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The study objective was to identify changes of prevalence and resistance of important pathogens in specimens of cystic fibrosis (CF) patients within a decade.Samples of 94 patients, who attended 2 CF centers from 2001 to 2011 were retrospectively analyzed.Staphylococcus aureus was the most prevalent organism (74.5% in 2011) with an increase of methicillin-resistant S. aureus in patients (0% vs. 9.6%, n = 9). Resistance of S. aureus to gentamicin decreased (41.8% vs. 21%; P < 0.001), whereas resistance to rifampicin and trimethoprim/sulfamethoxazole (P < 0.05) increased significantly with a trend to increased resistance to clindamycin and erythromycin (P = 0.063). Methicillin-resistant S. aureus isolates belonged to 6 spa types (t003, t008, t011, t034, t045, t548). There was a significant increase of Pseudomonas aeruginosa prevalence (63.8% in 2011 vs. 46.8% in 2001, P = 0.019). Resistance of P. aeruginosa increased significantly to imipenem, gentamicin, amikacin, tobramycin, ciprofloxacin and fosfomycin, whereas resistance to piperacillin–tazobactam, meropenem and aztreonam decreased. Significantly fewer Stenotrophomonas maltophilia isolates were susceptible to all the analyzed antibiotics (trimethoprim/sulfamethoxazole, ciprofloxacin and colistin) in 2011 compared with 2001 (13.5% vs. 42.1%; P = 0.023), whereas the resistance to colistin increased significantly (11.1% vs. 62.2%; P < 0.001). Burkholderia cepacia complex and nontuberculous mycobacteria were not detected in 2001 but in 2011 in 7.4% (n = 9) and 7.4% (n = 9) of patients, respectively. B. cepacia complex isolates belonged to 8 multilocus sequence types.Our retrospective analysis revealed an increase of important CF-related pathogens, the emergence of new pathogens and a substantial increase of multidrug-resistant CF-specific isolates. Our findings are of importance to clinicians for the alertness of local epidemiology, which may be useful for prevention and treatment strategies.