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.

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.

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.

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.

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.

A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae.

Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

Induction of the reactive chlorine-responsive transcription factor RclR in Escherichia coli following ingestion by neutrophils.

Neutrophils generate hypochlorous acid (HOCl) and related reactive chlorine species as part of their defence against invading microorganisms. In isolation, bacteria respond to reactive chlorine species by upregulating responses that provide defence against oxidative challenge. Key questions are whether these responses are induced when bacteria are phagocytosed by neutrophils, and whether this provides them with a survival advantage. We investigated RclR, a transcriptional activator of the rclABC operon in Escherichia coli that has been shown to be specifically activated by reactive chlorine species. We first measured induction by individual reactive chlorine species, and showed that HOCl itself activates the response, as do chloramines (products of HOCl reacting with amines) provided they are cell permeable. Strong RclR activation was seen in E. coli following phagocytosis by neutrophils, beginning within 5 min and persisting for 40 min. RclR activation was suppressed by inhibitors of NOX2 and myeloperoxidase, providing strong evidence that it was due to HOCl production in the phagosome. RclR activation demonstrates that HOCl, or a derived chloramine, enters phagocytosed bacteria in sufficient amount to induce this response. Although RclR was induced in wild-type bacteria following phagocytosis, we detected no greater sensitivity to neutrophil killing of mutants lacking genes in the rclABC operon.

The Cu(II) Reductase RclA Protects Escherichia coli against the Combination of Hypochlorous Acid and Intracellular Copper.

Enterobacteria, including Escherichia coli, bloom to high levels in the gut during inflammation and strongly contribute to the pathology of inflammatory bowel diseases. To survive in the inflamed gut, E. coli must tolerate high levels of antimicrobial compounds produced by the immune system, including toxic metals like copper and reactive chlorine oxidants such as hypochlorous acid (HOCl). Here, we show that extracellular copper is a potent detoxifier of HOCl and that the widely conserved bacterial HOCl resistance enzyme RclA, which catalyzes the reduction of copper(II) to copper(I), specifically protects E. coli against damage caused by the combination of HOCl and intracellular copper. E. coli lacking RclA was highly sensitive to HOCl when grown in the presence of copper and was defective in colonizing an animal host. Our results indicate that there is unexpected complexity in the interactions between antimicrobial toxins produced by innate immune cells and that bacterial copper status is a key determinant of HOCl resistance and suggest an important and previously unsuspected role for copper redox reactions during inflammation.IMPORTANCE During infection and inflammation, the innate immune system uses antimicrobial compounds to control bacterial populations. These include toxic metals, like copper, and reactive oxidants, including hypochlorous acid (HOCl). We have now found that RclA, a copper(II) reductase strongly induced by HOCl in proinflammatory Escherichia coli and found in many bacteria inhabiting epithelial surfaces, is required for bacteria to resist killing by the combination of intracellular copper and HOCl and plays an important role in colonization of an animal host. This finding indicates that copper redox chemistry plays a critical and previously underappreciated role in bacterial interactions with the innate immune system.

A DedA Family Membrane Protein Is Required for Burkholderia thailandensis Colistin Resistance.

Colistin is a "last resort" antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations (MIC) often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in Burkholderia thailandensis (DbcA; DedA of Burkholderia required for colistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of ΔdbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis ΔdbcA was observed by expression of dbcA, E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis ΔdbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species.