Inflammatory markers in cystic fibrosis patients with transmissible Pseudomonas aeruginosa

The major cause of morbidity and mortality for individuals with cystic fibrosis (CF) is pulmonary disease due tochronic infection and a damaging host inflammatory response. The major bacterial pathogen involved is Pseudomonas aeruginosa 1. Recently, there have been a number of reports of P. aeruginosa cross-infection outbreaks at CF centres and holiday camps 2–8. Isolates of many of the transmissible strains commonly exhibit unusual phenotypic features; morphotypes of the most prevalent transmissible strain at the Manchester Adult CF Centre, P. aeruginosa strain MA, are associated with an unusual pyocin type, are nonpigmented, nonmotile and are often resistant to the majority of usual anti-pseudomonal antibiotics 3. The clinical significance of acquisition of a transmissible strain for patients who already harbour their own sporadic strain ofP. aeruginosa is unclear, although some clinicians have reported an increase in morbidity 2, 8, 9 and mortality 2 among CF patients infected with a transmissible strain of P.aeruginosa.

Airway inflammation is a major factor in the pathogenesis of CF lung disease. Inflammatory mediators as markers of the host response to infection may reflect the intensity of lung injury and relate to changes in clinical status. When clinically stable, CF patients with chronic P. aeruginosa infection are known to have increased levels of inflammatory markers compared to the non‐CF population 10, 11. If transmissible strains of P. aeruginosa are more virulent they may provoke an enhanced inflammatory response. In this study, levels ofinflammatory markers in clinically stable adult CF patientswho harbour a transmissible P. aeruginosa have been compared with those harbouring sporadic strains of P.aeruginosa.

Methods

CF patients with chronic P. aeruginosa infection who attend the Manchester Adult CF Centre were invited to participate in the study if they were clinically stable (defined as an absence of new symptoms and stable pulmonary function) and had received a course of i.v. antibiotic treatment 4–6 weeks earlier. The course of i.v. antibiotic treatment was of at least 7 days duration and consisted of a combination of at least two different classes of intravenous anti-pseudomonal antibiotics. The in vitro sensitivity of the P.aeruginosa from the most recently available sputum isolate is used to guide antibiotic choice and adjusted where appropriate when a current culture is available. Approval for the study was given by South Manchester Research Ethics Committee and written informed consent was obtained from each patient.

Patients were grouped on the basis of results of prospective bacterial fingerprinting of P. aeruginosa isolates from sputa, as previously reported 3. Patients were grouped into those who have harboured a transmissible strain of P. aeruginosa (P. aeruginosa strain MA) for at ≥12 months and those whoharbour their own sporadic P. aeruginosa. The two groups were prospectively matched by per cent predicted forced expiratory volume in one second (%FEV₁). Patients co-infected with Burkholderia cepacia complex or other transmissible strains of P. aeruginosa were excluded from the study. Baseline patient demographics in the study were age, sex, CF genotype, presence of pancreatic insufficiency and/or diabetes mellitus, and use of nebulised antibiotics, deoxyribonuclease, long-term oral macrolide antibiotics, inhaled corticosteroids, and oral corticosteroids. %FEV₁, per cent predicted forced vital capacity (%FVC) and body mass index (BMI) were measured. The total duration of the previous course of i.v. antibiotics and time from completion to the study visit were recorded. The in vitro phenotypic appearance (nonmucoid or mucoid) and antibiotic sensitivity patterns of the P. aeruginosa isolates were noted. For thepurpose of this study, multiresistance was defined as resistance to two of the three major classes of usual antipseudomonal antibiotics (aminoglycosides, quinolones, β‐lactams). Antibiotic resistance was assessed by the British Society for Antimicrobial Chemotherapy standardised disc testing method. Susceptibility and resistance of isolates was based on standard breakpoint values for each antibiotic (British Society for Antimicrobial Chemotherapy).

A sample of whole blood was analysed the same day for total white cell and differential cell counts. Samples of sputum, urine, plasma and serum were obtained for measurement of inflammatory markers. Sputum was diluted weight for weight with 0.9% (weight/volume) saline in a 1:5 ratio, centrifuged at 3000×g for 30 min and the supernatant collected. All samples were stored in aliquots at −80°C pending the measurement of inflammatory mediators.

Inflammatory markers were analysed as a single batch byablinded investigator. The levels of the following markerswere measured: sputum neutrophil elastase (NE), interleukin (IL)‐8 and tumour necrosis factor (TNF)‐α, plasma IL‐6 andNE/α₁‐antitrypsin complexes, serum C‐reactive protein (CRP), and urine TNF receptor 1. NE was detected by spectrophotometric assay, utilising the substrate N‐Methoxysuccinyl-Ala‐Ala‐Pro‐Val‐p‐nitroanilide. All other parameters were measured by commercially available enzyme-linked immunosorbent assays (ELISA), with the exception ofNE/α₁‐antitrypsin complexes, which were detected byan ELISA developed within the authors' laboratory, andrelevant performance and quality control checks were conducted.

Results

For the transmissible group there were 22 eligible patients (i.e. patients who had chronic infection with the transmissible P. aeruginosa strain MA for >12 months); 20 out of 22 patients were recruited. Two were not recruited, as they did not receive a course of i.v. antibiotics over the study period. The 20 patients with the transmissible strain were matched by %FEV₁ with 20 CF patients harbouring sporadic P. aeruginosa. The two groups (sporadic versus transmissible P.aeruginosa strains) were well matched in terms of mean age, %FEV₁, %FVC, BMI and demographic features (table 1⇓). Patients received i.v. antibiotic therapy for a median (range) of 14 (9–37) and 14 (8–28) days (p=0.68) in the sporadic and transmissible P. aeruginosa groups, respectively. The study visit was a mean (se) 35.8 (1.2) and 33.7 (0.9) days (p=0.20) after the final day of i.v. antibiotic treatment for the sporadic and transmissible P. aeruginosa groups, respectively. There were no differences between the sporadic and transmissible P.aeruginosa groups in median change in %FEV₁ (−2.2 (−11.5–15.4) and −2.5 (−23.5–6.3); p=0.57), %FVC (−3.9 (−14.1–6.6) and −5.0 (−50.7–6.4); p=0.37) or BMI (0.15 (1.2–−1.3) and 0.15 (−0.8–1.2); p=0.36) from the end of the course of i.v. antibiotics to the study day.

Whole blood, plasma and serum samples were obtained from all patients. Two patients in the transmissible P. aeruginosa group were unable to produce a sputum sample. Two patients in the sporadic P. aeruginosa group were unable to produce a urine sample because of time restraints on the study day. The results of whole blood white cell and neutrophil counts and the levels of inflammatory markers are given in table 2⇓. There was no difference between the twogroups for the levels of white cell counts and the inflammatory markers.

Discussion

This study examines differences in levels of inflammatory markers between two groups of clinically stable CF patients with chronic P. aeruginosa infection; those who harbour their own sporadic P. aeruginosa and those who harbour a transmissible strain (P. aeruginosa strain MA). There were no differences in levels of inflammatory markers between thetwo groups.

There are an increasing number of reports of P. aeruginosa cross-infection outbreaks among individuals with CF 2–7, 12. The significance of P. aeruginosa cross-infection and the provision of resources for microbiological surveillance and implementation of infection control measures remains controversial 13–16. Whilst new acquisition of P. aeruginosa bypreviously “Pseudomonas-naïve” CF patients is associated with a worsening of their clinical status 1, 17–21, the effect ofacquisition of a different strain for patients who already harbour P. aeruginosa is unclear. However, there are reasons for CF physicians to be concerned about transmissible strains of P. aeruginosa. Isolates belonging to transmissible strains of P. aeruginosa often exhibit unusual phenotypic characteristics 3, including in vitro antibiotic resistance 2, 3, 6. In the present study, a higher number (16 out of 20) of patients in the transmissible P. aeruginosa group were found to harbour a multi-resistant P. aeruginosa isolate than patients with sporadic strains (three out of 20) (p<0.001). Transmissible strains have also been associated with super-infection of CF patients who already harbour other P. aeruginosa 4. The authors have previously documented an increase in treatment requirements for adult CF patients who harbour a transmissible strain of P. aeruginosa 9. More worrying is a report from Nixon et al. 2 of an increased morbidity and mortality for CF children in association with P. aeruginosa cross-infection. A recent publication by Armstrong et al. 8 observed a lower %FEV₁ among patients at a paediatric CF centre who harboured a transmissible P. aeruginosa; however, the two groups were not prospectively matched.

There is a current lack of published data as to whether transmissible P. aeruginosa are more virulent than sporadic strains, but such information is needed by CF clinicians. The current study was designed to investigate whether adult CF patients who harbour transmissible P. aeruginosa strain MA demonstrate a more florid host inflammatory response than those who harbour sporadic strains of P. aeruginosa. Patients with chronic P. aeruginosa infection experience episodes of respiratory exacerbations that are associated with a decrease in lung function and a systemic inflammatory response, with increased levels of inflammatory markers, including TNF‐α, NE/α₁‐antitrypsin complexes, IL‐6, IL‐8 and CRP 11, 22–24. I.v. antibiotic therapy leads to a reduction in host inflammatory responses 11, 23. However, even at times of apparent clinical stability, CF patients with chronic P. aeruginosa infection still demonstrate increased levels of inflammatory markers indicating chronic inflammatory activity and accompanying lung injury 11, 24–26. The persistent inflammatory response to chronic pulmonary infection also leads to an increased resting energy expenditure and patient metabolism 23, adversely affecting nutritional status. If some strains of P. aeruginosa are more virulent they should be more likely to provoke an enhanced host inflammatory response. However, in this study, no differences in levels of inflammatory markers were shown between CF patients with infection by transmissible and sporadic strains of P. aeruginosa at times of clinical stability.

Although these findings provide a degree of reassurance forclinicians that care for CF patients who harbour transmissible P. aeruginosa, the study has a number of drawbacks. First, as the number of CF patients identified as harbouring transmissible P. aeruginosa is small, subjects for such studies are limited and it is, therefore, difficult to fully exclude a type‐2 error. Secondly, some patients who harbour the transmissible strain are co‐infected with a sporadic P. aeruginosa strain. However, although inclusion of these patients might bias results, particularly if a transmissible strain was less virulent, these subjects make the group more representative ofthe CFpatients in clinical practice. Thirdly, in addition tobacterial virulence factors, DNA polymorphisms in host genes regulating inflammatory responses to respiratory pathogens may influence levels of inflammatory markers 27. It is difficult both to assess and make allowances for such a potential confounding variable in an in‐vivo study. Finally, measuring levels of host inflammatory markers may not provide an accurate prediction of future clinical outcome. A previous study did not find a difference in levels of blood cytokines between CF patients with P. aeruginosa and B. cepacia complex 22, despite a well-recognised increased pathogenicity of B. cepacia complex in clinical practice 28,29. However, that study had smaller numbers of subjects andmeasured circulating inflammatory markers, which may not be as representative of inflammatory activity within the lung as those in bronchoalveolar lavage fluid or sputa. The present study recruited a greater number of patients andexamined inflammatory markers in sputa and the circulation.

In conclusion, at times of clinical stability, cystic fibrosis patients infected with transmissible Pseudomonas aeruginosa do not have a heightened inflammatory response above those who harbour sporadic strains. Further prospective studies are needed to assess the long-term clinical effects of spread of transmissible Pseudomonas aeruginosa strains among individuals with cystic fibrosis.