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1.
Sci Rep ; 10(1): 6903, 2020 04 23.
Article in English | MEDLINE | ID: mdl-32327704

ABSTRACT

Diagnostically informative microbial cell-free DNA (cfDNA) can be detected from blood plasma during fulminant infections such as sepsis. However, the potential for DNA from airway pathogens to enter the circulation of cystic fibrosis (CF) patients during chronic infective states has not yet been evaluated. We assessed whether patient blood contained measurable quantities of cfDNA from CF respiratory microorganisms by sequencing plasma from 21 individuals with CF recruited from outpatient clinics and 12 healthy controls. To account for possible contamination with exogenous microbial nucleic acids, statistical significance of microbe-derived read counts from CF patients was determined relative to the healthy control population. In aggregate, relative abundance of microbial cfDNA was nearly an order of magnitude higher in CF patients than in healthy subjects (p = 8.0×10-3). 15 of 21 (71%) CF patients demonstrated cfDNA from one or more relevant organisms. In contrast, none of the healthy subjects evidenced significant microbial cfDNA for any of the organisms examined. Concordance of cfDNA with standard microbiological culture of contemporaneously collected patient sputum was variable. Our findings provide evidence that cfDNA from respiratory pathogens are present in the bloodstream of most CF patients, which could potentially be exploited for the purposes of noninvasive clinical diagnosis.


Subject(s)
Bacteria/genetics , Cell-Free Nucleic Acids/blood , Cystic Fibrosis/blood , Cystic Fibrosis/microbiology , Lung/microbiology , Adult , Base Sequence , Female , Humans , Male , Middle Aged , Sputum/microbiology , Young Adult
2.
PLoS Pathog ; 15(3): e1007511, 2019 03.
Article in English | MEDLINE | ID: mdl-30893371

ABSTRACT

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis.


Subject(s)
Cell Proliferation/physiology , Pseudomonas aeruginosa/growth & development , Wound Healing/physiology , Adult , Animals , Bacteria/growth & development , Bacterial Infections/metabolism , Biofilms/growth & development , Disease Models, Animal , Female , Genetic Fitness , Host Microbial Interactions/physiology , Humans , Male , Mice , Pseudomonas Infections , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Virulence/physiology , Wound Infection/metabolism , Wound Infection/microbiology
3.
mBio ; 8(5)2017 10 31.
Article in English | MEDLINE | ID: mdl-29089424

ABSTRACT

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.IMPORTANCE Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.


Subject(s)
Anti-Bacterial Agents/pharmacology , Aztreonam/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Evolution, Molecular , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/pathogenicity , Animals , Chromosomes, Bacterial/genetics , Chronic Disease , Cystic Fibrosis/complications , Cystic Fibrosis/microbiology , Directed Molecular Evolution/methods , Disease Models, Animal , Genetic Fitness , Humans , Membrane Transport Proteins , Mice , Microbial Sensitivity Tests , Mutation , Phenotype , Pneumonia/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development , Whole Genome Sequencing
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