Predictive modeling of antibiotic eradication therapy success for new-onset Pseudomonas aeruginosa pulmonary infections in children with cystic fibrosis.

Chronic Pseudomonas aeruginosa (Pa) lung infections are the leading cause of mortality among cystic fibrosis (CF) patients; therefore, the eradication of new-onset Pa lung infections is an important therapeutic goal that can have long-term health benefits. The use of early antibiotic eradication therapy (AET) has been shown to clear the majority of new-onset Pa infections, and it is hoped that identifying the underlying basis for AET failure will further improve treatment outcomes. Here we generated machine learning models to predict AET outcomes based on pathogen genomic data. We used a nested cross validation design, population structure control, and recursive feature selection to improve model performance and showed that incorporating population structure control was crucial for improving model interpretation and generalizability. Our best model, controlling for population structure and using only 30 recursively selected features, had an area under the curve of 0.87 for a holdout test dataset. The top-ranked features were generally associated with motility, adhesion, and biofilm formation.

Pseudomonas aeruginosa Strain-sharing in Early Infection Among Children With Cystic Fibrosis.

BACKGROUND: We previously identified Pseudomonas aeruginosa isolates with characteristics typical of chronic infection in some early infections in children with cystic fibrosis (CF), suggesting that these isolates may have been acquired from other patients. Our objective was to define the extent of P. aeruginosa strain-sharing in early CF infections and its impact on antibiotic eradication treatment failure rates. METHODS: We performed whole genome sequencing on isolates from early pediatric CF pulmonary infections and from the following comparator groups in the same hospital: chronic CF infection, sink drains, sterile site infections, and asymptomatic carriage. Univariate logistic regression was used to assess factors associated with treatment failure. RESULTS: In this retrospective, observational study, 1029 isolates were sequenced. The CF clones strain B and clone C were present. In 70 CF patients with early infections, 14 shared strains infected 29 (41%) patients over 5 years; 16% (n = 14) of infections had mixed strains. In the 70 children, approximately one-third of shared-strain infections were likely due to patient-to-patient transmission. Mixed-strain infections were associated with strain-sharing (odds ratio, 8.50; 95% confidence interval, 2.2-33.4; P = .002). Strain-sharing was not associated with antibiotic eradication treatment failure; however, nosocomial strain transmission was associated with establishment of chronic infection in a CF sibling pair. CONCLUSIONS: Although early P. aeruginosa CF infection is thought to reflect acquisition of diverse strains from community reservoirs, we identified frequent early CF strain-sharing that was associated with the presence of mixed strains and instances of possible patient-to-patient transmission.

Ecological Succession of Polymicrobial Communities in the Cystic Fibrosis Airways.

Antimicrobial therapies against cystic fibrosis (CF) lung infections are largely aimed at the traditional, well-studied CF pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex, despite the fact that the CF lung harbors a complex and dynamic polymicrobial community. A clinical focus on the dominant pathogens ignores potentially important community-level interactions in disease pathology, perhaps explaining why these treatments are often less effective than predicted based on in vitro testing. A better understanding of the ecological dynamics of this ecosystem may enable clinicians to harness these interactions and thereby improve treatment outcomes. Like all ecosystems, the CF lung microbial community develops through a series of stages, each of which may present with distinct microbial communities that generate unique host-microbe and microbe-microbe interactions, metabolic profiles, and clinical phenotypes. While insightful models have been developed to explain some of these stages and interactions, there is no unifying model to describe how these infections develop and persist. Here, we review current perspectives on the ecology of the CF airway and present the CF Ecological Succession (CFES) model that aims to capture the spatial and temporal complexity of CF lung infection, address current challenges in disease management, and inform the development of ecologically driven therapeutic strategies.

Microbiome networks and change-point analysis reveal key community changes associated with cystic fibrosis pulmonary exacerbations.

Over 90% of cystic fibrosis (CF) patients die due to chronic lung infections leading to respiratory failure. The decline in CF lung function is greatly accelerated by intermittent and progressively severe acute pulmonary exacerbations (PEs). Despite their clinical impact, surprisingly few microbiological signals associated with PEs have been identified. Here we introduce an unsupervised, systems-oriented approach to identify key members of the microbiota. We used two CF sputum microbiome data sets that were longitudinally collected through periods spanning baseline health and PEs. Key taxa were defined based on three strategies: overall relative abundance, prevalence, and co-occurrence network interconnectedness. We measured the association between changes in the abundance of the key taxa and changes in patient clinical status over time via change-point detection, and found that taxa with the highest level of network interconnectedness tracked changes in patient health significantly better than taxa with the highest abundance or prevalence. We also cross-sectionally stratified all samples into the clinical states and identified key taxa associated with each state. We found that network interconnectedness most strongly delineated the taxa among clinical states, and that anaerobic bacteria were over-represented during PEs. Many of these anaerobes are oropharyngeal bacteria that have been previously isolated from the respiratory tract, and/or have been studied for their role in CF. The observed shift in community structure, and the association of anaerobic taxa and PEs lends further support to the growing consensus that anoxic conditions and the subsequent growth of anaerobic microbes are important predictors of PEs.

Penicillin-binding protein 3 is a common adaptive target among Pseudomonas aeruginosa isolates from adult cystic fibrosis patients treated with ß-lactams.

OBJECTIVE: Determining the mechanisms that modulate ß-lactam resistance in clinical Pseudomonas aeruginosa (P. aeruginosa) isolates can be challenging, as the molecular profiles identified in mutation-based or expression-based resistance determinant screens may not correlate with in vitro phenotypes. One of the lesser studied resistance mechanisms in P. aeruginosa is the modification of penicillin-binding protein 3 (pbpB/ftsI). This study reported that nonsynonymous polymorphisms within pbpB frequently occur among ß-lactam resistant sputum isolates, and are associated with unique antibiotic susceptibility patterns. METHODS: Longitudinally collected isolates (n = 126) from cystic fibrosis (CF) patients with or without recent ß-lactam therapy or of non-clinical origin were tested for susceptibility to six ß-lactams (aztreonam, ceftazidime, cefsulodin, cefepime, meropenem, and piperacillin). Known ß-lactam resistance mechanisms were characterised by polymerase chain reaction (PCR)-based methods, and polymorphisms in the transpeptidase-encoding domain of pbpB identified by sequencing. RESULTS: Twelve nonsynonymous polymorphisms were detected among 86 isolates (67%) from five CF patients with a history of ß-lactam therapy, compared with one polymorphism in 30 (3.3%) from three patients who had not received ß-lactam treatments. No nonsynonymous polymorphisms were found in ten environmental isolates. Multiple pbpB alleles, often with different combinations of polymorphisms, were detected within the population of strains from each CF patient for up to 2.6 years. Traditional patterns of ampC or mexA de-repression reduced expression of oprD or the presence of extended-spectrum ß-lactamases were not observed in resistant isolates with nonsynonymous polymorphisms in pbpB. CONCLUSION: This study's findings suggest that pbpB is a common adaptive target, and may contribute to the development of ß-lactam resistance in P. aeruginosa.

A genome-wide association analysis reveals a potential role for recombination in the evolution of antimicrobial resistance in Burkholderia multivorans.

Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomics study of 111 B. multivorans sputum isolates from one CF patient through three stages of infection including an early incident isolate, deep sampling of a one-year period of chronic infection occurring weeks before a lung transplant, and deep sampling of a post-transplant infection. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found the incident isolate was basally related to the rest of the strains and more susceptible to antibiotics from three classes (ß-lactams, aminoglycosides, quinolones). The chronic infection isolates diversified into multiple, distinct genetic lineages and showed reduced antimicrobial susceptibility to the same antibiotics. The post-transplant reinfection isolates derived from the same source as the incident isolate and were genetically distinct from the chronic isolates. They also had a level of susceptibility in between that of the incident and chronic isolates. We identified numerous examples of potential parallel pathoadaptation, in which multiple mutations were found in the same locus or even codon. The set of parallel pathoadaptive loci was enriched for functions associated with virulence and resistance. Our GWAS analysis identified statistical associations between a polymorphism in the ampD locus with resistance to ß-lactams, and polymorphisms in an araC transcriptional regulator and an outer membrane porin with resistance to both aminoglycosides and quinolones. Additionally, these three loci were independently mutated four, three and two times, respectively, providing further support for parallel pathoadaptation. Finally, we identified a minimum of 14 recombination events, and observed that loci carrying putative parallel pathoadaptations and polymorphisms statistically associated with ß-lactam resistance were over-represented in these recombinogenic regions.

Epidemiology of Clonal Pseudomonas aeruginosa Infection in a Canadian Cystic Fibrosis Population.

RATIONALE: The extent of the genetic relatedness among Pseudomonas aeruginosa isolates and its impact on clinical outcomes in the cystic fibrosis (CF) population is poorly understood. OBJECTIVES: The objectives of this study were to determine the prevalence of clonal P. aeruginosa infection in Canada and to associate P. aeruginosa genotypes with clinical outcomes. METHODS: This was an observational study of adult and pediatric patients with CF across Canada. Isolates were typed using multilocus sequence typing. A clone was defined as sharing at least six of seven alleles. Genotyping results were associated with clinical outcomes, including forced expiratory volume in 1 second, body mass index, rate of pulmonary exacerbation, and death/transplant. RESULTS: A total of 1,537 P. aeruginosa isolates were genotyped to 403 unique sequence types (STs) in 402 individuals with CF. Although 39% of STs were shared, most were shared only among a small number of subjects, and the majority (79%) of the genetic diversity in P. aeruginosa isolates was observed between patients. There were no significant differences in clinical outcomes according to genotype. However, patients with a dynamic, changing ST infection pattern had both a steeper decline in forced expiratory volume in 1 second (-2.9% predicted change/yr, 95% confidence interval [CI] = -3.8 to -1.9 compared with 0.4, 95% CI = -0.3 to 1.0; P < 0.001) and body mass index (-1.0 percentile change/yr, 95% CI = -1.6 to -0.3 compared with -0.1, 95% CI = -0.7 to 0.5; P = 0.047) than those with a stable infection with the same ST. CONCLUSIONS: There was no widespread sharing of dominant clones in our CF population, and the majority of the genetic diversity in P. aeruginosa was observed between patients. Changing genotypes over time within an individual was associated with worse clinical outcomes.

Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation.

The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.

Selective Sweeps and Parallel Pathoadaptation Drive Pseudomonas aeruginosa Evolution in the Cystic Fibrosis Lung.

UNLABELLED: Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage originated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with recurrent selective sweeps. While we cannot identify the specific mutations responsible for the origins of the clonal lineages, we find that the majority of mutations occur in loci previously associated with virulence and resistance. Additionally, approximately one-third of all mutations occur in loci that are mutated multiple times, highlighting the importance of parallel pathoadaptation. One such locus is the gene encoding penicillin-binding protein 3, which received three independent mutations. Our functional analysis of these alleles shows that they provide differential fitness benefits dependent on the antibiotic under selection. These data reveal that bacterial populations can undergo extensive and dramatic changes that are not revealed by lower-resolution analyses. IMPORTANCE: Pseudomonas aeruginosa is a bacterial opportunistic pathogen responsible for significant morbidity and mortality in cystic fibrosis (CF) patients. Once it has colonized the lung in CF, it is highly resilient and rarely eradicated. This study presents a deep sampling examination of the fine-scale evolutionary dynamics of P. aeruginosa in the lungs of a chronically infected CF patient. We show that diversity of P. aeruginosa is driven by recurrent clonal emergence and expansion within this patient and identify potential adaptive variants associated with these events. This high-resolution sequencing strategy thus reveals important intraspecies dynamics that explain a clinically important phenomenon not evident at a lower-resolution analysis of community structure.

Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis.

Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to ß-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample.

Lung microbiota across age and disease stage in cystic fibrosis.

Understanding the significance of bacterial species that colonize and persist in cystic fibrosis (CF) airways requires a detailed examination of bacterial community structure across a broad range of age and disease stage. We used 16S ribosomal RNA sequencing to characterize the lung microbiota in 269 CF patients spanning a 60 year age range, including 76 pediatric samples from patients of age 4-17, and a broad cross-section of disease status to identify features of bacterial community structure and their relationship to disease stage and age. The CF lung microbiota shows significant inter-individual variability in community structure, composition and diversity. The core microbiota consists of five genera - Streptococcus, Prevotella, Rothia, Veillonella and Actinomyces. CF-associated pathogens such as Pseudomonas, Burkholderia, Stenotrophomonas and Achromobacter are less prevalent than core genera, but have a strong tendency to dominate the bacterial community when present. Community diversity and lung function are greatest in patients less than 10 years of age and lower in older age groups, plateauing at approximately age 25. Lower community diversity correlates with worse lung function in a multivariate regression model. Infection by Pseudomonas correlates with age-associated trends in community diversity and lung function.

Genomic analysis of the Kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease.

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries--even millennia--ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions.

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

Pulmonary bacterial communities in surgically resected noncystic fibrosis bronchiectasis lungs are similar to those in cystic fibrosis.

Background. Recurrent bacterial infections play a key role in the pathogenesis of bronchiectasis, but conventional microbiologic methods may fail to identify pathogens in many cases. We characterized and compared the pulmonary bacterial communities of cystic fibrosis (CF) and non-CF bronchiectasis patients using a culture-independent molecular approach. Methods. Bacterial 16S rRNA gene libraries were constructed from lung tissue of 10 non-CF bronchiectasis and 21 CF patients, followed by DNA sequencing of isolates from each library. Community characteristics were analyzed and compared between the two groups. Results. A wide range of bacterial diversity was detected in both groups, with between 1 and 21 bacterial taxa found in each patient. Pseudomonas was the most common genus in both groups, comprising 49% of sequences detected and dominating numerically in 13 patients. Although Pseudomonas appeared to be dominant more often in CF patients than in non-CF patients, analysis of entire bacterial communities did not identify significant differences between these two groups. Conclusions. Our data indicate significant diversity in the pulmonary bacterial community of both CF and non-CF bronchiectasis patients and suggest that this community is similar in surgically resected lungs of CF and non-CF bronchiectasis patients.

RAS, FLT3, and TP53 mutations in therapy-related myeloid malignancies with abnormalities of chromosomes 5 and 7.

Oncogenic mutations in the KRAS2, NRAS, or FLT3 gene are detected in more than 50% of patients with de novo acute myeloid leukemia (AML). RAS mutations are also prevalent in de novo myelodysplastic syndrome (MDS), especially chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia. However, few studies have examined these genetic lesions in therapy-related myeloid malignancies. Monosomy 7/del(7q) and monosomy 5/del(5q) represent the most common cytogenetic abnormalities in therapy-related MDS and AML (t-MDS/t-AML) and are strongly associated with prior exposure to alkylating agents. Mutational analysis of bone marrow specimens from a well-characterized cohort of 26 t-MDS/t-AML patients with abnormalities of chromosomes 5 and/or 7 revealed 3 with RAS mutations. Further analyses of 23 of these cases uncovered one FLT3 internal tandem duplication and five TP53 mutations. The four patients with RAS or FLT3 mutations had monosomy 7, including one with abnormalities of chromosomes 5 and 7. One specimen demonstrated mutations in both KRAS2 and TP53. RAS and FLT3 mutations, which are thought to stimulate the proliferation of leukemia cells, appear to be less common in t-MDS/t-AML than in de novo AML, whereas TP53 mutations are more frequent.