Peter J. Cole

Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro.

Pseudomonas aeruginosa culture filtrates varied in their ability to slow human ciliary beat frequency (7-71%). This activity did not correlate with known virulence factors. However, a close correlation (r = 0.97) existed between ciliary slowing and pigment content. In a prolonged culture, the increase in activity correlated (r = 0.94) with pigment accumulation. Gel filtration of lyophilized filtrate yielded a single peak of activity corresponding to the pigment fraction. Pyocyanin extracted from an active strain, and 1-hydroxyphenazine were purified by high performance liquid chromatography, and characterized by ultraviolet absorbance spectra and mass spectrometry. Both slowed cilia in a dose-dependent manner, and were synthesized and shown to be indistinguishable from the biological compounds. Pyocyanin caused gradual onset of slowing and ultimate widespread ciliostasis with epithelial disruption. 1-hydroxyphenazine caused rapid onset of ciliary slowing associated with dyskinesia and ciliostasis. Pyocyanin assayed within filtrates accounted for a significant proportion of the bioactivity present.

Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth

AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors. METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined. Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis. Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy. Antifungal activity against C albicans and A fumigatus was determined in a well plate assay. RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity. Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml. These phenazines were active against nine other yeast species pathogenic for man. Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin. CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa.

Effect of sputum bacteriology on the quality of life of patients with bronchiectasis

Bronchiectatic patients have impaired health-related quality of life (QoL) and are prone to chronic lower respiratory tract infections. We have investigated whether impaired QoL is related to sputum bacteriology. Eighty seven patients with non-cystic fibrosis (non-CF) bronchiectasis, in a stable phase of their illness, completed three QoL measures, underwent a computed tomography (CT) scan and lung function tests, and provided a fresh sputum sample for microscopy and culture. The QoL of patients colonized by Pseudomonas aeruginosa (Pa group) was significantly worse than all other patients grouped together (non-Pa group), and specifically those infected by Haemophilus influenzae (Hi group) or who had no bacterial growth (NG group) (p<0.05), but not those infected by other bacterial species (O group). The Pa group had worse lung function, but no significant differences were found between the groups for forced expiratory volume in one second (FEV1) and peak expiratory flow rate. The Pa group had significantly worse bronchiectasis scores than the O, NG and non-Pa groups, but not the Hi group. There were no significant differences between the groups with respect to the number of infective exacerbations in the last year, but the Pa group had significantly more hospital admissions. Patients infected by P. aeruginosa for more than 3 yrs had significantly worse FEV1 (p<0.03) and bronchiectasis scores (p<0.05) than those infected with P. aeruginosa for less time, but not significantly worse QoL. We conclude that, overall, patients infected with P. aeruginosa have worse quality of life, and that P. aeruginosa is associated with a greater extent of disease and worse lung function. Although patients infected with H. influenzae had extensive bronchiectasis their quality of life was better than the P. aeruginosa infected group.

Increased levels of exhaled carbon monoxide in bronchiectasis: a new marker of oxidative stress

BACKGROUND Bronchiectasis is a chronic inflammatory lung disease associated with increased production of oxidants due mostly to neutrophilic inflammation. Induction of heme oxygenase (HO-1) by reactive oxygen species is a general cytoprotective mechanism against oxidative stress. HO-1 catabolises heme to bilirubin, free iron and carbon monoxide (CO). Exhaled CO measurements may therefore reflect an oxidative stress and be clinically useful in the detection and management of inflammatory lung disorders. METHODS The levels of exhaled CO of 42 non-smoking patients with bronchiectasis treated or not treated with inhaled corticosteroids were compared with CO levels in 37 normal non-smoking subjects. RESULTS Levels of exhaled CO were raised in patients with bronchiectasis, both those treated with inhaled corticosteroids (n = 27, median 5.5 ppm, 95% CI 5.16 to 7.76) and those not treated with inhaled corticosteroids (n = 15, median 6.0 ppm, 95% CI 4.74 to 11.8), compared with normal subjects (n = 37, median 3.0 ppm, 95% CI 2.79 to 3.81, p = 0.0024). There was no correlation between exhaled CO and HbCO levels (r = 0.42, p = 0.12) in normal subjects (n = 7), nor between the urine cotinine concentration and exhaled CO levels (r = 0.2, p = 0.12). CONCLUSIONS Increased levels of exhaled CO may reflect induction of HO-1 and oxidative stress in bronchiectasis. Measurement of exhaled CO may be useful in the management of bronchiectasis and possibly other chronic inflammatory lung disorders.

Effect of bacterial products on human ciliary function in vitro.

Ciliary activity protects the respiratory tract against inhaled particles, including bacteria, by transporting them trapped in mucus towards the pharynx. We have studied the effect of bacteria (Haemophilus influenzae, Staphylococcus aureus, and Pseudomonas aeruginosa) on human nasal cilia, measuring their in vitro ciliary beat frequency by a photometric technique. Supernatant fluids were obtained from 18 hour broth cultures by centrifugation alone, by filtration, and by lysis. Supernatants obtained from Ps aeruginosa and H influenzae caused a significantly lower ciliary beat frequency than controls (broth alone). Slowed cilia were dyskinetic and at times of maximal slowing ciliostasis occurred in some areas of the epithelium. A dose related effect was demonstrated. Abrogation of cilioinhibitory properties was achieved by heating the lysate to 56 degrees C for 30 minutes and by allowing the filtrate to stand at 37 degrees C for 120 minutes. Staphylococcal products were not cilioinhibitory. It is concluded that Ps aeruginosa and H influenzae release a factor (or factors) which causes slowing of human nasal cilia in vitro. The role of this factor in the pathogenesis of infection is discussed.

Nasal nitric oxide measurements for the screening of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) presents to general practitioners with symptoms pertinent to a variety of specialists because of the involvement of ciliated epithelium in the upper/lower respiratory tract, ears, eyes and genital tract. There is no easy, reliable screening test for PCD, and thus, the majority of patients remain undiagnosed. Nitric oxide (NO) is measurable in nasal air of normal subjects and found to be low in cystic fibrosis (CF) and very low in PCD. Recently, it was suggested to play an important role in regulating ciliary motility. The aim of this study was to evaluate whether measurements of nasal NO could be used to screen for PCD. Nasal NO was measured from the nasal cavity by a chemiluminescence analyser in subjects with PCD, healthy controls, CF, idiopathic bronchiectasis, Youngs syndrome and lone sinusitis. Nasal NO was significantly lower in PCD (64.0±36.6) compared with normal controls (759±145.8), idiopathic bronchiectasis (734±163.7), CF (447.5±162.6), lone sinusitis (1487±734) and Youngs syndrome (644±129.9). Nasal NO was also significantly lower in PCD than CF patients. Measurement of nasal nitric oxide may therefore be used clinically in various specialities to screen suspected patients for primary ciliary dyskinesia.

Sodium chloride increases the ciliary transportability of cystic fibrosis and bronchiectasis sputum on the mucus-depleted bovine trachea.

Mucus retention in the lungs is an important feature of several respiratory diseases (Regnis, J.A., M. Robinson, D.L. Bailey, P. Cook, P. Hooper, H.K. Chan, I. Gonda, G. Bautovich, and P.T.P. Bye. 1994. Am. J. Respir. Crit. Care Med. 150:66-71 and Currie, D.C., D. Pavia, J.E. Agnew, M.T. Lopez-Vidriero, P.D. Diamond, P.J. Cole, and S.W. Clarke. 1987. Thorax. 42:126-130). On the mucus-depleted bovine trachea, the ciliary transport rate of sputum from patients with cystic fibrosis and bronchiectasis of other causes was slow, but the rate was doubled by increasing the sodium chloride content by 90 mM. Increasing the sputum osmolality by inspissation or by the addition of nonelectrolytes had a similar effect. The viscoelasticity of sputum, but not the bovine ciliary beat frequency, was markedly saline dependent over the pathophysiological range. This suggests that low mucus salinity, not (as is generally assumed) its under-hydration, contributes to its retention in bronchiectasis due to cystic fibrosis and other causes, probably by affecting its rheology. It also indicates how the genetic defect in cystic fibrosis might lead to impaired mucus clearance. Therapies that increase the osmolality of lung mucus might benefit patients with mucus retention.

RELATIVE IMPORTANCE OF ANTIBIOTIC AND IMPROVED CLEARANCE IN TOPICAL TREATMENT OF CHRONIC MUCOPURULENT RHINOSINUSITIS: A Controlled Study

50 patients with chronic mucopurulent rhinosinusitis were randomly allocated to treatment with nasal sprays of dexamethasone, tramazoline, and neomycin, dexamethasone and tramazoline with no antibiotic, or matched placebo (propellant alone) four times daily to both nostrils for 2 weeks. The patients were assessed in a double-blind manner for symptomatic response and improvement in nasal mucociliary clearance, nasal airway resistance, sinus radiographs, and intranasal bacteriology and appearance. Both active preparations (with antibiotic 14 of 20 patients responded; without antibiotic 12 of 20 patients responded) were more effective than the placebo (2 of 10 patients responded). There was no significant difference in response between the active preparations with and without antibiotic. Thus, in treatment of chronic mucopurulent rhinosinusitis, reduction of the inflammatory response and decongestion make topical antibiotic unnecessary, probably by allowing host clearance mechanisms to recover.

The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro

Streptococcus pneumoniae culture filtrates and pneumolysin both slow human ciliary beating and damage respiratory epithelium in vitro. A polyclonal pneumolysin antibody bound to sepharose beads removed pneumolysin from culture filtrates and showed that pneumolysin alone was responsible for the effects on epithelium. In a 48-h organ culture pneumolysin caused ciliary slowing and epithelial disruption in a dose-dependent manner down to 5 ng/ml. Comparison of the ciliary slowing activity and pneumolysin concentration in filtrates in a continuous broth culture showed a maximal effect at 16 h (pneumolysin 7.5 micrograms/ml). Later the activity decreased while the pneumolysin concentration increased (8.8 micrograms/ml). This loss of activity was prevented by neutralisation of the acid pH of the culture medium. Eight different culture filtrates produced significant (P less than 0.05) ciliary slowing which correlated (r = 0.95) with simultaneously measured haemolytic (pneumolysin) activity. Substitution of tryptophan (position 433) by phenylalanine reduced the haemolytic and ciliary slowing activity of pneumolysin, but did not affect its ability to activate complement. There was no correlation between the ciliary slowing produced by the culture filtrate and that produced by the autolysate of a particular strain, nor between ciliary slowing and the extent of autolysis or the serotype of the strain.

Fertility in men with primary ciliary dyskinesia presenting with respiratory infection.

BACKGROUND--Primary ciliary dyskinesia is characterised by chronic rhinosinusitis, chronic bronchial sepsis (usually with bronchiectasis), dextrocardia in approximately 50% of cases, and male infertility. The latter, described in patients attending infertility clinics, results from immotile but viable spermatozoa. Experience in a respiratory clinic suggests that infertility in men is not invariable. METHODS--The seminal fluid of 12 men with primary ciliary dyskinesia, six with dextrocardia, who presented consecutively with upper and lower respiratory tract sepsis was examined. Nasal ciliary beating was dyskinetic or absent in all cases, and nasal ciliary ultrastructure was abnormal in those 11 patients examined. RESULTS--Viable but immotile spermatozoa with abnormal tail ultrastructure were found in the ejaculate of only two patients. Two other patients had apparently fathered children; seminology in both these cases showed a normal spermatozoa count, one with normal spermatozoal motility and normal ultrastructure, the other with moderately reduced spermatozoal motility and abnormal ultrastructure (dynein arm deficiency on the peripheral microtubule doublets). A further two patients had normal spermatozoa counts, normal spermatozoa tail ultrastructure, and normal or only moderately reduced motility of spermatozoa. The spermatozoa of one patient were normally motile but there was severe oligozoospermia, and five patients were azoospermic. CONCLUSIONS--Not all men with primary ciliary dyskinesia have immotile spermatozoa. Seminal analysis is recommended in men with primary ciliary dyskinesia so that accurate counselling about reproductive capability may be given.