A cohort study of the Copenhagen CF Centre eradication strategy against Staphylococcus aureus in patients with CF

Journal of Cystic Fibrosis 12 (2013) 42 48 www.elsevier.com/locate/jcf Original Article A cohort study of the Copenhagen CF Centre eradication strategy against Staphylococcus aureus in patients with CF Christina Schjellerup Dalbøge a,, Tacjana Pressler b, Niels Høiby a,c, Kim Gjerum Nielsen b, Helle Krogh Johansen a a Department of Clinical Microbiology, afsnit 9301 Rigshospitalet, Juliane Maries Vej 22, DK-2100 Copenhagen Ø, Denmark b CF Centre Copenhagen and Paediatric Pulmonary Service, Department of Paediatrics and Adolescent Medicine, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark c Institut of International Health, Immunology, and Microbiology, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark Received 23 January 2012; received in revised form 5 June 2012; accepted 5 June 2012 Available online 10 July 2012 Abstract Background: Staphylococcus aureus is an important pathogen in CF. Centre prevalence of intermittent colonization and chronic S. aureus infections and the effectiveness of an anti-s. aureus eradication strategy was assessed. Methods: All airway cultures from 300 patients in a 2-year period were retrieved and all anti S. aureus antibiotic treatments were evaluated for treatment success. Chronic infection was defined as a positive culture of S. aureus in 50% or more of the months each year. Change in FEV 1 following 2 weeks of treatment of S. aureus was assessed in clinically stable patients. Primary outcome was S. aureus eradication at next clinical visit and number of patients chronically infected. Results: The yearly prevalence of S. aureus intermittent and chronic infection was 47% and 14%, respectively. Eradication was successful at the next clinical visit in 61% of the standard treatment and 53% of the prolonged treatments, respectively. FEV 1 improved significantly following anti- S. aureus treatment (3.3%, pb0.0001). Conclusion: Our anti-s. aureus eradication strategy in CF patients resulted in a low prevalence of chronic infections and high treatment efficacy. Furthermore, anti-staphylococcal treatment may be associated with a short-term improvement in lung function. 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: MRSA; MSSA; Anti-staphylococcus treatment; Treatment effectiveness; Cystic fibrosis 1. Introduction Infections with Pseudomonas aeruginosa and other Gramnegative rods are associated with deterioration in lung function Part of this work was presented at the 24th Annual North American Cystic Fibrosis Conference (NACFC) as a poster Effect of Antibiotic Treatment of Staphylococcus aureus Infection in Patients with Cystic Fibrosis at the Copenhagen CF Centre and as an oral presentation and a poster Impact of Prophylactic Antibiotics for Respiratory Tract Colonization with Staphylococcus aureus on Lung Function and BMI in CF Patients Without Acute Exacerbation. Corresponding author at: Department of Clinical Microbiology, afsnit 9301, Rigshospitalet, Juliane Maries Vej 22, DK-2100 Copenhagen Ø, Denmark. Tel.: +45 4013 6503. E-mail address: christinadalboege@gmail.com (C.S. Dalbøge). and increased morbidity and mortality eventually leading to end stage lung disease [1]. Several studies have demonstrated that pulmonary inflammation is evident soon after diagnosis and that known CF airway pathogens such as, S. aureus and P. aeruginosa, are the most commonly detected bacteria [2]. Aggressive treatment of infections, in particular those due to P. aeruginosa, has been shown to decrease pulmonary inflammation [3]. Furthermore a significant association between early lower respiratory tract infection with S. aureus and increased inflammation together with decreased body mass index (BMI) z-scores has been reported [4]. While treatment of P. aeruginosa infection is clearly important, infection with S. aureus and efforts to treat this organism more aggressively need to be investigated as well as the clarification of risk for the 1569-1993/$ -see front matter 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2012.06.005

C.S. Dalbøge et al. / Journal of Cystic Fibrosis 12 (2013) 42 48 43 selection of Gram negative bacteria such as P. aeruginosa from such a treatment [5,6]. A particular aspect of chronic infection with S. aureus is the appearance of small colony variants (SCV). There is evidence that exoproducts of P. aeruginosa enhance SCV formation as well as long-term systemic aminoglycoside and trimethoprim sulfamethoxazole treatments [7]. SCV are associated with higher rates of antimicrobial resistance and more advanced lung disease in CF [8,9]. The purpose of this study was to assess the prevalence of intermittent and chronic S. aureus infections, including the incidence of MRSA, and evaluate the effectiveness of the anti-s. aureus treatment regimens currently used in our center. This strategy has been unchanged since the establishment of the CF centre in 1968 and the first results were reported 30 years ago [10]. Since then the survival of CF patients has improved and the age distribution has changed significantly. New antibiotics with anti-s. aureus activity have been introduced. It is therefore important to evaluate this treatment strategy as well as the short-term effect of anti-staphylococcal antibiotic treatment on lung function and BMI. 2. Materials and methods 2.1. Patients and study design This study is a single-centre cohort study, which was conducted from 01.01.2008 to 31.12.2009. All CF patients treated at the Copenhagen CF Centre were eligible for the study; 302 in 2008 and 298 in 2009. All samples of airway secretions showing a positive culture of S. aureus and the corresponding antibiotic treatment was retrieved from the CF database. Ethics: according to Danish regulations this study did not need approval from an ethics committee. 2.2. General care All CF patients were seen once a month in the outpatient clinic where the clinical status and microbiological status were evaluated as previously described [11]. 2.3. Microbiological methods The methods have been described in details elsewhere [12]. In brief, a sputum or lower respiratory tract secretion sample obtained by endolaryngeal suction or bronchoalveolar lavage (BAL) was examined. The specimens were evaluated by microscopy of Gram-stained smears and cultured on chocolate agar, 5% horse blood agar, 7.5% NaCl glucose agar, modified Conradi/Drigalski agar selective for Gram-negative rods, BSSA agar (Biomerieux), Sabouraud agar ph 4.0, and a 15 cm primary antibiotic susceptibility 5% horse blood agar plate with the following antibiotic containing tablets: colistin, ciprofloxacin, tobramycin, piperacillin tazobactam, aztreonam, imipenem, meropenem, ceftazidime (Neosensitabs, Rosco, Copenhagen, Denmark) which also functions as a selective medium for 48 72h at 37 C. The 5% blood agar and the chocolate agar plates were incubated in 5% CO 2. Specimens which by microscopy contained areas with bacteria in relationship to mucus, cylinder epithelial cells and leukocytes were evaluated as originating from the lower respiratory tract, whereas specimens containing only squamous epithelial cells were evaluated as originating from the pharynx and not representative of the lower respiratory tract. Different phenotypes of each CF pathogenic species including S. aureus and different antibiotic susceptibility patterns on the primary susceptibility plate were further examined and identified and a secondary susceptibility to antibiotics was done on the isolates. 2.4. Eradication strategy against S. aureus: chemotherapy and Spa-typing No prophylactic anti-staphylococcal antibiotic chemotherapy is used at the Copenhagen CF centre. Treatment indication for S. aureus is the detection of S. aureus both by microscopy and culture in secretions from the lower respiratory tract (sputum, endolaryngeal suction or BAL) regardless of the patient's clinical condition [10]. However if the patient was showing signs of increased symptoms the treatment will be initiated on culture results only. Susceptibility testing is always used to guide the treatment. Treatment regimens: 1) Standard regimen: this regimen was used in the first and second time a S. aureus isolate was cultured, and consists of the following oral treatment: two weeks of oral fusidic acid in combination with dicloxacillin or with amoxicillin+ clavulanic acid if indicated by the presence of other pathogens such as H. influenzae. To delay the renal excretion of the beta-lactam antibiotics probenecid is added to the regimen. 2) Prolonged regimen: should the standard treatment fail to eradicate the S. aureus infection, oral antibiotic is prescribed for more than 14 days, and most often consists of one month of fusidic acid in combination with dicloxacillin followed by two months of dicloxacillin as a monotherapy and probenecid, or one month of fusidic acid or rifampicin or clindamycin or linezolide in combination with dicloxacillin and probenecid followed by two months of dicloxacillin as a monotherapy and probenecid. 3) Penicillin allergy regimen: if the patient is allergic to penicillin one of two 14 day oral regimens are considered. Either a combination of clindamycin and fusidic acid or azithromycin, or a combination of clarithromycin and rifampicin. If necessary, this regimen is prolonged. 4) Other regimens: If the patients receive antibiotics against other bacteria e.g. P. aeruginosa, other combinations are prescribed, e.g. nebulized tobramycin in addition with a broad-spectrum beta-lactam antibiotic with or without fusidic acid according to antibiotic susceptibility. 5) Regimen in case of MRSA: treatment consists of a combination of two antibiotics according to susceptibility testing in most cases fusidic acid and rifampicin or clindamycin or linezolide.

44 C.S. Dalbøge et al. / Journal of Cystic Fibrosis 12 (2013) 42 48 Spa-typing: All S. aureus isolates were Spa-typed at the Department of Hospital Infection at the State Serum Institut, Copenhagen [13]. Spa-typing replaced phage typing in 2007, after a one year period where both typing systems were used in order to maintain concordance between the two typing systems [14]. In all cases the results were in accordance. 2.5. Definition of frequency/prevalence/incidence, chronic infection and therapy outcomes The patient was defined as intermittent infected with S. aureus if one or more, but less than 50% of monthly cultures per year were positive. The yearly prevalence of infected patients was calculated as the number of patients with one or more positive S. aureus samples divided by the total number of patients treated at the centre the corresponding year. The yearly incidence of new chronically infected patients was calculated as the number of patients who became chronically infected that year, and were not chronically infected the year before, divided by the number of patients at risk=the number of patients without chronic S. aureus infection at the beginning of the year+the number of new CF patients without chronic S. aureus infection who were admitted to the CF Centre that year. The yearly prevalence of chronically infected patients was calculated as the number of patients who harbored the bacteria chronically that year divided by the total number of patients treated at the center that year. If S. aureus was absent from the lower respiratory tract at the next visit to the clinic or if the Spa-type was different the treatment was regarded successful even if re-infection with another Spa-type had occurred. If S. aureus with the same Spa-type was present, the treatment was considered unsuccessful. These principles previously relying on phage typing were unchanged since the founding of the CF Centre [10]. Patients were defined as chronically infected if S. aureus was present in 50% or more of the monthly cultures of the particular year, which is a modified Leeds criterion used for P. aeruginosa infection [15]. Only patients with at least 4 yearly samples were evaluated for being chronically infected. 2.6. Clinical effectiveness of anti-staphylococcal antibiotic chemotherapy Patients were included in this part of the study if S. aureus was the only microorganism cultured and if clinical data on lung function, weight and height from 7 days before and up to 30 days after the end of the oral S. aureus treatment, was available. During 2009, the clinical effectiveness of antistaphylococcal antibiotic chemotherapy was assessed in a subgroup of otherwise clinically stable patients not exhibiting any exacerbations according to Fuchs criteria [16] and who fulfilled the previously described treatment criteria. The percentage change in the absolute values of FEV 1 and FVC [17,18], and z-score BMI was calculated. 2.7. Statistics One-way ANOVA was used for the between group comparison, and the t-test for paired samples was used for the within group comparison. Level of significance is p 0.05 (two-tailed). To see if there was a difference in the number of patients in different age groups who received anti-s. aureus chemotherapy we used the χ 2 test. All statistical analyses were made using GraphPad Prism version 5 for Windows 7 or SPSS 12.0 for Windows. 3. Results 3.1. S. aureus prevalence The prevalence of CF patients with S. aureus positive cultures during the study period was unchanged; 142 out of 302 patients (47%) in 2008 and 141/298 (47%) in 2009, respectively. A total of 168 CF patients, with a median (range) age of 16 (0 to 47) years were culture positive for S. aureus at one or more occasions over the two year period. Sixty percent of them were children under the age of 18 years. The median (range) number of positive cultures per patient per year was 4 [1 13] positive cultures per patient per year. A total of 1200 positive S. aureus cultures were found, of which 540 samples were confirmed by microscopy in 136 patients with a median (range) of 3 [1 14] per patient in the two year period. Ninety-three percent of all children treated at the CF Centre (100 children out of 108) had at least one positive S. aureus culture in contrast to only 36% of all adult patients (68 patients out of 190 patients over 18 years of age). No significant difference was found among the groups according to the number of positive cultures, but significantly more patients aged 0 to 18 were infected with S. aureus compared to the adult patients (pb0.0001). Small colony variants (SCV) have not been found in the present study. Demographic data is summarized in Table 1. 3.2. Susceptibility of S. aureus isolates A total of 1200 S. aureus isolates, divided in 170 different strains were found in the 168 patients based on Spa-types. The majority (88.7%) of the isolates were penicillin resistant; resistance to other antibiotics was rare (Table 2). MRSA was only found in two sputum samples from two different patients, these were only cultured once from each patient and did not belong to the same Spa-type. 3.3. Treatment effectiveness A total of 577 anti-staphylococcus treatments were prescribed to 138 patients, with a median (range) of 3 [1 13] treatments per patient in the two year period. In total the S. aureus infection was eradicated at the next clinic visit in 49% of the treatments and in 12% of cases the initial strain was eradicated but the patient was re-infected with another strain

C.S. Dalbøge et al. / Journal of Cystic Fibrosis 12 (2013) 42 48 45 Table 1 The demographic data of the whole CF population treated at the center, the patients with S. aureus positive cultures and the 65 CF patients evaluated for the clinical effect of the antibiotic treatment on S. aureus cultures. Copenhagen CF population 2009 S. aureus culture positive patients 2008 2009 Treatment group available for clinical evaluation Gender M/F 146/152 79/89 41/24 Genotype Delta F 508 homozygote 202 (68%) 118 (70%) 44 (68%) Delta F 508/other 85 42 18 Other/other 8 4 2 Unknown 3 4 1 Age median (range) years 24 (0 to 62) 16years (0 to 47) 16.5years (4.8 to 39.2) Age 0 18years 108 (36%) 100 (60%) 39 (60%) Age N18years 190 (64%) 68 (40%) 26 (40%) FEV1% median (range) a 85.3% (39.9 to 117.2) FVC% median (range) a 95.2% (64.8 to 135) z-score BMI median (range) 0.21 ( 3.8 to 2.4) a Percentage of predicted value. when cultured at the next visit to the clinic (1 month later). The penicillin allergy regimen was significantly more effective in eradicating S. aureus (p=0.012) (Table 3). 3.4. Chronic S. aureus infection Repeated and prolonged treatments eventually resulted in the eradication of S. aureus in majority of the patients. However, 41 patients (14%), 95%CI; 4.1 to 10.0 fulfilled the criteria of chronic infection with S. aureus in 2008 and 42 patients or 14%, (95% CI; 6.1 to 12.9) in 2009. Only 24 (8%) of the patients were chronically infected during the entire study period and the yearly incidence of new chronically infected patients in 2009 was 7% (17 patients). Only 7 and 9% of the patients (respectively each year during the study) were infected with a strain with an unchanged Spa-type and as such confirmed to be truly chronically infected according to our definition. Only 5% of the infected patients were infected with a strain with an unchanged Spa-type during the entire study period. Table 2 Susceptibility of S. aureus strains. Number of cultures (%) Distributed by number of patients (%) Number of Spa-types Fully susceptible 81 (7) 38 (23) 30 P 1065 (89) 154 (92) 156 P+T 37 (3) 14 (8) 13 Azi/Cli 312 (26) 51 (30) 53 P+T+Azi/Cli 12 (1) 4 (2) 6 P+R 50 (4) 17 (10) 16 P+R+Azi/Cli 30 (3) 8 (5) 9 P+R+Azi/Cli+F 6 (0.5) 1 (0.6) 2 P+F 21 (2) 9 (5) 9 P+G+Azi/Cli 2 (0.2) 2 (1) 2 P+T+R+Cli/Azi 6 (0.5) 2 (1) 2 Met 1 (0.1) 1 (0.6) 1 Met+Cli/Azi 1 (0.1) 1 (0.6) 1 * Some of the strains were resistant not only to penicillin and thereby were counted more than once. Penicillin (P), tetracycline (T), rifampicin (R), gentamicin (G), fusidic acid (F), azithromycin (Azi), clindamycin (Cli), methicillin (Met). 3.5. Effectiveness of anti-staphylococcal antibiotic chemotherapy During 2009, 146 anti-s. aureus treatments in 65 patients were available for clinical evaluation which amounted to a median (range) of 2 [1 5] treatments per patient. The median (range) FEV 1 of the patients were 85.3% of the predicted (39.9 to 117.2%). Sixty percent of the treated patients were children under 18 years of age (Table 1). FEV 1 increased after treatment in 103 patients (70%) with a median of 5.6%, while the remaining 43 patients (30%) showed a decrease, with a median of 3.8% (Fig. 1). In total, anti-staphylococcal antibiotic chemotherapy accomplished a small, but statistically significant median (range) improvements in both FEV 1 % predicted of 3.3% ( 25% to 36%, pb0.0001), and FVC of 2.2% ( 39 to 40%, p= 0.001). z score BMI was unchanged. 4. Discussion During the two year period the prevalence of S. aureus was stable at around 47%, with 14% of patients being chronically infected. In general S. aureus was highly susceptible and the Table 3 Number of antibiotic treatment regimens, percentage of successful treatments in the standard, prolonged, allergy and other regimens. Standard regimen Prolonged regimen Penicillin allergy regimen Others Total Number of treatments 205 156 81 137 577 Number of patients 83 62 44 72 138 Successful eradication (%) 97 (47) 69 (44) 44 (54) 75 (55) 285 (49) Successful eradication of 125 (61) 83 (53) 57 (70) 88 (64) 353 (61) original strains (%) a Re-infection with another 28 (14) 14 (9) 13 (16) 13 (9) 68 (12) strain (%) b a If S. aureus was absent from the lower respiratory tract at the next visit to the clinic or if the Spa-type of cultured strain was different the treatment was regarded successful. b Original strain eradicated, but re-infected with another strain.

46 C.S. Dalbøge et al. / Journal of Cystic Fibrosis 12 (2013) 42 48 Fig. 1. Effect of anti-staphylococcal antibiotic therapy on FEV1 (% predicted). Change in lung function is shown on the x axis and each single treatment is shown as a horizontal bar on the y axis. eradication strategies eventually, after initial, repeated or prolonged treatments resulted in eradication of the strain. In 2009 the prevalence of any S. aureus isolate from the respiratory tract of CF patients in USA was 65.8%, of which 23.7% were MRSA [19,20]. A recently published Belgian study reported 67% of their patient population as chronically infected with S. aureus and 18% harboring MRSA strains [21]. This is in contrast to findings in our patient population with only 0.3% of patients harboring MRSA strains and none with chronic MRSA infection. The low prevalence of MRSA in our CF population corresponds well with the low prevalence of MRSA in the general Danish population [22]. The prevalence of macrolide and penicillin resistant S. aureus strains was higher in our CF Centre patients compared with the prevalence of isolates from Danish bacteraemic non-cf patients. Penicillin resistant S. aureus was seen in 77% of the strains from non-cf patients vs. 89% of the cultures in the CF population. The difference was even more marked for azithromycin resistant S. aureus strains; 4% in non-cf patients vs. 26% in CF patients [23]. High macrolide resistance has been previously reported and ascribed to macrolide antibiotics used in chronic P. aeruginosa lung infection [24,25]. In the present study the majority of S. aureus positive cultures were found in the children population (60% in age group 0 18 years). Since the prevalence of chronic P. aeruginosa infected CF children is very low in Denmark (6%) [26] the high macrolide resistance cannot be explained by macrolide use for chronic P. aeruginosa treatment but may be caused by the use of macrolides and clindamycin to treat e.g. S. aureus infections in CF children. There is some experimental evidence that exoproducts of P. aeruginosa enhance SCV formation and conversely SCV growth provides a survival advantage for S. aureus in the presence of P. aeruginosa infection [9]. The very low prevalence of chronic P. aeruginosa infection among CF children in our study population together with the minimal usage of sulphamethoxazole+trimetoprim can probably explain why small colony variants (SCV) are not seen in the present study since our methods are adequate for culturing SCV. The treatment strategy (initial, repeated and prolonged treatments) has proved to be very efficient since less than 15% of the patient population became chronically infected with S. aureus. This result is similar to previously published results by our CF Centre three decades ago [10] and similar to the prevalence of chronic S. aureus reported from the UK where prophylactic anti-staphylococcal treatment is recommended [27]. The prophylactic antibiotics for S. aureus are not used in our centre since all patients are investigated each month and all patients with a positive S. aureus culture from the secretion obtained from the lower respiratory tract are prescribed anti-s. aureus treatments. After 12% of the treatments re-infection with another strain occurred at the next visit at the clinic and treatment was then repeated. Prophylactic treatment of S. aureus has been associated with an increased prevalence of Gram negative bacteria such as P. aeruginosa [10,11] and yet, without employing prophylactic antibiotics for S. aureus and despite frequent use of anti-s. aureus antibiotics, the prevalence of chronic P. aeruginosa infection in our population is very low [26]. S. aureus isolates may persist in CF patients over years. In a large survey of S. aureus in six French hospitals, 85 of 238 patients (30%) who were culture positive for S. aureus, had three or more consecutive isolates over at least 6 months and 48% of the subjects harbored the identical isolate as shown by pulse field gel electrophoresis up to 28 months. Data from two large randomized controlled trials noted the persistence of S. aureus for a median of 37 months with most of the patients harboring a single clone [28,29]. In the present study 14% (7% with persistent Spa-type) of our patients fulfilled the criteria of chronic infection and the chronic infection persisted in only 8% (5% with the same Spa-type) of the patients, indicating that chronic S. aureus infection persisted only in a few patients whereas a few new chronic infections occurred every year. Antibiotic treatment of any relevant microorganism, ignoring the presence or absence of respiratory symptoms has been our policy since the establishment of our CF centre in 1968 [10]. However, the overall short term influence on lung function and BMI of our early treatment policy has not previously been reported. We investigated the short term effectiveness of the treatment in otherwise clinically stable patients with positive S. aureus infections, and found a significant effect on pulmonary function for FEV 1 and FVC, respectively, similar to the results of early eradication of P. aeruginosa colonization [30]. Furthermore, a BAL study assessing inflammation as a reflection of

C.S. Dalbøge et al. / Journal of Cystic Fibrosis 12 (2013) 42 48 47 S. aureus infection supports the use of specific surveillance and eradication programs for Saureus[31]. Ranganathan et al. [4] reported an association between infection with S. aureus and lower BMI, and that the use of anti-staphylococcal antibiotics lead to increased BMI and furthermore, increase in childhood BMI has been shown to be associated with better lung function [32] supporting the policy to treat S. aureus infection even in apparently asymptomatic CF patients. The present study has several limitations. The study period is only two years and the patient population is small but homogenous with respect to examination and treatment policy. The effect on lung function and BMI is only evaluated after a single two week treatment. Furthermore, the results cannot be compared across the different CF Centres due to the lack of a commonly appreciated definition of chronic infection, different frequency of sputum culture once a year against once a month and different treatment strategies [6,12,33]. However, we have used the Leeds definition of chronic infection in CF, which was introduced for the description of P. aeruginosa infections [15] which gives nearly the same results as our previously published definition (continuous presence of S. aureus for 6months) [10]. A prospective study of longer duration in a larger patient population is needed to confirm possible long-term benefits of the eradication treatment in a clinically stable CF patient. In conclusion, S. aureus was frequently found in the lower respiratory tract of Danish CF patients. Use of a stepwise two-drug antibiotic combination therapy strategy results in a high level of eradication with a prevalence of chronic S. aureus infection b15%, which has been unchanged for decades as well as a few resistance problems and significant short term effects on lung function. Conflict of interest statement Competing interest: none. The following are the financial/nonfinancial disclosures of the authors. CSD: none. TP: none. NH: none. KGN: none. HKJ: none. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jcf.2012.06.005. References [1] Walters SMA. Epidemilogy of cystic fibrosis. In: Hodson ME GD, Bush A, editors. Cystic fibrosis. London: Hodder Arnold; 2007. p. 21 45. [2] Armstrong DS, Hook SM, Jamsen KM, et al. Lower airway inflammation in infants with cystic fibrosis detected by newborn screening. Pediatr Pulmonol Dec 2005;40(6):500 10. [3] Douglas TA, Brennan S, Gard S, et al. Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis. Eur Respir J Feb 2009;33(2):305 11. [4] Ranganathan SC, Parsons F, Gangell C, Brennan S, Stick SM, Sly PD. Evolution of pulmonary inflammation and nutritional status in infants and young children with cystic fibrosis. Thorax May 2011;66(5):408 13. [5] Ratjen F, Comes G, Paul K, Posselt HG, Wagner TO, Harms K. Effect of continuous antistaphylococcal therapy on the rate of P. aeruginosa acquisition in patients with cystic fibrosis. Pediatr Pulmonol Jan 2001;31(1): 13 6. [6] Smyth A, Walters S. Prophylactic antibiotics for cystic fibrosis. Cochrane Database Syst Rev 2003(3) (CD001912). [7] Schneider M, Muhlemann K, Droz S, Couzinet S, Casaulta C, Zimmerli S. Clinical characteristics associated with isolation of small-colony variants of Staphylococcus aureus and Pseudomonas aeruginosa from respiratory secretions of patients with cystic fibrosis. J Clin Microbiol May 2008;46(5):832 4. [8] Besier S, Smaczny C, von Mallinckrodt C, et al. Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J Clin Microbiol Jan 2007;45(1):168 72. [9] Hoffman LR, Deziel E, D'Argenio DA, et al. Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A Dec 26 2006;103(52):19890 5. [10] Szaff M, Hoiby N. Antibiotic treatment of Staphylococcus aureus infection in cystic fibrosis. Acta Paediatr Scand Sep 1982;71(5):821 6. [11] Høiby N, Pressler T. Emerging pathogens in cystic fibrosis. In: Webb AK, Ratjen F, editors. Cystic Fibrosis. European Respiratory Monograph. European Respiratory Society Journals Ltd; 2006. p. 66 78. [12] Høiby N, Frederiksen B. Microbiology of cystic fibrosis. In: Hodson M, Geddes D, editors. 2nd ed. London: Arnold; 2000. p. 83 107. [13] Larsen AR, Stegger M, Sorum M. Spa typing directly from a meca, spa and pvl multiplex PCR assay a cost-effective improvement for methicillin-resistant Staphylococcus aureus surveillance. Clin Microbiol Infect Jun 2008;14(6):611 4. [14] Hallin M, Friedrich AW, Struelens MJ. Spa typing for epidemiological surveillance of Staphylococcus aureus. Methods Mol Biol 2009;551: 189 202. [15] Lee TW, Brownlee KG, Conway SP, Denton M, Littlewood JM. Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros Mar 2003;2(1):29 34. [16] Fuchs HJ, Borowitz DS, Christiansen DH, et al. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. N Engl J Med Sep 8 1994;331(10):637 42. [17] Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general U.S. population. Am J Respir Crit Care Med Jan 1999;159(1):179 87. [18] Wang X, Dockery DW, Wypij D, Fay ME, Ferris Jr BG. Pulmonary function between 6 and 18 years of age. Pediatr Pulmonol Feb 1993;15(2): 75 88. [19] Cystic Fibrosis Foundation Patient Registry. Annual Data Report, Bethesda, Maryland; 2009 [ 2011 Cystic Fibrosis Foundation]. [20] Dasenbrook EC, Checkley W, Merlo CA, Konstan MW, Lechtzin N, Boyle MP. Association between respiratory tract methicillin-resistant Staphylococcus aureus and survival in cystic fibrosis. JAMA Jun 16 2010;303(23):2386 92. [21] Vanderhelst E, De Meirleir L, Verbanck S, Pierard D, Vincken W, Malfroot A. Prevalence and impact on FEV(1) decline of chronic methicillin-resistant Staphylococcus aureus (MRSA) colonization in patients with cystic fibrosis a single-center, case control study of 165 patients. J Cyst Fibros Jan 2012;11(1):2 7. [22] DANMAP. Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark; 2010. p. 1 156. http://www.danmap.org. [23] Annual Report On Staphylococcus Aureus Bacteraemia Cases in Denmark. Cases in Denmark; 2008. [24] Hansen CR, Pressler T, Hoiby N, Johansen HK. Long-term, low-dose azithromycin treatment reduces the incidence but increases macrolide

48 C.S. Dalbøge et al. / Journal of Cystic Fibrosis 12 (2013) 42 48 resistance in Staphylococcus aureus in Danish CF patients. J Cyst Fibros Jan 2009;8(1):58 62. [25] Tramper-Stranders GA, Wolfs TF, Fleer A, Kimpen JL, van der Ent CK. Maintenance azithromycin treatment in pediatric patients with cystic fibrosis: long-term outcomes related to macrolide resistance and pulmonary function. Pediatr Infect Dis J Jan 2007;26(1):8 12. [26] Olesen Hanne, et al. ECFS patient registry report on 2007 data; 2010. [27] UK CF Registry. Annual Data Report; 2009 [ Cystic Fibrosis Trust 2011]. [28] Branger C, Gardye C, Lambert-Zechovsky N. Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time. J Med Microbiol Oct 1996;45(4):294 301. [29] Kahl BC, Duebbers A, Lubritz G, et al. Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study. J Clin Microbiol Sep 2003;41(9):4424 7. [30] Frederiksen B, Koch C, Hoiby N. Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol May 1997;23(5):330 5. [31] Gangell C, Gard S, Douglas T, et al. Inflammatory responses to individual microorganisms in the lungs of children with cystic fibrosis. Clin Infect Dis Sep 2011;53(5):425 32. [32] McPhail GL, Acton JD, Fenchel MC, Amin RS, Seid M. Improvements in lung function outcomes in children with cystic fibrosis are associated with better nutrition, fewer chronic Pseudomonas aeruginosa infections, and dornase alfa use. J Pediatr Dec 2008;153(6):752 7. [33] Stone A, Saiman L. Update on the epidemiology and management of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, in patients with cystic fibrosis. Curr Opin Pulm Med 2007; Nov;13(6).