Cystic fibrosis pathogens persist in the upper respiratory tract following initiation of elexacaftor/tezacaftor/ivacaftor therapy.

Elexacaftor/tezacaftor/ivacaftor (ETI) therapy has revolutionized the treatment of cystic fibrosis (CF) for most affected individuals but the effects of treatment on sinus microbiota are still unknown. Changes to the airway microbiota in CF are associated with disease state and alterations to the bacterial community after ETI initiation may require changes to clinical management regimens. We collected sinus swab samples from the middle meatus in an observational study of 38 adults with CF and chronic rhinosinusitis (CRS) from 2017 to 2021 and captured the initiation of ETI therapy. We performed 16S and custom amplicon sequencing to characterize the sinus microbiota pre- and post-ETI. Real-time quantitative PCR (RT-qPCR) was performed to estimate total bacterial abundance. Sinus samples from people with CF (pwCF) clustered into three community types, dependent on the dominant bacterial organism: a Pseudomonas-dominant, Staphylococcus-dominant, and mixed dominance cluster. Shannon's diversity index was low and not significantly altered post-ETI. Total bacterial load was not significantly lowered post-ETI. Pseudomonas spp. abundance was significantly reduced post-ETI, but eradication was not observed. Staphylococcus spp. became the dominant organism in most individuals post-ETI and we showed the presence of methicillin-resistant Staphylococcus aureus (MRSA) in the sinus both pre- and post-ETI. We also demonstrated that the sinus microbiome is predictive of the presence of Pseudomonas spp., Staphylococcus spp., and Serratia spp. in the sputum. Pseudomonas spp. and Staphylococcus spp., including MRSA, persist in the sinuses of pwCF after ETI therapy, indicating that these pathogens will continue to be important in CF airway disease management in the era of highly effective modulator therapies (HEMT).IMPORTANCEHighly effective modulator therapies (HEMT), such as elexacaftor/tezacaftor/ivacaftor (ETI), for cystic fibrosis (CF) have revolutionized patient care and quality of life for most affected individuals. The effects of these therapies on the microbiota of the airways are still unclear, though work has already been published on changes to microbiota in the sputum. Our study presents evidence for reduced relative abundance of Pseudomonas spp. in the sinuses following ETI therapy. We also show that Staphylococcus spp. becomes the dominant organism in the sinus communities of most individuals in this cohort after ETI therapy. We identified methicillin-resistant Staphylococcus aureus (MRSA) in the sinus microbiota both pre- and post-therapy. These findings demonstrate that pathogen monitoring and treatment will remain a vital part of airway disease management for people with cystic fibrosis (pwCF) in the era of HEMT.

Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm.

Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF.

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.

Persistence and evolution of Pseudomonas aeruginosa following initiation of highly effective modulator therapy in cystic fibrosis.

Today, more than 90% of people with cystic fibrosis (pwCF) are eligible for the highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy called elexacaftor/tezacaftor/ivacaftor (ETI) and its use is widespread. Given the drastic respiratory symptom improvement experienced by many post-ETI, clinical studies are already underway to reduce the number of respiratory therapies, including antibiotic regimens, that pwCF historically relied on to combat lung disease progression. Early studies suggest that bacterial burden in the lungs is reduced post-ETI, yet it is unknown how chronic Pseudomonas aeruginosa populations are impacted by ETI. We found that pwCF remain infected throughout their upper and lower respiratory tract with their same strain of P. aeruginosa post-ETI, and these strains continue to evolve in response to the newly CFTR-corrected airway. Our work underscores the continued importance of CF airway microbiology in the new era of highly effective CFTR modulator therapy. IMPORTANCE: The highly effective cystic fibrosis transmembrane conductance regulator modulator therapy Elexakaftor/Tezacaftor/Ivacaftor (ETI) has changed cystic fibrosis (CF) disease for many people with cystic fibrosis. While respiratory symptoms are improved by ETI, we found that people with CF remain infected with Pseudomonas aeruginosa. How these persistent and evolving bacterial populations will impact the clinical manifestations of CF in the coming years remains to be seen, but the role and potentially changing face of infection in CF should not be discounted in the era of highly effective modulator therapy.

Effects of highly effective modulator therapy on the dynamics of the respiratory mucosal environment and inflammatory response in cystic fibrosis.

BACKGROUND: While the widespread initiation of elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic clinical improvements among persons with cystic fibrosis (pwCF), little is known about how ETI affects the respiratory mucosal inflammatory and physiochemical environment, or how these changes relate to lung function. METHODS: We performed a prospective, longitudinal study of adults with CF and chronic rhinosinusitis (CF-CRS) followed at our CF center (n = 18). Endoscopic upper respiratory tract (paranasal sinus) aspirates from multiple visit dates, both pre- and post-ETI initiation, were collected and tested for cytokines, metals, pH, and lactate levels. Generalized estimating equations were used to identify relationships between ETI and upper respiratory tract (URT) biomarker levels, and between URT biomarkers and lung function or clinical sinus parameters. RESULTS: ETI was associated with decreased upper respiratory mucosal cytokines B-cell activating factor (BAFF), IL-12p40, IL-32, IL-8, IL-22 and soluble tumor necrosis factor-1 (sTNFR1), and an increase in a proliferation-inducing ligand (APRIL) and IL-19. ETI was also associated with decreased URT levels of copper, manganese, and zinc. In turn, lower URT levels of BAFF, IL-8, lactate, and potassium were each associated with ~1.5% to 4.3% improved forced expiratory volume in 1 s (FEV1), while higher levels of IFNγ, iron, and selenium were associated with ~2% to 10% higher FEV1. CONCLUSIONS: Our observations suggest a dampening of inflammatory signals and restriction in microbial nutrients in the upper respiratory tract with ETI. These findings improve our understanding of how ETI impacts the mucosal environment in the respiratory tract, and may give insight into the improved infectious and inflammatory status and the resulting clinical improvements seen in pwCF.

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.

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

Bacterial respiratory inhibition triggers dispersal of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa grows as a biofilm under many environmental conditions, and the bacterium can disperse from biofilms via highly regulated, dynamic processes. However, physiologic triggers of biofilm dispersal remain poorly understood. Based on prior literature describing dispersal triggered by forms of starvation, we tested bacterial respiratory inhibitors for biofilm dispersal in two models resembling chronic airway infections. Our underlying hypothesis was that respiratory inhibitors could serve as a model for the downstream effects of starvation. We used two experimental conditions. In the first condition, biofilms were grown and dispersed from the surface of airway epithelial cells, and the second condition was a model where biofilms were grown on glass in cell culture media supplemented with host-relevant iron sources. In both biofilm models, the respiratory inhibitors potassium cyanide and sodium azide each triggered biofilm dispersal. We hypothesized that cyanide-induced dispersal was due to respiratory inhibition rather than signaling via an alternative mechanism, and, indeed, if respiration was supported by overexpression of cyanide-insensitive oxidase, dispersal was prevented. Dispersal required the activity of the cyclic-di-GMP regulated protease LapG, reinforcing the role of matrix degradation in dispersal. Finally, we examined the roles of individual phosphodiesterases, previously implicated in dispersal to specific triggers, and found signaling to be highly redundant. Combined deletion of the phosphodiesterases dipA, bifA, and rbdA was required to attenuate the dispersal phenotype. In summary, this work adds insight into the physiology of biofilm dispersal under environmental conditions in which bacterial respiration is abruptly limited. IMPORTANCE The bacterium Pseudomonas aeruginosa grows in biofilm communities that are very difficult to treat in human infections. Growing as a biofilm can protect bacteria from antibiotics and the immune system. Bacteria can leave a biofilm through a process called "dispersal." Dispersed bacteria seed new growth areas and are more susceptible to killing by antibiotics. The triggers for biofilm dispersal are not well understood, and if we understood dispersal better it might lead to the development of new treatments for infection. In this paper, we find that inhibiting P. aeurginosa's ability to respire (generate energy) can trigger dispersal from a biofilm grown in association with human respiratory epithelial cells in culture. The dispersal process requires a protease which is previously known to degrade the biofilm matrix. These findings give us a better understanding of how the biofilm dispersal process works so that future research can discover better ways of clearing bacteria growing in biofilms.

Bacterial Outer Membrane Vesicles and Immune Modulation of the Host.

This article reviews the role of outer membrane vesicles (OMVs) in mediating the interaction between Gram-negative bacteria and their human hosts. OMVs are produced by a diverse range of Gram-negative bacteria during infection and play a critical role in facilitating host-pathogen interactions without requiring direct cell-to-cell contact. This article describes the mechanisms by which OMVs are formed and subsequently interact with host cells, leading to the transport of microbial protein virulence factors and short interfering RNAs (sRNA) to their host targets, exerting their immunomodulatory effects by targeting specific host signaling pathways. Specifically, this review highlights mechanisms by which OMVs facilitate chronic infection through epigenetic modification of the host immune response. Finally, this review identifies critical knowledge gaps in the field and offers potential avenues for future OMV research, specifically regarding rigor and reproducibility in OMV isolation and characterization methods.

Antiviral epithelial-macrophage crosstalk permits secondary bacterial infections.

IMPORTANCE: Miscommunication of antiviral and antibacterial immune signals drives worsened morbidity and mortality during respiratory viral-bacterial coinfections. Extracellular vesicles (EVs) are a form of intercellular communication with broad implications during infection, and here we show that epithelium-derived EVs released during the antiviral response impair the antibacterial activity of macrophages, an innate immune cell crucial for bacterial control in the airway. Macrophages exposed to antiviral EVs display reduced clearance of Staphylococcus aureus as well as altered inflammatory signaling and anti-inflammatory metabolic reprogramming, thus revealing EVs as a source of dysregulated epithelium-macrophage crosstalk during coinfection. As effective epithelium-macrophage communication is critical in mounting an appropriate immune response, this novel observation of epithelium-macrophage crosstalk shaping macrophage metabolism and antimicrobial function provides exciting new insight and improves our understanding of immune dysfunction during respiratory coinfections.

Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression.

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift.

Staphylococcal secreted cytotoxins are competition sensing signals for Pseudomonas aeruginosa.

Coinfection with two notorious opportunistic pathogens, the Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus , dominates chronic pulmonary infections. While coinfection is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. Here, we dissected molecular mechanisms of interspecies sensing between P. aeruginosa and S. aureus . We discovered that P. aeruginosa senses S. aureus secreted peptides and, counterintuitively, moves towards these toxins. P. aeruginosa tolerates such a strategy through "competition sensing", whereby it preempts imminent danger/competition by arming cells with type six secretion (T6S) and iron acquisition systems. Intriguingly, while T6S is predominantly described as weaponry targeting Gram-negative and eukaryotic cells, we find that T6S is essential for full P. aeruginosa competition with S. aureus , a previously undescribed role for T6S. Importantly, competition sensing was activated during coinfection of bronchial epithelia, including T6S islands targeting human cells. This study reveals critical insight into both interspecies competition and how antagonism may cause collateral damage to the host environment.

Microbial and Immune Regulation of the Gut-Lung Axis during Viral-Bacterial Coinfection.

Viral-bacterial coinfections of the respiratory tract have long been associated with worsened disease outcomes. Clinical and basic research studies demonstrate that these infections are driven via complex interactions between the infecting pathogens, microbiome, and host immune response, although how these interactions contribute to disease progression is still not fully understood. Research over the last decade shows that the gut has a significant role in mediating respiratory outcomes, in a phenomenon known as the "gut-lung axis." Emerging literature demonstrates that acute respiratory viruses can modulate the gut-lung axis, suggesting that dysregulation of gut-lung cross talk may be a contributing factor during respiratory coinfection. This review will summarize the current literature regarding modulation of the gut-lung axis during acute respiratory infection, with a focus on the role of the microbiome, secondary infections, and the host immune response.

A Novel Provider Education Module to Enhance Detection of Alpha-1 Antitrypsin Deficiency.

Background: Alpha-1 antitrypsin deficiency (AATD) is the most common genetic risk factor for early-onset emphysema. However, AATD continues to be underrecognized and underdiagnosed. Provider awareness about AATD, concerns with testing costs, and limited understanding about therapeutic options contribute to its underdiagnosis. We hypothesized that provider education would improve awareness of AATD and improve screening. Objective: To evaluate the impact of a targeted provider education module on AATD screening. Methods: We developed a web-based education module to address barriers to screening for AATD, deployed the education module using the Medscape Education platform, assessed perceived healthcare provider confidence in AATD screening, and conducted a prospective pre and postintervention study of AATD testing practices at a high-volume academic outpatient subspecialty pulmonary clinic. Results: A total of 11,385 healthcare providers, including eight pulmonologists at our institution, completed the web-based education module. Confidence in identifying patients at high risk for AATD improved after completing the module ("not confident" in AATD screening was 7.7% postintervention compared with 19.4% preintervention). The rate of screening patients at high risk for AATD improved more than twofold (AATD screening rate 9.7% preintervention vs. 20.4% postintervention; P = 0.004). Among patients screened for AATD in our cohort, 27.2% had a genotype/phenotype or low alpha-1 antitrypsin concentration consistent with AATD. Conclusion: Targeted healthcare provider education can improve the confidence in testing for AATD. Improvements in provider confidence corresponded to improvements in AATD screening in a subspecialty pulmonary clinic. More than one-fourth of screening tests suggested AATD, underpinning the value of testing in high-risk individuals.

Low Diversity and Instability of the Sinus Microbiota over Time in Adults with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1ß [IL-1ß]). Increased IL-1ß was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1ß in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1ß. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Novel Requirement for Staphylococcal Cell Wall-Anchored Protein SasD in Pulmonary Infection.

Staphylococcus aureus can complicate preceding viral infections, including influenza virus. A bacterial infection combined with a preceding viral infection, known as superinfection, leads to worse outcomes than a single infection. Most of the pulmonary infection literature focuses on the changes in immune responses to bacteria between homeostatic and virally infected lungs. However, it is unclear how much of an influence bacterial virulence factors have in single or superinfection. Staphylococcal species express a broad range of cell wall-anchored proteins (CWAs) that have roles in host adhesion, nutrient acquisition, and immune evasion. We screened the importance of these CWAs using mutants lacking individual CWAs in vivo in both bacterial pneumonia and influenza superinfection. In bacterial pneumonia, the lack of individual CWAs leads to various decreases in bacterial burden, lung damage, and immune infiltration into the lung. However, the presence of a preceding influenza infection partially abrogates the requirement for CWAs. In the screen, we found that the uncharacterized CWA S. aureus surface protein D (SasD) induced changes in both inflammatory and homeostatic lung markers. We further characterized a SasD mutant (sasD A50.1) in the context of pneumonia. Mice infected with sasD A50.1 have decreased bacterial burden, inflammatory responses, and mortality compared to wild-type S. aureus. Mice also have reduced levels of interleukin-1ß (IL-1ß), likely derived from macrophages. Reductions in IL-1ß transcript levels as well as increased macrophage viability point at differences in cell death pathways. These data identify a novel virulence factor for S. aureus that influences inflammatory signaling within the lung. IMPORTANCE Staphylococcus aureus is a common commensal bacterium that can cause severe infections, such as pneumonia. In the lung, viral infections increase the risk of staphylococcal pneumonia, leading to combined infections known as superinfections. The most common virus associated with S. aureus pneumonia is influenza, and superinfections lead to worse patient outcomes than either infection alone. While there is much known about how the immune system differs between healthy and virally infected lungs, the role of bacterial virulence factors in single and superinfection is less understood. The significance of our research is identifying bacterial components that play a role in the initiation of lung injury, which could lead to future therapies to prevent pulmonary single or superinfection with S. aureus.

Genomic characterization of lytic bacteriophages targeting genetically diverse Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa infections can be difficult to treat and new therapeutics are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and used them to screen and isolate over a dozen P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. We evolved bacterial mutants that were resistant to phage infection for four different phages, and used genome sequencing and functional analysis to study them further. We also tested phages for their ability to kill P. aeruginosa grown in biofilms in vitro and ex vivo on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

SprayNPray: user-friendly taxonomic profiling of genome and metagenome contigs.

BACKGROUND: Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. RESULTS: While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semi-automated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. CONCLUSIONS: The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.

Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis.

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution.