Pharmacokinetics of omega-3 fatty acids in patients with severe sepsis compared with healthy volunteers: A prospective cohort study.

BACKGROUND: Pharmacokinetics (PK) of pharmaceuticals and pharmaconutrients are poorly understood in critically ill patients, and dosing is often based on healthy subject data. This might be particularly problematic with enteral medications due to metabolic abnormalities and impaired gastrointestinal tract absorption common in critically ill patients. Utilizing enteral fish oil, this study was undertaken to better understand and define PK of enteral omega-3 fatty acids (eicospentaenoic acid [EPA] and docosahexaenoic acid [DHA]) in critically ill patients with severe sepsis. MATERIALS AND METHODS: Healthy volunteers (n = 15) and mechanically ventilated (MV) adults with severe sepsis (n = 10) were recruited and received 9.75 g EPA and 6.75 g DHA daily in two divided enteral doses of fish oil for 7 days. Volunteers continued their normal diet without other sources of fish oil, and sepsis patients received standard enteral feeding. Blood was collected at frequent intervals during the 14-day study period. Peripheral blood mononuclear cells (PMBCs) and neutrophils were isolated and analyzed for membrane fatty acid (FA) content. Mixed linear models and t-tests were used to analyze changes in FA levels over time and FA levels at individual time points, respectively. PK parameters were obtained based on single compartment models of EPA and DHA kinetics. RESULTS: Healthy volunteers were 41.1 ± 10.3 years; 67% were women. In patients with severe sepsis (55.6 ± 13.4 years, 50% women), acute physiologic and chronic health evaluation (APACHE) II score was 27.2 ± 8.8 at ICU admission and median MV duration was 10.5 days. Serum EPA and DHA were significantly lower in sepsis vs. healthy subjects over time. PBMC EPA concentrations were generally not different between groups over time, while PBMC DHA was higher in sepsis patients. Neutrophil EPA and DHA concentrations were similar between groups. The half-life of EPA in serum and neutrophils was significantly shorter in sepsis patients, whereas other half-life parameters did not vary significantly between healthy volunteers and sepsis patients. CONCLUSIONS: While incorporation of n-3 FAs into PBMC and neutrophil membranes was relatively similar between healthy volunteers and sepsis patients receiving identical high doses of fish oil for one week, serum EPA and DHA were significantly lower in sepsis patients. These findings imply that serum concentrations and EPA and DHA may not be the dominant driver of leukocyte membrane incorporation of EPA and DHA. Furthermore, lower serum EPA and DHA concentrations suggest that either these n-3 FAs were being metabolized rapidly in sepsis patients or that absorption of enteral medications and pharmaconutrients, including fish oil, may be impaired in sepsis patients. If enteral absorption is impaired, doses of enteral medications administered to critically ill patients may be suboptimal.

In Vivo Proteome of Pseudomonas aeruginosa in Airways of Cystic Fibrosis Patients.

Chronic airway infection with P. aeruginosa (PA) is a hallmark of cystic fibrosis (CF) disease. The mechanisms producing PA persistence in CF therapies remain poorly understood. To gain insight on PA physiology in patient airways and better understand how in vivo bacterial functioning differs from in vitro conditions, we investigated the in vivo proteomes of PA in 35 sputum samples from 11 CF patients. We developed a novel bacterial-enrichment method that relies on differential centrifugation and detergent treatment to enrich for bacteria to improve identification of PA proteome with CF sputum samples. Using two nonredundant peptides as a cutoff, a total of 1304 PA proteins were identified directly from CF sputum samples. The in vivo PA proteomes were compared with the proteomes of ex vivo-grown PA populations from the same patient sample. Label-free quantitation and proteome comparison revealed the in vivo up-regulation of siderophore TonB-dependent receptors, remodeling in central carbon metabolism including glyoxylate cycle and lactate utilization, and alginate overproduction. Knowledge of these in vivo proteome differences or others derived using the presented methodology could lead to future treatment strategies aimed at altering PA physiology in vivo to compromise infectivity or improve antibiotic efficacy.

Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa.

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.

Regional Isolation Drives Bacterial Diversification within Cystic Fibrosis Lungs.

Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting ten CF lung pairs and studying ∼12,000 regional isolates, we were able to investigate whether clonally related Pseudomonas aeruginosa inhabiting different lung regions evolve independently and differ functionally. Phylogenetic analysis of genome sequences showed that regional isolation of P. aeruginosa drives divergent evolution. We investigated the consequences of regional evolution by studying isolates from mildly and severely diseased lung regions and found evolved differences in bacterial nutritional requirements, host defense and antibiotic resistance, and virulence due to hyperactivity of the type 3 secretion system. These findings suggest that bacterial intermixing is limited in CF lungs and that regional selective pressures may markedly differ. The findings also may explain how specialized bacterial variants arise during infection and raise the possibility that pathogen diversification occurs in other chronic infections characterized by spatially heterogeneous conditions.

Effects of obesity and bariatric surgery on airway hyperresponsiveness, asthma control, and inflammation.

BACKGROUND: Asthma in obese subjects is poorly understood, and these patients are often refractory to standard therapy. OBJECTIVES: We sought to gain insights into the pathogenesis and treatment of asthma in obese subjects by determining how obesity and bariatric surgery affect asthma control, airway hyperresponsiveness (AHR), and markers of asthmatic inflammation. METHODS: We performed a prospective study of (1) asthmatic and nonasthmatic patients undergoing bariatric surgery compared at baseline and (2) asthmatic patients followed for 12 months after bariatric surgery. RESULTS: We studied 23 asthmatic and 21 nonasthmatic patients undergoing bariatric surgery. At baseline, asthmatic patients had lower FEV(1) and forced vital capacity and lower numbers of lymphocytes in bronchoalveolar lavage fluid. After surgery, asthmatic participants experienced significant improvements in asthma control (asthma control score, 1.55 to 0.74; P < .0001) and asthma quality of life (4.87 to 5.87, P < .0001). Airways responsiveness to methacholine improved significantly (methacholine PC(20), 3.9 to 7.28, P = .03). There was a statistically significant interaction between IgE status and change in airways responsiveness (P for interaction = .01). The proportion of lymphocytes in bronchoalveolar lavage fluid and the production of cytokines from activated peripheral blood CD4(+) T cells increased significantly. CONCLUSIONS: Bariatric surgery improves AHR in obese asthmatic patients with normal serum IgE levels. Weight loss has dichotomous effects on airway physiology and T-cell function typically involved in the pathogenesis of asthma, suggesting that obesity produces a unique phenotype of asthma that will require a distinct therapeutic approach.