Stability Considerations for Bacteriophages in Liquid Formulations Designed for Nebulization.

Pulmonary bacterial infections present a significant health risk to those with chronic respiratory diseases (CRDs) including cystic fibrosis (CF) and chronic-obstructive pulmonary disease (COPD). With the emergence of antimicrobial resistance (AMR), novel therapeutics are desperately needed to combat the emergence of resistant superbugs. Phage therapy is one possible alternative or adjunct to current antibiotics with activity against antimicrobial-resistant pathogens. How phages are administered will depend on the site of infection. For respiratory infections, a number of factors must be considered to deliver active phages to sites deep within the lung. The inhalation of phages via nebulization is a promising method of delivery to distal lung sites; however, it has been shown to result in a loss of phage viability. Although preliminary studies have assessed the use of nebulization for phage therapy both in vitro and in vivo, the factors that determine phage stability during nebulized delivery have yet to be characterized. This review summarizes current findings on the formulation and stability of liquid phage formulations designed for nebulization, providing insights to maximize phage stability and bactericidal activity via this delivery method.

Pseudomonas aeruginosa modulates neutrophil granule exocytosis in an in vitro model of airway infection.

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 ß 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.

Bacteriophage: A new therapeutic player to combat neutrophilic inflammation in chronic airway diseases.

Persistent respiratory bacterial infections are a clinical burden in several chronic inflammatory airway diseases and are often associated with neutrophil infiltration into the lungs. Following recruitment, dysregulated neutrophil effector functions such as increased granule release and formation of neutrophil extracellular traps (NETs) result in damage to airway tissue, contributing to the progression of lung disease. Bacterial pathogens are a major driver of airway neutrophilic inflammation, but traditional management of infections with antibiotic therapy is becoming less effective as rates of antimicrobial resistance rise. Bacteriophages (phages) are now frequently identified as antimicrobial alternatives for antimicrobial resistant (AMR) airway infections. Despite growing recognition of their bactericidal function, less is known about how phages influence activity of neutrophils recruited to sites of bacterial infection in the lungs. In this review, we summarize current in vitro and in vivo findings on the effects of phage therapy on neutrophils and their inflammatory mediators, as well as mechanisms of phage-neutrophil interactions. Understanding these effects provides further validation of their safe use in humans, but also identifies phages as a targeted neutrophil-modulating therapeutic for inflammatory airway conditions.

Development and validation of a miniaturized bacteriophage host range screening assay against antibiotic resistant Pseudomonas aeruginosa.

Antimicrobial resistance is a current global health crisis, and the increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Prior to clinical application, phage suitability is assessed, via susceptibility testing and breadth of host range to bacteriophage, however, these are both large-scale manual processes and labor-intensive. The aim of the study was to establish and validate a scaled down methodology for high-throughput screening to reduce procedural footprint. In this paper, we describe a scaled-down adapted methodology that can successfully screen bacteriophages, isolated and purified from wastewater samples. Furthermore, we describe a miniaturized host range assay against clinical Pseudomonas aeruginosa isolates using a spot test (2 µL/ drop) that was found to be both sensitive (94.6%) and specific (94.7%). It also demonstrated a positive predictive value (PPV) of 86.4% and negative predictive value (NPV) of 98%. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates was corroborated using the scaled down 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.

Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.

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.

Changes in airway inflammation with pseudomonas eradication in early cystic fibrosis.

BACKGROUND: Neutrophil elastase is a significant risk factor for structural lung disease in cystic fibrosis, and Pseudomonas aeruginosa airway infection is linked with neutrophilic inflammation and substantial respiratory morbidity. We aimed to evaluate how neutrophil elastase (NE) activity changes after P. aeruginosa eradication and influences early disease outcomes. METHODS: We assessed participants in the AREST CF cohort between 2000 and 2018 who had P. aeruginosa cultured from their routine annual bronchoalveolar lavage (BAL) fluid and who underwent eradication treatment and a post eradication BAL. Factors associated with persistent P. aeruginosa infection, persistent neutrophilic inflammation following eradication and worse structural lung disease one year post-eradication were evaluated. RESULTS: Eighty-eight episodes (3 months to 6 years old) of P. aeruginosa infection were studied. Eradication was successful in 84.1% of episodes. Median activity of NE was significantly reduced post-eradication from 9.15 to 3.4 nM (p = 0.008) but persisted in 33 subjects. High post-eradication NE levels were associated with an increased risk for P. aeruginosa infection in the next annual visit (odds ratio=1.7, 95% confidence interval 1.1-2.7, p = 0.014). Post-eradication NE levels (difference, 0.8; 95% confidence interval, 0.1-1.5) and baseline bronchiectasis computed tomography (CT) score (difference, 0.4; 95% confidence interval, 0.1-0.8) were the best predictors of bronchiectasis progression within 1 year (backward stepwise linear regression model, R2= 0.608, P<0.001), independent of eradication. CONCLUSION: In children with CF, NE activity may persist following successful P. aeruginosa eradication and is significantly associated with bronchiectasis progression. Evaluating strategies to diminish neutrophilic inflammation is essential for improving long-term outcomes.

Progress in Model Systems of Cystic Fibrosis Mucosal Inflammation to Understand Aberrant Neutrophil Activity.

In response to recurrent infection in cystic fibrosis (CF), powerful innate immune signals trigger polymorphonuclear neutrophil recruitment into the airway lumen. Exaggerated neutrophil proteolytic activity results in sustained inflammation and scarring of the airways. Consequently, neutrophils and their secretions are reliable clinical biomarkers of lung disease progression. As neutrophils are required to clear infection and yet a direct cause of airway damage, modulating adverse neutrophil activity while preserving their pathogen fighting function remains a key area of CF research. The factors that drive their pathological behavior are still under investigation, especially in early disease when aberrant neutrophil behavior first becomes evident. Here we examine the latest findings of neutrophils in pediatric CF lung disease and proposed mechanisms of their pathogenicity. Highlighted in this review are current and emerging experimental methods for assessing CF mucosal immunity and human neutrophil function in the laboratory.

Human milk oligosaccharides, milk microbiome and infant gut microbiome modulate neonatal rotavirus infection.

Neonatal rotavirus infections are predominantly asymptomatic. While an association with gastrointestinal symptoms has been described in some settings, factors influencing differences in clinical presentation are not well understood. Using multidisciplinary approaches, we show that a complex interplay between human milk oligosaccharides (HMOs), milk microbiome, and infant gut microbiome impacts neonatal rotavirus infections. Validating in vitro studies where HMOs are not decoy receptors for neonatal strain G10P[11], population studies show significantly higher levels of Lacto-N-tetraose (LNT), 2'-fucosyllactose (2'FL), and 6'-siallylactose (6'SL) in milk from mothers of rotavirus-positive neonates with gastrointestinal symptoms. Further, these HMOs correlate with abundance of Enterobacter/Klebsiella in maternal milk and infant stool. Specific HMOs also improve the infectivity of a neonatal strain-derived rotavirus vaccine. This study provides molecular and translational insight into host factors influencing neonatal rotavirus infections and identifies maternal components that could promote the performance of live, attenuated rotavirus vaccines.

Glycan recognition in globally dominant human rotaviruses.

Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P[6] RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut.

Milk Oligosaccharides Inhibit Human Rotavirus Infectivity in MA104 Cells.

Background: Oligosaccharides in milk act as soluble decoy receptors and prevent pathogen adhesion to the infant gut. Milk oligosaccharides reduce infectivity of a porcine rotavirus strain; however, the effects on human rotaviruses are less well understood.Objective: In this study, we determined the effect of specific and abundant milk oligosaccharides on the infectivity of 2 globally dominant human rotavirus strains.Methods: Four milk oligosaccharides-2'-fucosyllactose (2'FL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and galacto-oligosaccharides-were tested for their effects on the infectivity of human rotaviruses G1P[8] and G2P[4] through fluorescent focus assays on African green monkey kidney epithelial cells (MA104 cells). Oligosaccharides were added at different time points in the infectivity assays. Infections in the absence of oligosaccharides served as controls.Results: When compared with infections in the absence of glycans, all oligosaccharides substantially reduced the infectivity of both human rotavirus strains in vitro; however, virus strain-specific differences in effects were observed. Compared with control infections, the maximum reduction in G1P[8] infectivity was seen with 2'FL when added after the onset of infection (62% reduction, P < 0.01), whereas the maximum reduction in G2P[4] infectivity was seen with the mixture of 3'SL + 6'SL when added during infection (73% reduction, P < 0.01). The mixture of 3'SL + 6'SL at the same ratio as is present in breast milk was more potent in reducing G2P[4] infectivity (73% reduction, P < 0.01) than when compared with 3'SL (47% reduction) or 6'SL (40% reduction) individually. For all oligosaccharides the reduction in infectivity was mediated by an effect on the virus and not on the cells.Conclusions: Milk oligosaccharides reduce the infectivity of human rotaviruses in MA104 cells, primarily through an effect on the virus. Although breastfed infants are directly protected, the addition of specific oligosaccharides to infant formula may confer these benefits to formula-fed infants.

Rotavirus Serum IgA Immune Response in Children Receiving Rotarix Coadministered With bOPV or IPV.

BACKGROUND: Vaccine schedules including bivalent oral and inactivated poliovirus vaccines will replace trivalent oral poliovirus vaccines in 2016. METHODS: We evaluated rotavirus immunoglobulin A seroresponses when the second dose of Rotarix at 16 weeks was given concomitantly with inactivated or bivalent oral poliovirus vaccines. RESULTS: Rotavirus immunoglobulin A seroresponse rate at week 28 was 15% lower in recipients of bivalent oral poliovirus vaccines compared with inactivated poliovirus vaccines. CONCLUSION: Bivalent oral poliovirus vaccine decreases rotavirus IgA seroresponse rates when coadministered at 16 weeks of age.

Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.

Group A Streptococcus (GAS) causes an exceptionally broad range of infections in humans, from relatively mild pharyngitis and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome. An epidemic of severe invasive human infections caused by type emm59 GAS, heretofore an exceedingly rare cause of disease, spread west to east across Canada over a 3-year period (2006 to 2008). By sequencing the genomes of 601 epidemic, historic, and other emm59 organisms, we discovered that a recently emerged, genetically distinct emm59 clone is responsible for the Canadian epidemic. Using near-real-time genome sequencing, we were able to show spread of the Canadian epidemic clone into the United States. The extensive genome data permitted us to identify patterns of geographic dissemination as well as links between emm59 subclonal lineages that cause infections. Mouse and nonhuman primate models of infection demonstrated that the emerged clone is unusually virulent. Transmission of epidemic emm59 strains may have occurred primarily by skin contact, as suggested by an experimental model of skin transmission. In addition, the emm59 strains had a significantly impaired ability to persist in human saliva and to colonize the oropharynx of mice, and seldom caused human pharyngitis. Our study contributes new information to the rapidly emerging field of molecular pathogenomics of bacterial epidemics and illustrates how full-genome data can be used to precisely illuminate the landscape of strain dissemination during a bacterial epidemic.

Polymorphisms in regulator of protease B (RopB) alter disease phenotype and strain virulence of serotype M3 group A Streptococcus.

Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.

Perinatal changes in mitral and aortic valve structure and composition.

At birth, the mechanical environment of valves changes radically as fetal shunts close and pulmonary and systemic vascular resistances change. Given that valves are reported to be mechanosensitive, we investigated remodeling induced by perinatal changes by examining compositional and structural differences of aortic and mitral valves (AVs, MVs) between 2-day-old and 3rd fetal trimester porcine valves using immunohistochemistry and Movat pentachrome staining. Aortic valve composition changed more with birth than the MV, consistent with a greater change in AV hemodynamics. At 2 days, AV demonstrated a trend of greater versican and elastin (P  =  0.055), as well as greater hyaluronan turnover (hyaluronan receptor for endocytosis, P  =  0.049) compared with the 3rd-trimester samples. The AVs also demonstrated decreases in proteins related to collagen synthesis and fibrillogenesis with birth, including procollagen I, prolyl 4-hydroxylase, biglycan (all P ≤ 0.005), and decorin (P  =  0.059, trend). Both AVs and MVs demonstrated greater delineation between the leaflet layers in 2-day-old compared with 3rd-trimester samples, and AVs demonstrated greater saffron-staining collagen intensity, suggesting more mature collagen in 2-day-old compared with 3rd-trimester samples (each P < 0.05). The proportion of saffron-staining collagen also increased in AV with birth (P < 0.05). The compositional and structural changes that occur with birth, as noted in this study, likely are important to proper neonatal valve function. Furthermore, normal perinatal changes in hemodynamics often do not occur in congenital valve disease; the corresponding perinatal matrix maturation may also be lacking and could contribute to poor function of congenitally malformed valves.