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that hypermutation play an important role during diversification and adaptation of Achromobacter sp. Methods: The mutation frequency of Achromobacter sp. isolates is estimated by plating isolates onto LB agar and LB agar containing rifampicin. Mutation frequency values are reported as the number of rifampicin-resistant colonies in proportion to the total viable count. Genes of the MMR-system of Achromobacter will be amplified by PCR and subsequently sequenced. Results: In this study we investigate the prevalence of hypermutator strains of Achromobacter sp. among patients affiliated with the CF Centre at Aarhus University Hospital. MMR gene sequences of potential hypermutators and normomutators will be compared in order to decipher the genetics of hypermutation in Achromobacter sp. Conclusion: Hypermutation might be a key element of genetic adaptation of Achromobacter sp. in the CF lung. Adaptation of Achromobacter sp. to changing environments is essential for persistence in the CF lung.

Objectives The spectrum of bacteria cultured from the airways of patients with cystic fibrosis (CF) shows a characteristic association with patient age. However, surveillance of pathogens is usually limited to investigations of throat swabs in young children and other patients with limited disease that do not produce sputum. Therefore widely cited prevalence rates may not be representative for young patients with CF. Microbial surveillance of Danish CF patients is performed by laryngeal suction if patients do not produce sputum. We calculated the prevalence rates of selected bacteria in young patients from a Danish CF center. Methods 5588 samples from 149 patients aged 0–24 years were cultured during a 5 year period. The proportion of samples with growth of Haemophilus influenzae, Staphylococcus aureus , and Pseudomonas aeruginosa was calculated for different age groups. Results Haemophilus influenzae is the dominant pathogen cultured in respiratory samples in infants (0–1 years, 39%) and small children (2–5 years, 49%), but is rarely seen in adolescents (12–17 years, 9%) and young adults (18–24 years, 8%). S. aureus is relatively common in infants and small children, and becomes the dominant pathogen in childhood and throughout adolescence and young adulthood. P. aeruginosa is rarely cultured from infants and young children. Conclusion Examination of respiratory secretions secured by laryngeal suction reveals H. influenzae as the dominant pathogen in CF patients below 6 years of age. The significance of H. influenzae in early stages of CF is poorly understood. The dynamics of colonisation and displacement of strains by antimicrobial therapy are presently being investigated.

Objective To investigate species distribution, antimicrobial susceptibility and possible transmission of non-tuberculous mycobacteria among Danish cystic fibrosis patients. Methods Thirty-five non-tuberculous mycobacteria cultured from 2006 to 2013 from Danish cystic fibrosis patients from two centres were investigated (one isolate per patient). The isolates were subjected to antimicrobial susceptibility testing to 15 antimicrobial agents by broth microdilution and to PCR for the erm(41) gene. Partial sequencing of the rpoB gene was used to determine species identification of the isolates. Results Twenty-three isolates were identified as Mycobacterium abscessus subspecies abscessus , 10 isolates as Mycobacterium abscessus subspecies bolletii (six isolates as subtype massiliense and four as subtype bolletii ), and two as Mycobacterium chelonae . Conclusion Strain relatedness was assessed by a Variable Number Tandem Repeat (VNTR) typing scheme which only revealed a few small clusters (2–4 patients) of related strains within the patient population. These clusters will be further investigated by whole-genome sequencing.

Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as major cause of therapeutic failure of chronic lung infection in cystic fibrosis patients. The infection is characterized by antibiotic-tolerant biofilms in mucus with zones of oxygen (O 2 ) depletion mainly due to polymorphonuclear leukocytic activity. In contrast to other bactericidal antibiotics, the bactericidal effect of colistin on planktonic P. aeruginosa does not depend on the formation of cytotoxic amounts of hydroxyl radicals (OH • ) suggesting the activity of colistin is independent of the presence of O 2 . Therefore we investigated biofilm cultures of wildtype PAO1, catalase deficient mutant ( ΔkatA ) and colistin resistant CF isolate grown in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate for 1 or 3 days before 3 hours of colistin treatment. The killing of bacteria after colistin treatment was measured by CFU counts and the OH • formation was measured by induction of 3'-(p-hydroxyphenyl) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogenperoxide treatment. Aerobic colistin treatment did not induce HPF fluorescence in any of the three strains and catalase deficiency was not associated with increased susceptibility, indicating that colistin acts independently of OH • formation. We demonstrated increased susceptibility of PAO1 biofilms towards colistin during anaerobic conditions. The maximum enhancement of killing by anaerobic conditions exceeded 4 logs using 8 mg/L of colistin compared to killing at aerobic conditions. We highly recommend considering colistin treatment of biofilms in the anaerobic zones of CF mucus and paranasal sinuses.