Pathogenesis of Achromobacter xylosoxidans respiratory infections: colonization, persistence, and transcriptome profiling in synthetic cystic fibrosis sputum medium.

Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long infections of the respiratory mucosa caused by a diverse array of opportunists, which are leading causes of morbidity and mortality. In recent years, there has been increased appreciation for the range and diversity of microbes causing CF-related respiratory infections. The introduction of new therapeutics and improved detection methodology has revealed CF-related opportunists such as Achromobacter xylosoxidans (Ax). Ax is a Gram-negative bacterial species which is widely distributed in environmental sources and has been increasingly observed in sputa and other samples from pwCF, typically in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors including flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized cultures of CFBE41o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization of cell layers. Ax colonized and persisted in mouse lungs for up to 72 h post infection, with inflammatory consequences that include increased neutrophil influx in the lung, lung damage, cytokine production, and mortality. We also identified genes that are differentially expressed in synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF.

Acute polymicrobial airway infections: analysis in cystic fibrosis mice.

Cystic fibrosis (CF) is a genetic disorder affecting epithelial ion transport, which among other impacts results in defective mucociliary clearance and innate defenses in the respiratory tract. Consequently, people with CF experience lifelong infections of the respiratory mucosa that are chronic and polymicrobial in nature. Young children with CF are initially colonized by opportunists like nontypeable Haemophilus influenzae (NTHi), which normally resides within the microbiome of the nasopharynx and upper airways and can also cause infections of the respiratory mucosa that include bronchitis and otitis media. NTHi is typically supplanted by other microbes as patients age; for example, people with CF are often chronically infected with mucoid strains of Pseudomonas aeruginosa, which prior work in our laboratory has shown to promote colonization and persistence by other opportunists that include Stenotrophomonas maltophilia. Our previous work has shown that polymicrobial infection impacts host colonization and persistence of incoming microbes via diverse mechanisms that include priming of host immunity that can promote microbial clearance, and cooperativity within polymicrobial biofilms, which can promote persistence. In infection studies with BALB/c Cftrtm1UNC mice, results showed, as previously observed for WT BALB/c mice, preceding infection with NTHi decreased colonization and persistence by P. aeruginosa. Likewise, polymicrobial infection of BALB/c Cftrtm1UNC and C57BL/6 Cftrtm1UncTg(FABPhCFTR)1Jaw/J mice showed correlation between S. maltophilia and P. aeruginosa, with increased bacterial colonization and lung pathology. Based on these results, we conclude that our previous observations regarding polymicrobial infections with CF opportunists in WT mice are also validated using CF mice.

Comparative genomics of clinical Stenotrophomonas maltophilia isolates reveals genetic diversity which correlates with colonization and persistence in vivo.

Stenotrophomonas maltophilia is a Gram-negative emerging opportunistic pathogen often present in people with respiratory diseases such as cystic fibrosis (CF). People with CF (pwCF) experience lifelong polymicrobial infections of the respiratory mucosa. Our prior work showed that Pseudomonas aeruginosa promotes persistence of S. maltophilia in mouse respiratory infections. As is typical for environmental opportunistic pathogens, S. maltophilia has a large genome and a high degree of genetic diversity. In this study, we evaluated the genomic content of S. maltophilia, combining short and long read sequencing to construct nearly complete genomes of 10 clinical isolates. The genomes of these isolates were then compared with all publicly available S. maltophilia genome assemblies, and each isolate was then evaluated for colonization/persistence in vivo, both alone and in coinfection with P. aeruginosa. We found that while the overall genome size and GC content were fairly consistent between strains, there was considerable variability in both genome structure and gene content. Similarly, there was significant variability in S. maltophilia colonization and persistence in experimental mouse respiratory infections in the presence or absence of P. aeruginosa. Ultimately, this study gives us a greater understanding of the genomic diversity of clinical S. maltophilia isolates, and how this genomic diversity relates to both interactions with other pulmonary pathogens and to host disease progression. Identifying the molecular determinants of infection with S. maltophilia can facilitate development of novel antimicrobial strategies for a highly drug-resistant pathogen.

Nontypeable Haemophilus influenzae Infection Impedes Pseudomonas aeruginosa Colonization and Persistence in Mouse Respiratory Tract.

Patients with cystic fibrosis (CF) experience lifelong respiratory infections, which are a significant cause of morbidity and death. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted, and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa. In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of P. aeruginosa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater P. aeruginosa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by P. aeruginosa resulted in significantly lower levels of P. aeruginosa, compared to infection with P. aeruginosa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines, compared with P. aeruginosa-infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide endotoxin prior to P. aeruginosa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent P. aeruginosa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early-life infections in patients with CF.

Pathogenesis of Achromobacter xylosoxidans respiratory infections: colonization and persistence of airway epithelia and differential gene expression in synthetic cystic fibrosis sputum medium.

Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long respiratory mucosal infections caused by a diverse array of opportunists, and these infections are a leading cause of morbidity and mortality for pwCF. In recent years, there has been increased appreciation for the range and diversity of microbes in CF-related respiratory infections. Introduction of new therapeutics and improved detection methodology has revealed CF related opportunists such as Achromobacter xylosoxidans (Ax). Ax is a Gram-negative bacterial species that is widely distributed in the environment and has been increasingly observed in sputa and other samples from pwCF; typically Ax infections occur in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors involved in flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized CFBE14o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization. Ax colonized and persisted in mouse lung for up to 72 hours post infection, with inflammatory consequences that include increased neutrophilia, lung damage, cytokine production, and mortality. Transcript profiling reveled differential expression of Ax genes during growth in SCFM2 synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF.

Comparative genomics of clinical Stenotrophomonas maltophilia isolates reveals regions of diversity which correlate with colonization and persistence in vivo.

Stenotrophomonas maltophilia is a Gram-negative emerging opportunistic pathogen often found in respiratory diseases such as cystic fibrosis (CF). Patients with CF experience lifelong polymicrobial infections of the respiratory mucosa. Our prior work showed that P. aeruginosa promotes persistence of S. maltophilia mouse respiratory infections. As is typical for environmental opportunistic pathogens, S. maltophilia has a large genome and a high degree of genetic diversity. In this study, we evaluated the genomic content of S. maltophilia, combining short and long read sequencing to construct complete genomes of 10 clinical isolates which were then compared with the larger phylogeny of S. maltophilia genomic sequence data, and compared colonization/persistence in vivo, alone and in coinfection with P. aeruginosa. We found that while the overall genome size and GC content were fairly consistent, there was considerable variability in arrangement and gene content. Similarly, there was significant variability in S. maltophilia colonization and persistence in vivo in experimental mouse respiratory infection. Ultimately, this study gives us a greater understanding of the genomic diversity of S. maltophilia isolated from patients, and how this genomic diversity relates to interactions with other pulmonary pathogens, and to host disease progression. Identifying the molecular determinants of infection with S. maltophilia can facilitate development of novel antimicrobial strategies for a highly drug-resistant pathogen.

Pseudomonas aeruginosa Promotes Persistence of Stenotrophomonas maltophilia via Increased Adherence to Depolarized Respiratory Epithelium.

Stenotrophomonas maltophilia is an emerging opportunistic respiratory pathogen in people with cystic fibrosis (CF). S. maltophilia is frequently observed in polymicrobial infections, and we have previously shown that Pseudomonas aeruginosa promotes colonization and persistence of S. maltophilia in mouse respiratory infections. In this study, we used host and bacterial RNA sequencing to further understand the molecular underpinnings of this interaction. To evaluate S. maltophilia transcript profiles, we used a recently described method for selective capture of bacterial mRNA transcripts with strain-specific RNA probes. We found that factors associated with the type IV pilus, including the histidine kinase subunit of a chemotactic two-component signaling system (chpA), had increased transcript levels during dual-species infection. Using immortalized CF respiratory epithelial cells, we found that infection with P. aeruginosa increases adherence of S. maltophilia, at least in part due to disruption of epithelial tight junctions. In contrast, an isogenic S. maltophilia chpA mutant strain lacked cooperative adherence to CF epithelia and decreased bacterial burden in vivo in dual-species infections with P. aeruginosa. Similarly, P. aeruginosa lacking elastase (lasB) failed to promote S. maltophilia adherence or bacterial colonization and persistence in vivo. Based on these results, we propose that disruption of lung tissue integrity by P. aeruginosa facilitates adherence of S. maltophilia to the lung epithelia, likely in a type IV pilus-dependent manner. These data lend insight into S. maltophilia colonization and persistence in people in later stages of CF disease and may have implications for interactions with other bacterial opportunists. IMPORTANCE Despite advances in treatment options for people with CF, complications of bacterial infections remain the greatest driver of morbidity and mortality in this patient population. These infections often involve more than one bacterial pathogen, and our understanding of how interspecies interactions impact disease progression is lacking. Previous work in our lab found that two CF pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, can work together in the lung to cause more severe infection. In the present study, we found that infection with P. aeruginosa promotes persistence of S. maltophilia by interfering with epithelial barrier integrity. Depolarization of the epithelial cell layer by P. aeruginosa-secreted elastase increased S. maltophilia adherence, likely in a type IV pilus-dependent manner. Ultimately, this work sheds light on the molecular mechanisms governing an important multispecies interaction seen in pulmonary diseases such as CF.

SNSP113 (PAAG) improves mucociliary transport and lung pathology in the Scnn1b-Tg murine model of CF lung disease.

BACKGROUND: Mucus stasis, a hallmark of muco-obstructive disease, results from impaired mucociliary transport and leads to lung function decline and chronic infection. Although therapeutics that target mucus stasis in the airway, such as hypertonic saline or rhDNAse, show some therapeutic benefit, they do not address the underlying electrostatic defect apparent in mucins in CF and related conditions. We have previously shown poly (acetyl, arginyl) glucosamine (PAAG, developed as SNSP113), a soluble, cationic polymer, significantly improves mucociliary transport in a rat model of CF by normalizing the charge defects of CF mucin. Here, we report efficacy in the CFTR-sufficient, ENaC hyperactive, Scnn1b-Tg mouse model that develops airway muco-obstruction due to sodium hyperabsorption and airway dehydration. METHODS: Scnn1b-Tg mice were treated with either 250 µg/mL SNSP113 or vehicle control (1.38% glycerol in PBS) via nebulization once daily for 7 days and then euthanized for analysis. Micro-Optical Coherence Tomography-based evaluation of excised mouse trachea was used to determine the effect on the functional microanatomy. Tissue analysis was performed by routine histopathology. RESULTS: Nebulized treatment of SNSP113 significantly improved mucociliary transport in the airways of Scnn1b-Tg mice, without altering the airway surface or periciliary liquid layer. In addition, SNSP113 significantly reversed epithelial hypertrophy and goblet cell metaplasia. Finally, SNSP113 significantly ameliorated eosinophilic crystalline pneumonia and lung consolidation in addition to inflammatory macrophage influx in this model. CONCLUSION: Overall, this study extends the efficacy of SNSP113 as a potential therapeutic to alleviate mucus stasis in muco-obstructive diseases in CF and potentially in related conditions.