title: Denitrification by cystic fibrosis pathogens - Stenotrophomonas maltophilia is dormant in sputum.
abstract: 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 N(2)O 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 NO(3)(-) and NO(2)(-) was estimated by the Griess Assay. All isolates were assayed during 2 days of incubation in anaerobic LB broth with NO(3)(-) or NO(2)(-). 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 NO(3)(-) caused increased production of N(2)O by P. aeruginosa, A. xylosoxidans and B. multivorans and increased growth for all pathogens. Growth was, however, lowest for S. maltophilia. NO(3)(-) was metabolized by all pathogens, but only P. aeruginosa was able to remove NO(2)(-). 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 NO(3)(-) reduction. Denitrification as demonstrated by N(2)O 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.
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Kolpen
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Kasper Nørskov
authors.initials: KN
authors.lastname: Kragh
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bjarnsholt
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Laura
authors.initials: L
authors.lastname: Line
authors.affiliation: Department of Paediatrics, Copenhagen CF Centre, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Christine Rønne
authors.initials: CR
authors.lastname: Hansen
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Christina Schjellerup
authors.initials: CS
authors.lastname: Dalbøge
authors.affiliation: Department of Veterinary Disease Biology, Veterinary Clinical Microbiology, University of Copenhagen, 1870 Frederiksberg, Denmark.
authors.forename: Nana
authors.initials: N
authors.lastname: Hansen
authors.affiliation: Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Helsingør, Denmark; Plant Functional Biology and Climate Change Cluster, University of Technology, Sydney, Australia; Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore.
authors.forename: Michael
authors.initials: M
authors.lastname: Kühl
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Høiby
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark. Electronic address: Peter.Oestrup.Jensen@regionh.dk.
authors.forename: Peter Østrup
authors.initials: PØ
authors.lastname: Jensen
mesh_terms: D042441:Achromobacter denitrificans / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000693:Anaerobiosis; D058491:Bacterial Load; D042602:Burkholderia cepacia complex / Q000378:metabolism; D002648:Child; D003550:Cystic Fibrosis / Q000150:complications*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D058440:Denitrification; D005260:Female; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009609:Nitrous Oxide / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013183:Sputum / Q000382:microbiology*; D020615:Stenotrophomonas maltophilia / Q000378:metabolism*; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D009566:Nitrates; D009573:Nitrites; D012336:RNA, Ribosomal, 16S; D009609:Nitrous Oxide
keywords: Achromobacter xylosoxidans; Burkholderia multivorans and Stenotrophomonas maltophilia; Cystic fibrosis; Denitrification; N(2)O; Pseudomonas aeruginosa