic approaches that target the development of treatment resistance in cancers (e.g., via adaptive or extinction therapy) and antimicrobial resistance (e.g., via innovations in chemistry, antimicrobial usage, and phage therapy). With respect to public health, the insight that many modern human pathologies (e.g., obesity) result from mismatches between the ecologies in which we evolved and our modern environments has important implications for disease prevention. Life-history evolution can also shed important light on patterns of disease burden, for example in reproductive health. Experience during the COVID-19 (SARS-CoV-2) pandemic has underlined the critical role of evolutionary dynamics (e.g., with respect to virulence and transmissibility) in predicting and managing this and future pandemics, and in using evolutionary principles to understand and address aspects of human behavior that impede biomedical innovation and public health (e.g., unhealthy behaviors and vaccine hesitancy). In conclusion, greater interdisciplinary collaboration is vital to systematically leverage the insight-generating power of evolutionary medicine to better understand, prevent, and treat existing and emerging threats to human, animal, and planetary health.

Complete Genome Assembly and Annotation of Escherichia coli Bacteriophage 55 from Rivière la Quint in Gonaïves, Haiti.

We present the structural and functional annotation of Escherichia coli bacteriophage 55, which has a genome length of 170,393 bp, with 219 predicted genes.

Developing Phage Therapy That Overcomes the Evolution of Bacterial Resistance.

The global rise of antibiotic resistance in bacterial pathogens and the waning efficacy of antibiotics urge consideration of alternative antimicrobial strategies. Phage therapy is a classic approach where bacteriophages (bacteria-specific viruses) are used against bacterial infections, with many recent successes in personalized medicine treatment of intractable infections. However, a perpetual challenge for developing generalized phage therapy is the expectation that viruses will exert selection for target bacteria to deploy defenses against virus attack, causing evolution of phage resistance during patient treatment. Here we review the two main complementary strategies for mitigating bacterial resistance in phage therapy: minimizing the ability for bacterial populations to evolve phage resistance and driving (steering) evolution of phage-resistant bacteria toward clinically favorable outcomes. We discuss future research directions that might further address the phage-resistance problem, to foster widespread development and deployment of therapeutic phage strategies that outsmart evolved bacterial resistance in clinical settings.

Complete Genome Assembly and Annotation of Escherichia coli Bacteriophage 107.

We present the annotated genome sequence of Escherichia coli bacteriophage 107, a T4-like bacteriophage. Phage 107 has a genome length of 167,509 bp and 287 predicted genes.

Experimental Evolution of the TolC-Receptor Phage U136B Functionally Identifies a Tail Fiber Protein Involved in Adsorption through Strong Parallel Adaptation.

Bacteriophages have received recent attention for their therapeutic potential to treat antibiotic-resistant bacterial infections. One particular idea in phage therapy is to use phages that not only directly kill their bacterial hosts but also rely on particular bacterial receptors, such as proteins involved in virulence or antibiotic resistance. In such cases, the evolution of phage resistance would correspond to the loss of those receptors, an approach termed evolutionary steering. We previously found that during experimental evolution, phage U136B can exert selection pressure on Escherichia coli to lose or modify its receptor, the antibiotic efflux protein TolC, often resulting in reduced antibiotic resistance. However, for TolC-reliant phages like U136B to be used therapeutically, we also need to study their own evolutionary potential. Understanding phage evolution is critical for the development of improved phage therapies as well as the tracking of phage populations during infection. Here, we characterized phage U136B evolution in 10 replicate experimental populations. We quantified phage dynamics that resulted in five surviving phage populations at the end of the 10-day experiment. We found that phages from all five surviving populations had evolved higher rates of adsorption on either ancestral or coevolved E. coli hosts. Using whole-genome and whole-population sequencing, we established that these higher rates of adsorption were associated with parallel molecular evolution in phage tail protein genes. These findings will be useful in future studies to predict how key phage genotypes and phenotypes influence phage efficacy and survival despite the evolution of host resistance. IMPORTANCE Antibiotic resistance is a persistent problem in health care and a factor that may help maintain bacterial diversity in natural environments. Bacteriophages ("phages") are viruses that specifically infect bacteria. We previously discovered and characterized a phage called U136B, which infects bacteria through TolC. TolC is an antibiotic resistance protein that helps bacteria pump antibiotics out of the cell. Over short timescales, phage U136B can be used to evolutionarily "steer" bacterial populations to lose or modify the TolC protein, sometimes reducing antibiotic resistance. In this study, we investigate whether U136B itself evolves to better infect bacterial cells. We discovered that the phage can readily evolve specific mutations that increase its infection rate. This work will be useful for understanding how phages can be used to treat bacterial infections.

Differentiation of Spontaneous Bacterial Peritonitis from Secondary Peritonitis in Patients with Liver Cirrhosis: Retrospective Multicentre Study.

Ascitic fluid infection is a serious complication of liver cirrhosis. The distinction between the more common spontaneous bacterial peritonitis (SBP) and the less common secondary peritonitis in patients with liver cirrhosis is crucial due to the varying treatment approaches. This retrospective multicentre study was conducted in three German hospitals and analysed 532 SBP episodes and 37 secondary peritonitis episodes. Overall, >30 clinical, microbiological, and laboratory parameters were evaluated to identify key differentiation criteria. Microbiological characteristics in ascites followed by severity of illness and clinicopathological parameters in ascites were the most important predictors identified by a random forest model to distinguish between SBP and secondary peritonitis. To establish a point-score model, a least absolute shrinkage and selection operator (LASSO) regression model selected the ten most promising discriminatory features. By aiming at a sensitivity of 95% either to rule out or rule in SBP episodes, two cut-off scores were defined, dividing patients with infected ascites into a low-risk (score ≥ 45) and high-risk group (score < 25) for secondary peritonitis. Overall, the discrimination of secondary peritonitis from SBP remains challenging. Our univariable analyses, random forest model, and LASSO point score may help clinicians with the crucial differentiation between SBP and secondary peritonitis.

Inhaled Bacteriophage Therapy for Multi-Drug Resistant Achromobacter.

The rise of antimicrobial resistant (AMR) bacteria is a global public health threat. AMR Achromobacter bacteria pose a challenging clinical problem, particularly for those with cystic fibrosis (CF) who are predisposed to chronic bacterial lung infections. Lytic bacteriophages (phages) offer a potential alternative to treat AMR infections, with the possible benefit that phage selection for resistance in target bacteria might coincide with reduced pathogenicity. The result is a genetic "trade-off," such as increased sensitivity to chemical antibiotics, and/or decreased virulence of surviving bacteria that are phage resistant. Here, we show that two newly discovered lytic phages against Achromobacter were associated with stabilization of respiratory status when deployed to treat a chronic pulmonary infection in a CF patient using inhaled (nebulized) phage therapy. The two phages demonstrate traits that could be generally useful in their development as therapeutics, especially the possibility that the phages can select for clinically useful trade-offs if bacteria evolve phage resistance following therapy. We discuss the limitations of the current study and suggest further work that should explore whether the phages could be generally useful in targeting pulmonary or other Achromobacter infections in CF patients.

Paul E. Turner.

Interview with Paul E. Turner of Yale University, who studies the evolutionary genetics of microbes.

A Novel Machine Learning-Based Point-Score Model as a Non-Invasive Decision-Making Tool for Identifying Infected Ascites in Patients with Hydropic Decompensated Liver Cirrhosis: A Retrospective Multicentre Study.

This study is aimed at assessing the distinctive features of patients with infected ascites and liver cirrhosis and developing a scoring system to allow for the accurate identification of patients not requiring abdominocentesis to rule out infected ascites. A total of 700 episodes of patients with decompensated liver cirrhosis undergoing abdominocentesis between 2006 and 2020 were included. Overall, 34 clinical, drug, and laboratory features were evaluated using machine learning to identify key differentiation criteria and integrate them into a point-score model. In total, 11 discriminatory features were selected using a Lasso regression model to establish a point-score model. Considering pre-test probabilities for infected ascites of 10%, 15%, and 25%, the negative and positive predictive values of the point-score model for infected ascites were 98.1%, 97.0%, 94.6% and 14.9%, 21.8%, and 34.5%, respectively. Besides the main model, a simplified model was generated, containing only features that are fast to collect, which revealed similar predictive values. Our point-score model appears to be a promising non-invasive approach to rule out infected ascites in clinical routine with high negative predictive values in patients with hydropic decompensated liver cirrhosis, but further external validation in a prospective study is needed.

Arms race and fluctuating selection dynamics in Pseudomonas aeruginosa bacteria coevolving with phage OMKO1.

Experimental evolution studies have examined coevolutionary dynamics between bacteria and lytic phages, where two models for antagonistic coevolution dominate: arms-race dynamics (ARD) and fluctuating-selection dynamics (FSD). Here, we tested the ability for Pseudomonas aeruginosa to coevolve with phage OMKO1 during 10 passages in the laboratory, whether ARD versus FSD coevolution occurred, and how coevolution affected a predicted phenotypic trade-off between phage resistance and antibiotic sensitivity. We used a unique "deep" sampling design, where 96 bacterial clones per passage were obtained from the three replicate coevolving communities. Next, we examined phenotypic changes in growth ability, susceptibility to phage infection and resistance to antibiotics. Results confirmed that the bacteria and phages coexisted throughout the study with one community undergoing ARD, whereas the other two showed evidence for FSD. Surprisingly, only the ARD bacteria demonstrated the anticipated trade-off. Whole genome sequencing revealed that treatment populations of bacteria accrued more de novo mutations, relative to a control bacterial population. Additionally, coevolved bacteria presented mutations in genes for biosynthesis of flagella, type-IV pilus and lipopolysaccharide, with three mutations fixing contemporaneously with the occurrence of the phenotypic trade-off in the ARD-coevolved bacteria. Our study demonstrates that both ARD and FSD coevolution outcomes are possible in a single interacting bacteria-phage system and that occurrence of predicted phage-driven evolutionary trade-offs may depend on the genetics underlying evolution of phage resistance in bacteria. These results are relevant for the ongoing development of lytic phages, such as OMKO1, in personalized treatment of human patients, as an alternative to antibiotics.

Assembly and Annotation of Escherichia coli Bacteriophage U115.

We present the annotated genome sequence of Escherichia coli bacteriophage U115, a T4-like bacteriophage. Phage U115 has a genome length of 166,986 bp and has 286 predicted genes.

Decay and damage of therapeutic phage OMKO1 by environmental stressors.

Antibiotic resistant bacterial pathogens are increasingly prevalent, driving the need for alternative approaches to chemical antibiotics when treating infections. One such approach is bacteriophage therapy: the use of bacteria-specific viruses that lyse (kill) their host cells. Just as the effect of environmental conditions (e.g. elevated temperature) on antibiotic efficacy is well-studied, the effect of environmental stressors on the potency of phage therapy candidates demands examination. Therapeutic phage OMKO1 infects and kills the opportunistic human pathogen Pseudomonas aeruginosa. Here, we used phage OMKO1 as a model to test how environmental stressors can lead to damage and decay of virus particles. We assessed the effects of elevated temperatures, saline concentrations, and urea concentrations. We observed that OMKO1 particles were highly tolerant to different saline concentrations, but decayed more rapidly at elevated temperatures and under high concentrations of urea. Additionally, we found that exposure to elevated temperature reduced the ability of surviving phage particles to suppress the growth of P. aeruginosa, suggesting a temperature-induced damage. Our findings demonstrate that OMKO1 is highly tolerant to a range of conditions that could be experienced inside and outside the human body, while also showing the need for careful characterization of therapeutic phages to ensure that environmental exposure does not compromise their expected potency, dosing, and pharmacokinetics.