A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air–liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP‐10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin‐1α and β were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral–bacterial co‐infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV‐infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N‐formylmethionyl‐leucyl‐phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.

Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A−C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.

Antimicrobial resistance is a global health crisis, partly contributed by inappropriate use of antibiotics. The increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Current procedures assessing susceptibility and breadth of host range to bacteriophage are conducted using large-scale manual processes that are labor-intensive. The aim here was to establish and validate a scaled down methodology for high-throughput screening in order to reduce procedural footprint. Bacteriophages were isolated from wastewater samples and screened for specificity against 29 clinical Pseudomonas aeruginosa isolates and PA01 using a spot test (2 μL/ drop). Host range assessment was performed on four representative P. aeruginosa isolates using both double agar overlay assay on petri dishes and 24-well culture plates. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates were corroborated between the current standard practice of whole plate phage assay and 24-well phage assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

Introduction: Poor airway epithelial repair and deficient barrier integrity may contribute to reduced lung function after preterm birth. Azithromycin (AZM) has been shown to improve epithelial wound repair and barrier function in other chronic lung diseases. We hypothesised that AZM would improve repair as well as barrier function of primary nasal epithelial cells (NECs) of preterm birth survivors. Methods: Very preterm NECs (<32 weeks gestation, n=12, 1.4±0.1 years corrected postnatal age) were cultured as monolayers, wounded, AZM treated (10, 1 and 0.1 µg/ml), and imaged frequently over 72h to assess wound repair. Differentiated cultures were also established from term (n=8, 2.7±0.6 years) and preterm (n=12, 1.4±0.1 year) NECs and treated with AZM (0.4 µg/ml) for 10 days. Barrier integrity was measured via transepithelial electrical resistance (TEER) and cell permeability. Results: Preterm NECs showed variable wound repair and were classed as repairers (n=7, 98.6±3.5% repair) or non-repairers (n=5, 61.1±21.9% repair). AZM enhanced wound repair of non-repairers at all doses (p<0.01), with the greatest improvement seen at 1 µg/ml (29.3±7.4%, p<0.0001). Although TEER did not differ between cohorts, differentiated cultures were significantly more permeable in the preterm group (2.3 fold; p0.05). Conclusions: Preterm NECs have impaired wound repair and increased permeability. AZM improves the defective wound repair but does not resolve the permeability. AZM may be a candidate for future clinical studies to improve lung health in survivors of preterm birth, but further investigation is required.