1 Department of Immunology and Microbiology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Københavns Universitet2 Marine Biology, Department of Biology, Faculty of Science, Københavns Universitet3 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet4 Rigshospitalet5 unknown6 Institut for Veterinær Sygdomsbiologi, Københavns Universitet7 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet8 Department of Immunology and Microbiology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Københavns Universitet9 Marine Biology, Department of Biology, Faculty of Science, Københavns Universitet
OBJECTIVE: Chronic Pseudomonas aeruginosa lung infection is the most severe complication for cystic fibrosis (CF) patients. Infected endobronchial mucus of CF patients contains anaerobic zones mainly due to the respiratory burst of polymorphonuclear leukocytes. We have recently demonstrated ongoing denitrification in sputum from patients infected with P. aeruginosa. Therefore we aimed to investigate, whether the pathogenicity of several known CF pathogens is correlated to their ability to perform denitrification. METHODS: We measured denitrification with N2O microsensors in concert with anaerobic growth measurements by absorbance changes and colony counting in isolates from 32 CF patients chronically infected with the highly pathogenic bacteria P. aeruginosa, Achromobacter xylosoxidans, Burkholderia multivorans or the less pathogenic bacterium Stenotrophomonas maltophilia. Consumption of NO3(-) and NO2(-) was estimated by the Griess Assay. All isolates were assayed during 2 days of incubation in anaerobic LB broth with NO3(-) or NO2(-). PNA FISH staining of 16S rRNA was used to estimate the amount of ribosomes per bacterial cells and thereby the in situ growth rate of S. maltophilia in sputum. RESULTS: Supplemental NO3(-) caused increased production of N2O by P. aeruginosa, A. xylosoxidans and B. multivorans and increased growth for all pathogens. Growth was, however, lowest for S. maltophilia. NO3(-) was metabolized by all pathogens, but only P. aeruginosa was able to remove NO2(-). S. maltophilia had limited growth in sputum as seen by the weak PNA FISH staining. CONCLUSIONS: All four pathogens were able to grow anaerobically by NO3(-) reduction. Denitrification as demonstrated by N2O production was, however, not found in S. maltophilia isolates. The ability to perform denitrification may contribute to the pathogenicity of the infectious isolates since complete denitrification promotes faster anaerobic growth. The inability of S. maltophilia to proliferate by denitrification and therefore grow in the anaerobic CF sputum may explain its low pathogenicity in CF patients.
International Journal of Medical Microbiology, 2015, Vol 305, Issue 1, p. 1-10