Elevated exopolysaccharide levels in Pseudomonas aeruginosa flagellar mutants have implications for biofilm growth and chronic infections.

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.

Staphylococcus aureus small-colony variants are independently associated with worse lung disease in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is associated with diverse bacteria chronically infecting the airways. Slow-growing, antibiotic-resistant mutants of Staphylococcus aureus known as small-colony variants (SCVs) have been isolated from respiratory secretions from European adults and children with CF lung disease using specific but infrequently used culture techniques. Staphylococcus aureus SCVs can be selected either by exposure to specific antibiotics or by growth with another CF pathogen, Pseudomonas aeruginosa. We sought to determine the prevalence, clinical significance, and likely mechanisms of selection of S. aureus SCVs among a US cohort of children with CF. METHODS: We performed a 2-year study of 100 children with CF using culture techniques sensitive for S. aureus SCVs, and evaluated associations with clinical characteristics using multivariable regression models. RESULTS: Staphylococcus aureus SCV infection was detected among 24% of participants and was significantly associated with a greater drop in lung function during the study (P = .007, adjusted for age and lung function at enrollment). This association persisted after adjusting for infection with other known CF pathogens, including P. aeruginosa and methicillin-resistant S. aureus. Evidence indicated that S. aureus SCVs were likely selected in vivo by treatment with the antibiotic trimethoprim-sulfamethoxazole and possibly by coinfection with P. aeruginosa. CONCLUSIONS: Infection with SCV S. aureus was independently associated with worse CF respiratory outcomes in this pediatric cohort. As many clinical microbiology laboratories do not specifically detect S. aureus SCVs, validation and extension of these findings would require widespread changes in the usual laboratory and clinical approaches to these bacteria.

Randomized trial of biofilm testing to select antibiotics for cystic fibrosis airway infection.

RATIONALE: In cystic fibrosis (CF), conventional antibiotic susceptibility results correlate poorly with clinical outcomes. We hypothesized that biofilm testing would more accurately reflect the susceptibilities of bacteria infecting CF airways. METHODS: A multicenter randomized pilot trial was conducted to assess the efficacy and safety of using biofilm susceptibility testing of Pseudomonas aeruginosa sputum isolates to guide antibiotic regimens for chronic airway infections in clinically stable adolescent and adult CF patients. Thirty-nine participants were randomized to biofilm or conventional treatment groups; 14-day courses of two antibiotics were selected according to an activity-based algorithm using the corresponding susceptibility results. RESULTS: Of the agents tested, meropenem was most active against biofilm-grown bacteria, and was included in regimens for about half of each study group. For 19 of 39 randomized participants, randomization to the other study group would not have changed the antibiotic classes of the assigned regimen. Study groups were comparable at baseline, and had similar mean decreases in bacterial density, measured in log(10) colony forming units per gram of sputum (biofilm, -2.94 [SD 2.83] vs. conventional, -3.27 [SD 3.09]), and mean increases in forced expiratory volume in 1 sec, measured in liters (0.18 [SD 0.20] vs. 0.12 [SD 0.22]). CONCLUSIONS: In this pilot study, antibiotic regimens based on biofilm testing did not differ significantly from regimens based on conventional testing in terms of microbiological and clinical responses. The predictive value of biofilm testing may nonetheless warrant evaluation in an adequately powered clinical trial in younger CF patients or those experiencing acute pulmonary exacerbation.

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

Unique lipid a modifications in Pseudomonas aeruginosa isolated from the airways of patients with cystic fibrosis.

Three structural features of lipid A (addition of palmitate [C16 fatty acid], addition of aminoarabinose [positively charged amino sugar residue], and retention of 3-hydroxydecanoate [3-OH C10 fatty acid]) were determined for Pseudomonas aeruginosa isolates from patients with cystic fibrosis (CF; n=86), from the environment (n=13), and from patients with other conditions (n=14). Among P. aeruginosa CF isolates, 100% had lipid A with palmitate, 24.6% with aminoarabinose, and 33.3% retained 3-hydroxydecanoate. None of the isolates from the environment or from patients with other conditions displayed these modifications. These results indicate that unique lipid A modifications occur in clinical P. aeruginosa CF isolates.

Use of Pseudomonas biofilm susceptibilities to assign simulated antibiotic regimens for cystic fibrosis airway infection.

OBJECTIVES: Increasing evidence indicates that Pseudomonas aeruginosa grows as a biofilm in the lungs of cystic fibrosis (CF) patients. In contrast, the bacterial inoculum used in conventional susceptibility testing is composed of planktonic cells. As a prelude to a clinical trial of biofilm susceptibility testing in CF, simulated antibiotic regimens based on either biofilm or conventional susceptibility testing of CF patient isolates were compared. PATIENTS AND METHODS: Biofilm and conventional susceptibilities were determined for P. aeruginosa isolate sets from 40 CF patients. An algorithm was used to assign simulated regimens of two anti-pseudomonal antibiotics for each patient/susceptibility method dataset. For agents with equivalent activity, the algorithm included a drug selection hierarchy, the rationale for which was suppression of chronic infection. Substitution of an alternative hierarchy, based on treatment of acute exacerbation, was used to evaluate the robustness of the regimen assignments. RESULTS: For both drug-ranking schemes, all 40 simulated regimens based on conventional susceptibilities included a beta-lactam antibiotic. In contrast, based on biofilm testing, only 43% of chronic regimens and 65% of acute regimens included a beta-lactam. Moreover, the conventional and biofilm regimens assigned to individual patients were discordant, with only 20% and 40% of chronic and acute regimens, respectively, consisting of drugs in the same two mechanistic classes by both methods. CONCLUSIONS: Biofilm susceptibility testing of CF P. aeruginosa isolate sets leads to different antibiotic assignments than conventional testing, with no single two-drug regimen predicted to provide optimal anti-biofilm activity against the majority of isolate sets.

Analysis of sequential aliquots of hypertonic saline solution-induced sputum from clinically stable patients with cystic fibrosis.

STUDY OBJECTIVES: Sputum induction (SI) is a noninvasive tool for sampling inflamed airways. The purpose of this study was to determine the optimal duration of collection in patients with cystic fibrosis (CF). The hypothesis was that the duration of SI collection would quantitatively and qualitatively alter the content of the induced sputum. METHODS: In 10 clinically stable patients with CF (mean +/- SD age, 28 +/- 7 years; mean FEV(1), 2.6 +/- 0.7 L), SI was performed with 3% hypertonic saline solution at five time points over 20 min. RESULTS: SI was well tolerated, with an average maximum fall in FEV(1) of 7 +/- 7%. The sample volumes, urea concentrations, interleukin-8 concentrations, total cell counts, and nonsquamous cell counts remained constant (p > 0.05). The percentage of neutrophils decreased from 89 +/- 5% to 86 +/- 4% (p = 0.03), and the percentage of alveolar macrophages increased 5 +/- 2% to 8 +/- 4% (p < 0.01). The mean quantitative microbiological counts of nonmucoid Pseudomonas aeruginosa and Staphylococcus aureus decreased over the 20-min time period each by half a log (p = 0.05 and p < 0.01, respectively). Surfactant protein-A concentration increased from 1.6 +/- 0.3 to 2.4 +/- 0.4 ng/mL (log(10); p < 0.001). CONCLUSIONS: We conclude that aliquots of induced sputum are similar in clinically stable patients with CF during 4-min intervals, although there is more alveolar sampling after 20 min. When induced-sputum samples are fractionated for research monitoring of inflammatory or microbiologic indexes, power calculations accounting for these variations over time are required.