In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A.

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

A Randomized Controlled Trial of Atorvastatin in Patients With Bronchiectasis Infected With Pseudomonas Aeruginosa: A Proof of Concept Study.

BACKGROUND: There are no randomized controlled trials of statin therapy in patients with severe bronchiectasis who are chronically infected with Pseudomonas aeruginosa. METHODS: Thirty-two patients chronically infected with P aeruginosa were recruited in this double-blind cross-over randomized controlled trial. Sixteen patients were recruited in each arm, were given atorvastatin 80 mg or placebo for 3 months followed by a washout period for 6 weeks, and then crossed over and administered the alternative therapy for 3 months. RESULTS: Twenty-seven patients completed the study. Atorvastatin did not significantly improve the primary end point of cough as measured by the Leicester Cough Questionnaire (mean difference, 1.92; 95% CI for difference, -0.57-4.41; P = .12). However, atorvastatin treatment resulted in an improved St. Georges Respiratory Questionnaire (-5.62 points; P = .016) and reduced serum levels of CXCL8 (P = .04), tumor necrosis factor (P = .01), and intercellular adhesion molecule 1 (P = .04). There was a trend toward improvement in serum C-reactive protein and serum neutrophil counts (P = .07 and P = .06, respectively). We demonstrated in vitro that atorvastatin 10 µM reduced formyl-methionyl-leucyl phenylalanine-induced upregulation of CD11b expression and changes in calcium flux, reflecting an ability to decrease neutrophil activation. CONCLUSIONS: We demonstrated that atorvastatin reduced systemic inflammation and improved quality of life in patients with bronchiectasis who were infected with P aeruginosa. These effects may be due to an ability of atorvastatin to modulate neutrophil activation. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01299194; URL: www.clinicaltrials.gov.

Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium.

BACKGROUND: Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS). METHODOLOGY/PRINCIPAL FINDINGS: We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment. CONCLUSIONS/SIGNIFICANCE: The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.

Garlic revisited: antimicrobial activity of allicin-containing garlic extracts against Burkholderia cepacia complex.

The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.

Cathelicidin host defence peptide augments clearance of pulmonary Pseudomonas aeruginosa infection by its influence on neutrophil function in vivo.

Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.

Atorvastatin as a stable treatment in bronchiectasis: a randomised controlled trial.

BACKGROUND: Bronchiectasis is characterised by chronic cough, sputum production, and recurrent chest infections. Pathogenesis is poorly understood, but excess neutrophilic airway inflammation is seen. Accumulating evidence suggests that statins have pleiotropic effects; therefore, these drugs could be a potential anti-inflammatory treatment for patients with bronchiectasis. We did a proof-of-concept randomised controlled trial to establish if atorvastatin could reduce cough in patients with bronchiectasis. METHODS: Patients aged 18-79 years were recruited from a secondary-care clinic in Edinburgh, UK. Participants had clinically significant bronchiectasis (ie, cough and sputum production when clinically stable) confirmed by chest CT and two or more chest infections in the preceding year. Individuals were randomly allocated to receive either high-dose atorvastatin (80 mg) or a placebo, given orally once a day for 6 months. Sequence generation was done with a block randomisation of four. Random allocation was masked to study investigators and patients. The primary endpoint was reduction in cough from baseline to 6 months, measured by the Leicester Cough Questionnaire (LCQ) score, with a lower score indicating a more severe cough (minimum clinically important difference, 1·3 units). Analysis was done by intention-to-treat. The trial is registered with ClinicalTrials.gov, number NCT01299181. FINDINGS: Between June 23, 2011, and Jan 30, 2011, 82 patients were screened for inclusion in the study and 22 were excluded before randomisation. 30 individuals were assigned atorvastatin and 30 were allocated placebo. The change from baseline to 6 months in LCQ score differed between groups, with a mean change of 1·5 units in patients allocated atorvastatin versus -0·7 units in those assigned placebo (mean difference 2·2, 95% CI 0·5-3·9; p=0·01). 12 (40%) of 30 patients in the atorvastatin group improved by 1·3 units or more on the LCQ compared with five (17%) of 30 in the placebo group (difference 23%, 95% CI 1-45; p=0·04). Ten (33%) patients assigned atorvastatin had an adverse event versus three (10%) allocated placebo (difference 23%, 95% CI 3-43; p=0·02). No serious adverse events were recorded. INTERPRETATION: 6 months of atorvastatin improved cough on a quality-of-life scale in patients with bronchiectasis. Multicentre studies are now needed to assess whether long-term statin treatment can reduce exacerbations. FUNDING: Chief Scientist's Office.

Draft Genome Sequence of a Stable Mucoid Strain of Pseudomonas aeruginosa PAO581 with a mucA25 Mutation.

A mutation in the mucA gene, which encodes a negative regulator of alginate production in Pseudomonas aeruginosa, is the main mechanism underlying the conversion to mucoidy in clinical isolates from patients with cystic fibrosis (CF). Here, we announce the draft genome sequence of the stable alginate-overproducing mucoid strain P. aeruginosa PAO581 with a mucA25 mutation, a derivative from the nonmucoid strains P. aeruginosa PAO381 and PAO1.

A lung segmental model of chronic Pseudomonas infection in sheep.

BACKGROUND: Chronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep. METHODOLOGY/PRINCIPAL FINDINGS: Using local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>10(4) cfu/g). CONCLUSIONS/SIGNIFICANCE: The agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.

Vitamin-D deficiency is associated with chronic bacterial colonisation and disease severity in bronchiectasis.

INTRODUCTION: Vitamin-D deficiency has been linked to an increased risk of respiratory infections. The objective of this study was to determine the frequency and clinical importance of vitamin-D deficiency in patients with bronchiectasis. METHODS: 25-hydroxyvitamin-D was measured by immunoassay in 402 stable patients with bronchiectasis. Patients were classified as vitamin-D deficient (serum 25-hydroxyvitamin-D <25 nmol/l), insufficient (25 nmol/l-74 nmol/l) or sufficient (≥ 75 nmol/l). Disease severity was assessed, including exacerbation frequency, measurement of airway inflammatory markers, sputum bacteriology and lung function over 3 years follow-up. RESULTS: 50% of bronchiectasis patients were vitamin-D deficient, 43% insufficient and only 7% sufficient. This compared to only 12% of age and sex matched controls with vitamin-D deficiency (p<0.0001). Vitamin-D deficient patients were more frequently chronically colonised with bacteria (p<0.0001), 21.4% of vitamin-D deficient subjects were colonised with Pseudomonas aeruginosa compared to 10.4% of insufficient patients and 3.6% of sufficient patients, p=0.003. Vitamin-D deficient patients had lower FEV(1)% predicted (p=0.002), and more frequent pulmonary exacerbations (p=0.04). Vitamin-D deficient patients had higher sputum levels of inflammatory markers and demonstrated a more rapid decline in lung function over 3 years follow-up. Defects in neutrophil function and assessment of airway LL-37 levels did not provide a mechanistic explanation for these findings. Vitamin-D deficient patients had, however, higher levels of Vitamin-D Binding Protein in sputum sol. CONCLUSIONS: Vitamin-D deficiency is common in bronchiectasis and correlates with markers of disease severity. The mechanism of this association is unclear.

Mannose-binding lectin deficiency and disease severity in non-cystic fibrosis bronchiectasis: a prospective study.

BACKGROUND: Mannose-binding lectin (MBL) is a key component of innate immunity. MBL deficiency is common (10-30% of the general population depending on the definition used) and has been associated with disease progression in cystic fibrosis. We aimed to assess the effect of MBL deficiency on disease severity in non-cystic fibrosis bronchiectasis. METHODS: We recruited patients with non-cystic fibrosis bronchiectasis and age-matched and sex-matched controls at a specialist bronchiectasis clinic in Edinburgh, UK. We assessed MBL function with genotyping (low-expressing genotype [deficiency] defined as homozygosity for exon 1 mutations [YO/YO] or compound heterozygosity [XA/YO]; YA/YO and XA/XA genotypes were defined as intermediate-expressing with all other genotypes defined as high-expressing) and serum measurements (deficiency defined with two parameters: <500 ng/mL or <200 ng/mL). We assessed rates of exacerbation, chronic bacterial colonisation, and lung function during 4 years of follow-up. FINDINGS: We included 470 patients with bronchiectasis and 414 controls. MBL genotype frequencies and MBL serum concentrations did not differ between patients and controls. 55 (12%) patients with bronchiectasis had low-expressing genotypes. These patients had a mean of 2·7 exacerbations per year (SD 1·8), compared with 1·9 per year (1·2) for 135 patients with intermediate-expressing genotypes and 1·9 per year (1·3) for 280 patients with high-expressing genotypes (p<0·0001). Chronic colonisation with bacteria was most frequent in patients with low-expressing genotypes (47 [85%] patients vs 82 [61%] patients with intermediate-expressing genotypes and 183 [65%] patients with high-expressing genotypes; p=0·0041); especially P aeruginosa colonisation (19 [35%] patients vs 13 [10%] patients and 36 [13%] patients; p<0·0001). Patients with low-expressing genotypes were more likely to be admitted to hospital for severe exacerbations during follow-up (27 [49%] patients vs 42 [31%] patients and 87 [31%] patients; p=0·032). Patients with low-expressing genotypes also had increased scores for radiological severity and worse quality of life compared with the other two groups. MBL serum deficiency (<200 ng/mL) was associated with increased exacerbations, hospital admissions, and radiological severity. When <500 ng/mL was used as the definition of deficiency, the associations with exacerbation frequency and radiological severity were no longer significant. INTERPRETATION: MBL might be an important modifier of disease severity in non-CF bronchiectasis. FUNDING: UK Medical Research Council, UK Chief Scientists Office.

Short- and long-term antibiotic treatment reduces airway and systemic inflammation in non-cystic fibrosis bronchiectasis.

RATIONALE: The vicious cycle hypothesis of bronchiectasis argues that bacterial colonization leads to airway inflammation and progressive lung damage. The logical extension of this hypothesis is that acute or chronic antibiotic therapy should improve airway inflammation and clinical outcome. There are little data to support this hypothesis in patients with non-cystic fibrosis (CF) bronchiectasis. OBJECTIVES: To determine whether acute or chronic antibiotic therapy improves airway inflammation and clinical outcome in non-CF bronchiectasis. METHODS: The relationship between bacterial load and airway and systemic inflammation was investigated in 385 stable patients, 15 stable patients treated with intravenous antibiotics, and 34 patients with an exacerbation of bronchiectasis treated with intravenous antibiotics. Long-term antibiotic therapy was investigated using samples from a 12-month controlled trial of nebulized gentamicin. MEASUREMENTS AND MAIN RESULTS: In stable patients, there was a direct relationship between airway bacterial load and markers of airway inflammation (P < 0.0001 for all analyses). High bacterial loads were associated with higher serum intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 (P < 0.05 above bacterial load ≥1 × 10(7) cfu/ml). In stable patients, there was a direct relationship between bacterial load and the risk of subsequent exacerbations (odds ratio, 1.20; 95% confidence interval, 1.11-1.29; P < 0.0001) and severe exacerbations (odds ratio, 1.11; 95% confidence interval, 1.01-1.21; P = 0.02). Short- and long-term antibiotic treatments were associated with reductions in bacterial load, airways, and systemic inflammation. CONCLUSIONS: High airway bacterial loads in non-CF bronchiectasis are associated with airway and systemic inflammation and a greater risk of exacerbations. Short- and long-term antibiotic therapy reduce markers of airways and systemic inflammation.

Pseudomonas aeruginosa bacteraemia in an adult with cystic fibrosis and acute appendicitis.

Despite their high bacterial load, bacteraemia is rare in patients with cystic fibrosis (CF). We report an adult with CF who developed Pseudomonas aeruginosa bacteraemia during an episode of acute appendicitis. The Pseudomonas aeruginosa isolated from the blood culture was confirmed by molecular typing to be the same transmissible strain responsible for the patient's chronic pulmonary infection. We hypothesise that this patient's bacteraemia was caused by Pseudomonas aerunginosa in swallowed sputum, crossing the inflamed appendiceal wall and entering the blood stream.

A randomized controlled trial of nebulized gentamicin in non-cystic fibrosis bronchiectasis.

RATIONALE: Bronchiectasis is a chronic debilitating disease with few evidence-based long-term treatments. OBJECTIVES: A randomized controlled trial assessing the efficacy of nebulized gentamicin therapy over 1 year in patients with non-cystic fibrosis bronchiectasis. METHODS: Sixty-five patients were randomized to either twice-daily nebulized gentamicin, 80 mg, or nebulized 0.9% saline, for 12 months. All were reviewed at three-monthly intervals during treatment and at 3 months' follow-up. MEASUREMENTS AND MAIN RESULTS: At each review the following were assessed: quantitative and qualitative sputum bacteriology; sputum purulence and 24-hour volume; FEV(1), FVC, and forced expiratory flow, midexpiratory phase; exercise capacity; Leicester Cough Questionnaire and St. George's Respiratory Questionnaire; and exacerbation frequency. Fifty-seven patients completed the study. At the end of 12 months' treatment, compared with the saline group, in the gentamicin group there was reduced sputum bacterial density with 30.8% eradication in those infected with Pseudomonas aeruginosa and 92.8% eradication in those infected with other pathogens; less sputum purulence (8.7% vs. 38.5%; P < 0.0001); greater exercise capacity (510 [350-690] m vs. 415 [267.5-530] m; P = 0.03); and fewer exacerbations (0 [0-1] vs. 1.5 [1-2]; P < 0.0001) with increased time to first exacerbation (120 [87-161.5] d vs. 61.5 [20.7-122.7] d; P = 0.02). The gentamicin group had greater improvements in Leicester Cough Questionnaire (81.4% vs. 20%; P < 0.01) and St. George's Respiratory Questionnaire (87.5% vs. 19.2%; P < 0.004) score. No differences were seen in 24-hour sputum volume, FEV(1), FVC, or forced expiratory flow, midexpiratory phase. No P. aeruginosa isolates developed resistance to gentamicin. At follow-up, all outcome measures were similar to baseline. CONCLUSIONS: Regular, long-term nebulized gentamicin is of significant benefit in non-cystic fibrosis bronchiectasis but treatment needs to be continuous for its ongoing efficacy. Clinical trial registered with www.clinicaltrials.gov (NCT 00749866).

Can early Burkholderia cepacia complex infection in cystic fibrosis be eradicated with antibiotic therapy?

INTRODUCTION: Organisms of the Burkholderia cepacia complex (BCC) are important pathogens in cystic fibrosis (CF). The majority of those who acquire BCC develop chronic infection but it can also result in rapid decline in a significant minority. In addition, chronic infection with Burkholderia cenocepacia in particular is regarded as a contraindication to lung transplantation in many units. Whilst aggressive antibiotic therapy is employed in CF to eradicate Pseudomonas aeruginosa before infection becomes irreversibly established, no formal assessment of such strategies has been previously reported for BCC, despite the apparent widespread adoption of this practice. METHODS: UK adult CF centers were surveyed about their current approach to new BCC infection. Outcomes of eradication therapy were assessed in patients attending the Manchester Adult CF Center with new BCC isolates between 1st January 2002 and 1st May 2011. Patients with previous infection with the same strain of BCC were excluded. BCC were identified at the national reference laboratories and confirmed by species-specific PCR and RecA sequencing. RESULTS: Routine use of therapies to attempt eradication of new BCC is commonly used in the UK (12/17 centers who responded). This typically involves a combination of intravenous and nebulised antibiotics. Of 19 eligible cases of new BCC infection, the organism has been eradicated in 7 (37%). Three of these did not receive specific eradication therapy. Of 14 patients who have received eradication therapy and completed follow up, BCC were cleared in only 4 (29%). CONCLUSIONS: Attempted eradication of new BCC is a common practice in UK adult CF centers. A minority of patients clear the infection spontaneously and the effects of eradication therapies are at best modest. Early treatment may be associated with better outcomes, though there are insufficient data to support the use of any specific treatment regimen. A prospective, systematic evaluation of treatments and outcomes is required.

Do processing time and storage of sputum influence quantitative bacteriology in bronchiectasis?

This study aimed to establish whether the bacterial density of spontaneous sputum is affected by the time and mode of sample storage. Ten patients with bronchiectasis collected all sputum expectorated over 45 min. The samples were aliquoted and processed at 25 degrees C for qualitative and quantitative bacteriology at 1, 2, 4 and 6 h from expectoration. Further aliquots were stored at 25 degrees C, 4 degrees C and -20 degrees C for 24 and 48 h prior to processing. The species present was identified and median (interquartile range) sputum log(10) bacterial density (c.f.u. ml(-1)) calculated. All samples cultured grew Pseudomonas aeruginosa and for two patients Staphylococcus aureus additionally grew for all samples. There was no significant difference in P. aeruginosa density in samples processed at 1, 2, 4 and 6 h following expectoration [8.2 (7.8-8.3) c.f.u. ml(-1), 8.0 (7.8-8.3) c.f.u. ml(-1), 8.0 (7.9-8.2) c.f.u. ml(-1), 8.1 (7.9-8.2) c.f.u. ml(-1), respectively, P=0.392]. Storage for 24 and 48 h at 4 degrees C did not significantly change the bacterial load compared with processing at 1 h [8.03 (7.6-8.2) c.f.u. ml(-1), P=0.07, and 7.96 (7.49-8.22) c.f.u. ml(-1), P=0.09, respectively]. Storage for 24 and 48 h at -20 degrees C significantly reduced P. aeruginosa density [7.1 (6.1-7.7) c.f.u. ml(-1), P=0.005, and 6.9 (6.2-7.6) c.f.u. ml(-1), P=0.008, respectively]. Storage at 25 degrees C for 24 and 48 h was associated with a significant increase in bacterial load [8.3 (8.1-8.6) c.f.u. ml(-1), P=0.009, and 8.4 (8.1-8.5) c.f.u. ml(-1), P=0.03, respectively]. Bacterial density was not affected by storage for up to 6 h following expectoration at 25 degrees C; beyond this, storage at 4 degrees C is preferred.

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Clinical outcome for cystic fibrosis patients infected with transmissible pseudomonas aeruginosa: an 8-year prospective study.

BACKGROUND: Although there is now compelling evidence for cross-infection by strains of Pseudomonas aeruginosa at some specialist (cystic fibrosis [CF]) centers, the clinical impact of infection by transmissible strains is unclear. METHODS: In an 8-year prospective study, we compared the clinical outcome of two groups of patients with CF infected by transmissible (n = 28) and sporadic strains (n = 52) of P aeruginosa. RESULTS: There were no differences between the two groups in survival, annual changes in spirometry, or BMI. There were differences in requirements for IV antibiotic treatment (mean [SD]: 29.3 [21.9] days vs 53.1 [32.5] days) and hospitalization (median [range]: 11.6 [1.1, 49.3] days vs 23.3 [5.5, 103.6] days) between patients infected with sporadic and transmissible strains of P aeruginosa, respectively. CONCLUSIONS: We conclude that infection by transmissible P aeruginosa does not increase mortality but is associated with increased health-care and antibiotic use for patients with CF.

The human cathelicidin LL-37 preferentially promotes apoptosis of infected airway epithelium.

Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.