Transmission of viable Haemophilus ducreyi by Musca domestica.

Haemophilus ducreyi was historically known as the causative agent of chancroid, a sexually-transmitted disease causing painful genital ulcers endemic in many low/middle-income nations. In recent years the species has been implicated as the causative agent of nongenital cutaneous ulcers affecting children of the South Pacific Islands and West African countries. Much is still unknown about the mechanism of H. ducreyi transmission in these areas, and recent studies have identified local insect species, namely flies, as potential transmission vectors. H. ducreyi DNA has been detected on the surface and in homogenates of fly species sampled from Lihir Island, Papua New Guinea. The current study develops a model system using Musca domestica, the common house fly, as a model organism to demonstrate proof of concept that flies are a potential vector for the transmission of viable H. ducreyi. Utilizing a green fluorescent protein (GFP)-tagged strain of H. ducreyi and three separate exposure methods, we detected the transmission of viable H. ducreyi by 86.11% ± 22.53% of flies sampled. Additionally, the duration of H. ducreyi viability was found to be directly related to the bacterial concentration, and transmission of H. ducreyi was largely undetectable within one hour of initial exposure. Push testing, Gram staining, and PCR were used to confirm the identity and presence of GFP colonies as H. ducreyi. This study confirms that flies are capable of mechanically transmitting viable H. ducreyi, illuminating the importance of investigating insects as vectors of cutaneous ulcerative diseases.

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.

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.

Polymicrobial interactions in the urinary tract: is the enemy of my enemy my friend?

The vast majority of research pertaining to urinary tract infection has focused on a single pathogen in isolation, and predominantly Escherichia coli. However, polymicrobial urine colonization and infection are prevalent in several patient populations, including individuals with urinary catheters. The progression from asymptomatic colonization to symptomatic infection and severe disease is likely shaped by interactions between traditional pathogens as well as constituents of the normal urinary microbiota. Recent studies have begun to experimentally dissect the contribution of polymicrobial interactions to disease outcomes in the urinary tract, including their role in development of antimicrobial-resistant biofilm communities, modulating the innate immune response, tissue damage, and sepsis. This review aims to summarize the epidemiology of polymicrobial urine colonization, provide an overview of common urinary tract pathogens, and present key microbe-microbe and host-microbe interactions that influence infection progression, persistence, and severity.

Enterococcus faecalis Polymicrobial Interactions Facilitate Biofilm Formation, Antibiotic Recalcitrance, and Persistent Colonization of the Catheterized Urinary Tract.

Indwelling urinary catheters are common in health care settings and can lead to catheter-associated urinary tract infection (CAUTI). Long-term catheterization causes polymicrobial colonization of the catheter and urine, for which the clinical significance is poorly understood. Through prospective assessment of catheter urine colonization, we identified Enterococcus faecalis and Proteus mirabilis as the most prevalent and persistent co-colonizers. Clinical isolates of both species successfully co-colonized in a murine model of CAUTI, and they were observed to co-localize on catheter biofilms during infection. We further demonstrate that P. mirabilis preferentially adheres to E. faecalis during biofilm formation, and that contact-dependent interactions between E. faecalis and P. mirabilis facilitate establishment of a robust biofilm architecture that enhances antimicrobial resistance for both species. E. faecalis may therefore act as a pioneer species on urinary catheters, establishing an ideal surface for persistent colonization by more traditional pathogens such as P. mirabilis.

Clinical predictors of cystic fibrosis chronic rhinosinusitis severity.

BACKGROUND: Chronic rhinosinusitis (CRS) is a significant manifestation of cystic fibrosis (CF) with wide-ranging symptom and disease severity. The goal of the study was to determine clinical variables that correlate with outcome measures of disease severity. METHODS: A prospective, longitudinal, observational study of 33 adults with symptomatic CRS treated in a CF-focused otolaryngology clinic was performed. Symptom severity, the presence of rhinosinusitis exacerbations, and endoscopic appearance were assessed, and regression analysis was used to determine clinical predictors of disease outcome. RESULTS: Thirty-three adults with CF-CRS were included in the study and followed for a mean of 15 months. Rhinosinusitis exacerbations occurred in 61% of participants during the study, and female sex increased the odds of presenting with an exacerbation visit. Sinus disease exacerbations were associated with an odds ratio of 2.07 for presenting with a pulmonary exacerbation at the next visit. CF-related diabetes was found to be associated with worse symptoms and endoscopic appearance. Infection with Staphylococcus aureus predicted worsening of symptoms, whereas infections with Pseudomonas aeruginosa improved over time. Allergic rhinitis was associated with worse endoscopic appearance, and nasal steroid use was associated with improved endoscopic appearance. CONCLUSION: Sex, CF-related diabetes, sinonasal infection status, allergic rhinitis, and nasal steroid use may all modulate severity of CF-CRS in adults. Sinusitis exacerbation may be a precursor to pulmonary exacerbation.

Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium.

Ferroptosis is a death program executed via selective oxidation of arachidonic acid-phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa-associated diseases such as CF and persistent lower respiratory tract infections.

Staphylococcus aureus Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection.

Staphylococcus aureus is a major cause of chronic respiratory infection in patients with cystic fibrosis (CF). We recently showed that Pseudomonas aeruginosa exhibits enhanced biofilm formation during respiratory syncytial virus (RSV) coinfection on human CF airway epithelial cells (AECs). The impact of respiratory viruses on other bacterial pathogens during polymicrobial infections in CF remains largely unknown. To investigate if S. aureus biofilm growth in the CF airways is impacted by virus coinfection, we evaluated S. aureus growth on CF AECs. Initial studies showed an increase in S. aureus growth over 24 h, and microscopy revealed biofilm-like clusters of bacteria on CF AECs. Biofilm growth was enhanced when CF AECs were coinfected with RSV, and this observation was confirmed with S. aureus CF clinical isolates. Apical conditioned medium from RSV-infected cells promoted S. aureus biofilms in the absence of the host epithelium, suggesting that a secreted factor produced during virus infection benefits S. aureus biofilms. Exogenous iron addition did not significantly alter biofilm formation, suggesting that it is not likely the secreted factor. We further characterized S. aureus-RSV coinfection in our model using dual host-pathogen RNA sequencing, allowing us to observe specific contributions of S. aureus and RSV to the host response during coinfection. Using the dual host-pathogen RNA sequencing approach, we observed increased availability of nutrients from the host and upregulation of S. aureus genes involved in growth, protein translation and export, and amino acid metabolism during RSV coinfection.IMPORTANCE The airways of individuals with cystic fibrosis (CF) are commonly chronically infected, and Staphylococcus aureus is the dominant bacterial respiratory pathogen in CF children. CF patients also experience frequent respiratory virus infections, and it has been hypothesized that virus coinfection increases the severity of S. aureus lung infections in CF. We investigated the relationship between S. aureus and the CF airway epithelium and observed that coinfection with respiratory syncytial virus (RSV) enhances S. aureus biofilm growth. However, iron, which was previously found to be a significant factor influencing Pseudomonas aeruginosa biofilms during virus coinfection, plays a minor role in S. aureus coinfections. Transcriptomic analyses provided new insight into how bacterial and viral pathogens alter host defense and suggest potential pathways by which dampening of host responses to one pathogen may favor persistence of another in the CF airways, highlighting complex interactions occurring between bacteria, viruses, and the host during polymicrobial infections.

Pseudomonas aeruginosa Contact-Dependent Growth Inhibition Plays Dual Role in Host-Pathogen Interactions.

Microorganisms exist in a diverse ecosystem and have evolved many different mechanisms for sensing and influencing the polymicrobial environment around them, utilizing both diffusible and contact-dependent signals. Contact-dependent growth inhibition (CDI) is one such communication system employed by Gram-negative bacteria. In addition to CDI mediation of growth inhibition, recent studies have demonstrated CDI-mediated control of communal behaviors such as biofilm formation. We postulated that CDI may therefore play an active role in host-pathogen interactions, allowing invading strains to establish themselves at polymicrobial mucosal interfaces through competitive interactions while simultaneously facilitating pathogenic capabilities via CDI-mediated signaling. Here, we show that Pseudomonas aeruginosa produces two CDI systems capable of mediating competition under conditions of growth on a surface or in liquid. Furthermore, we demonstrated a novel role for these systems in contributing to virulence in acute infection models, likely via posttranscriptional regulation of beneficial behaviors. While we did not observe any role for the P. aeruginosa CDI systems in biofilm biogenesis, we did identify for the first time robust CDI-mediated competition during interaction with a mammalian host using a model of chronic respiratory tract infection, as well as evidence that CDI expression is maintained in chronic lung infections. These findings reveal a previously unappreciated role for CDI in host-pathogen interactions and emphasize their importance during infection. IMPORTANCE How bacteria compete and communicate with each other is an increasingly recognized aspect of microbial pathogenesis with a major impact on disease outcomes. Gram-negative bacteria have recently been shown to employ a contact-dependent toxin-antitoxin system to achieve both competition and regulation of their physiology. Here, we show that this system is vital for virulence in acute infection as well as for establishment of chronic infection in the multidrug-resistant pathogen Pseudomonas aeruginosa. Greater understanding of the mechanisms underlying bacterial virulence and infection is important for the development of effective therapeutics in the era of increasing antimicrobial resistance.

Molecular phylogenetic analysis of non-sexually transmitted strains of Haemophilus ducreyi.

Haemophilus ducreyi, the etiologic agent of chancroid, has been previously reported to show genetic variance in several key virulence factors, placing strains of the bacterium into two genetically distinct classes. Recent studies done in yaws-endemic areas of the South Pacific have shown that H. ducreyi is also a major cause of cutaneous limb ulcers (CLU) that are not sexually transmitted. To genetically assess CLU strains relative to the previously described class I, class II phylogenetic hierarchy, we examined nucleotide sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, which encompass approximately 1% of the H. ducreyi genome. Sequences for all 11 loci indicated that strains collected from leg ulcers exhibit DNA sequences homologous to class I strains of H. ducreyi. However, sequences for 3 loci, including a hemoglobin receptor (hgbA), serum resistance protein (dsrA), and a collagen adhesin (ncaA) contained informative amounts of variation. Phylogenetic analyses suggest that these non-sexually transmitted strains of H. ducreyi comprise a sub-clonal population within class I strains of H. ducreyi. Molecular dating suggests that CLU strains are the most recently developed, having diverged approximately 0.355 million years ago, fourteen times more recently than the class I/class II divergence. The CLU strains' divergence falls after the divergence of humans from chimpanzees, making it the first known H. ducreyi divergence event directly influenced by the selective pressures accompanying human hosts.