Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum

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Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum

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Mette Kolpen a,b , Kasper Nørskov Kragh b , Thomas Bjarnsholt a,b , Laura Line a , Christine Rønne Hansen c , Christina Schjellerup Dalbøge a , Nana Hansen d , Michael Kühl e,f,g , Niels Høiby a,b , Peter Østrup Jensen a,∗ a

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 c Department of Paediatrics, Copenhagen CF Centre, Rigshospitalet, 2100 Copenhagen, Denmark d Department of Veterinary Disease Biology, Veterinary Clinical Microbiology, University of Copenhagen, 1870 Frederiksberg, Denmark e Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Helsingør, Denmark f Plant Functional Biology and Climate Change Cluster, University of Technology, Sydney, Australia g Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore b

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Article history: Received 14 April 2014 Received in revised form 3 July 2014 Accepted 15 July 2014

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Keywords: Cystic fibrosis Pseudomonas aeruginosa Achromobacter xylosoxidans Burkholderia multivorans and Stenotrophomonas maltophilia N2 O Denitrification

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Introduction

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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 N2 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 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 N2 O 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 N2 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. © 2014 Elsevier GmbH. All rights reserved.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene affecting apical ion transport (Riordan et al., 1989). The

∗ Corresponding author at: Department of Clinical Microbiology, Rigshospitalet, Juliane Mariesvej 22, 2100 Copenhagen, Denmark. Tel.: +45 35 45 77 74. E-mail address: [email protected] (P.Ø. Jensen).

defective ion transport results in the formation of thick viscous mucus, which makes the lungs susceptible to chronic respiratory infections by preventing mucociliary clearance (Knowles and Boucher, 2002; Boucher, 2007). Gram-negative pathogens such as Pseudomonas aeruginosa, Achromobacter xylosoxidans, Burkholderia multivorans and Stenotrophomonas maltophilia may cause chronic infections in the respiratory tract in CF patients (Spicuzza et al., 2009; Ciofu et al., 2013). While the pulmonary function is severely worsened in CF patients after the onset of chronic infections with P. aeruginosa, A. xylosoxidans and B. multivorans (2012a; 2011; 2012b;

http://dx.doi.org/10.1016/j.ijmm.2014.07.002 1438-4221/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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Burns et al., 1998; Drevinek and Mahenthiralingam, 2010; Rønne et al., 2006; Ciofu et al., 2013), the deterioration of the lung function in chronic S. maltophilia infections is slower or even stable for long 46 time (Dalbøge et al., 2011; Waters et al., 2013). 47 The major bacterial pathogen causing chronic lung infection in 48 CF patients P. aeruginosa (Høiby, 2000), exists in biofilm aggregates 49 surrounded by polymorphonuclear leukocytes (PMNs) in the endo50 bronchial mucus (Bjarnsholt et al., 2009; Baltimore et al., 1989) 51 that contains anaerobic zones (Worlitzsch et al., 2002). This anaer52 obiosis is predominantly caused by host cells due to the intense 53 depletion of O2 by PMNs for production of superoxide (Kolpen 54 et al., 2010) and to a lesser extent for production of nitric oxide 55 (NO) (Kolpen et al., 2014a) and for respiration by the lung epithe56 lium (Worlitzsch et al., 2002), while O2 consumption by microbial 57 aerobic respiration is diminutive (Kolpen et al., 2010). In spite of 58 anaerobiosis, P. aeruginosa grows and persists in the endobronchial 59 60Q2 secretions (Yang et al., 2008; Kragh et al., 2014). The mechanisms providing energy for this anaerobic growth apparently include 61 denitrification since endobronchial secretions harbor ongoing pro62 duction of nitrous oxide (N2 O) (Kolpen et al., 2014b) as well as 63 the denitrification marker OprF porin (Yoon et al., 2002) and the 64 ammonium concentrations in both sputum and tracheal aspirates 65 decreased with antibacterial therapy (Gaston et al., 2002). Arginine 66 fermentation may also contribute to the survival of P. aeruginosa 67 (Vander et al., 1984) although the energy yield is much lower than 68 in aerobic or anaerobic respiration (Yoon et al., 2002; Hoboth et al., 69 2009). Denitrification may also occur during aerobic growth albeit 70 with a rate inversely correlated to the concentration of O2 (Chen et 71 al., 2003). 72 Denitrification may favor the biofilm growing mucoid pheno73 type of P. aeruginosa, which is characteristic for chronic infections 74 and therefore highly pathogenic in CF, and may promote growth, 75 and increased antibiotic tolerance (Williams et al., 1978; Hassett, 76 1996; Borriello et al., 2004). In addition, denitrification involves 77 the reduction of NO to N2 O by the enzyme nitric oxide reductase, 78 which has been shown to enhance the virulence of P. aeruginosa 79 in mouse macrophages (Kakishima et al., 2007) and silkworm 80 (Arai and Iiyama, 2013) possibly by detoxifying NO. Denitrifica81 tion may therefore contribute to the pathogenicity during chronic 82 P. aeruginosa lung infection in CF patients. 83 A. xylosoxidans contains the genetic setup for denitrification, 84 which enables the release of N2 O in anaerobic cultures sup85 plemented with nitrate (NO3 − ) (Jakobsen et al., 2013), while B. 86 multivorans exhibits nitric oxide reductase activity (Kimura et al., 87 2012). The ability to reduce NO3 − beyond nitrite (NO2 − ) is known 88 for the three highly pathogenic isolates used in this study (Zumft, 89 1997, 2005; Jakobsen et al., 2013; Yuhara et al., 2008), but not 90 for S. maltophilia. We therefore hypothesized that denitrification 91 is related to the pathogenicity of CF pathogens by comparing deni92 trification of the CF highly pathogenic P. aeruginosa, A. xylosoxidans, 93 B. multivorans to the low pathogenic S. maltophilia. Denitrification 94 was quantitated as the anoxic release of N2 O, which is a signa95 ture for denitrification (Zumft, 1997) with N2 O microelectrodes 96 in anaerobic cultures isolated from chronic infected CF patients 97 supplemented with NO3 − and NO2 − . Furthermore, the effect of 98 NO3 − and NO2 − for anaerobic growth was determined by tradi99 tional growth assays as well as a newly developed PNA FISH staining 100 technique (Hansen et al., 2014). 101 44 45

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Materials and methods

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Patients

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As defined by the “Danish Act on Research Ethics Review of Health Research Projects” Section 2 the project did not constitute a

health research project and was thus initiated without approval from The Committees on Health Research Ethics in the Capital Region of Denmark. The study was carried out with 37 bacterial isolates. Chronic infection with the four pathogens was defined as the presence of bacteria in the lower respiratory tract as detected in each monthly culture from sputum samples for >6 months, or for a shorter time in the presence of increased antibody response (>2 precipitating antibodies, normal: 0–1) (Høiby, 2000). All patients were characterized as chronically infected with P. aeruginosa, A. xylosoxidans, B. multivorans or S. maltophilia. Chronically infected patients with more than one Gram-negative bacterial infection were not included.

Sputum samples The study was performed on surplus sputum expectorated for routine bacteriology from 3 CF patients with chronic S. maltophilia infections after obtaining oral permission with waiver of informed consent.

Bacteria Twelve P. aeruginosa CF isolates, 9 A. xylosoxidans CF isolates, 9 B. multivorans CF isolates and 7 S. maltophilia CF isolates, the P. aeruginosa reference strain PAO1 (MH340), the A. xylosoxidans reference strain DSM 2402 isolated from an ear discharge (DSMZ, Braunschweig, Germany) (Jakobsen et al., 2013), the B. multivorans reference strain LMG 13010 isolated in Brussels in CF sputum (M. Struelens Erasmus Ziekenhuis Brussel, Belgium), and the S. maltophilia reference strain ATCC® 13637 isolated from an oropharyngeal region of a patient with mouth cancer (LGC Standards AB, Sweden) were streaked over blue agar plates selective for Gram negative cells (Statens Serum Institute (SSI), Denmark) and incubated for 24 h. One scrape of bacteria was diluted in 100 ml Luria Bertani (LB)-broth (5 g/l yeast extract (Oxoid, Roskilde, Denmark), 10 g/l tryptone (Oxoid) and 10 g/l NaCl (Merck, Rahway, NJ, US), pH 7.5) and grown for 24 h (150 rpm, 37 ◦ C). OD600 was recorded in a spectrophotometer (UVmini 1240, Shimadzu, USA) and 50 ml culture was washed two times in phosphate buffered saline (PBS) (Substrate Department, Panum Institute, Denmark) and spun down (5000 rpm, 4 ◦ C, 10 min). The pellet was resuspended in 5 ml PBS. The actual bacterial content was estimated by CFU counting of serial dilution in 0.9% NaCl plated on blue plates.

Media LB broth was supplemented with KNO3 (P8394, Sigma–Aldrich, Denmark) or NaNO2 (S2252, Sigma–Aldrich) to obtain a final concentration of 10 mM. Thereafter, the media was sterile filtrated (0.20 ␮l) into 50 ml tubes sealed with semi-permeable parafilm before being placed in an anoxic atmosphere (Concept 400, Fischer Scientific, Denmark) for 72 h at 37 ◦ C.

Anaerobic treatment of bacterial isolates from chronic infected CF patients Inoculums of overnight cultures were added under anoxic consitions to LB broth with 10 mM KNO3 (Sigma–Aldrich) or 10 mM NaNO2 (Sigma–Aldrich) to reach a final concentration of ≈106 CFU/ml in 2 ml. This was done in glass culture tubes (35 mm × 12 mm) closed with aluminium screw-caps (Schuett Biotec, Germany). The glass vials were positioned in metal racks for 24 and 48 h (150 rpm, 37 ◦ C).

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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Growth measurements

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Immediately before N2 O measurement, the glass vials were positioned in a double-beam spectrophotometer (UV-190, Shimadzu) and OD600 was measured. LB broth in another glass vial was used as a reference. After N2 O measurements, the colony forming units (CFUs) of samples were estimated by serial dilutions in 0.9% NaCl plated onto blue agar plates (SSI).

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Microsensor measurements of N2 O

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The glass vials were positioned in a heated metal rack, kept at 37 ◦ C. N2 O concentration measurements were recorded with an amperometric N2 O microsensor (N2 O-100, Unisense A/S, Århus, Denmark) (Andersen et al., 2001) mounted in a motorized PCcontrolled profiling setup (MM33 and MC-232, Unisense A/S). The microsensor (tip diameter 100 ␮m) was connected to a picoammeter (PA2000, Unisense A/S) and positioned manually in the sample. Profile measurements, taken by movement of the sensor in vertical steps of 1000 ␮m through the sample, were controlled and stored by dedicated software (Sensortrace Pro 2.0, Unisense). The software was set to wait 5 s for the N2 O-microprofile, before moving the sensors to the next measuring depth. Replicates of 6 repeated profiles were separated by 5 s waiting. The N2 O microsensor was linearly calibrated according to (Andersen et al., 2001) by measuring sensor signals in N2 O free PBS at experimental temperature and salinity and in PBS with sequential addition of a known volume of N2 O saturated PBS up to a final concentration of 100 ␮M N2 O. The N2 O concentration in saturated PBS was estimated according to N2 O solubility measured by microgasometry (Weiss and Price, 1980).

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NO3 − and NO2 − quantification

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After N2 O measurements and serial dilution, samples were sterile filtrated (0.20 ␮m) and stored at 20 ◦ C for later use. The NO3 − 189 and NO2 − levels in the samples were measured using the Griess 190 colorimetric reaction (780001, Cayman Chemicals, USA) accord191 ing to the manufacturer’s recommendations. Samples were diluted 192 100× and transferred to a 96 well microtiter plate prior to a two193 step process: the first step converted NO3 − to NO2 − utilizing NO3 − 194 reductase. After incubation for 2 h this was followed by the addi195 tion of the Griess Reagent, where NO2 − was converted into a purple 196 197 azo compound. After incubation with Griess Reagent for 10 min, 198 the absorbance at 540–550 nm was measured using an ELISA plate 199 reader (Thermo Scientific Multiskan EX, Thermo Fisher Scientific 200 Inc, BioImage, Denmark). A NO3 − standard curve was used for 201 determination of total NO3 − and NO2 − concentration, while a NO2 − 202 standard curve was used for determination of NO2 − alone. The con203 centration of NO2 − was subtracted from the total NO3 − and NO2 − 204 concentration to estimate the NO3 − concentration. Even though 205 manipulations are needed in the Griess-assay the applied method 206 has previously enabled us to obtain estimations of the concentra207 tion of NO3 − or NO2 − in CF sputum that corresponds well with 208 Q3 several other investigations (Linnane et al., 1998; Grasemann et al., 209 1998; Jones et al., 2000; Palmer et al., 2007; Kolpen et al., 2014b). 188

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Measurement of growth rates The growth rates of S. maltophilia isolates were estimated in order to correlate them to the intensity of the fluorescent signal from the PNA FISH probe. Pure culture growth rates of isolated S. maltophilia from expectorated sputum were obtained under aerobic conditions at 37 ◦ C in LB broth. Overnight cultures of S. maltophilia were diluted in 100 ml LB broth in 250 ml flasks at 200 rpm to OD600 = 0.05 and the cultures were allowed to proliferate (37 ◦ C, 200 rpm). When OD600 reached 0.5, cultures were diluted to an

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OD600 of 0.05, where after growth rates were measured by monitoring changes in OD600 over time. Growth rates were expressed as the specific growth rate (divisions per hour).

Fixation of S. maltophilia cells Twenty ␮l of the isolated S. maltophilia were fixed in a drop of Fixation solution (790226, Advandx, Denmark) and mixed on a microscope slide (Superfrost® plus, Thermo scientific). Fixed cells were incubated for 20 min at 65 ◦ C on an AccublockTM Digital Dry Bath (Labnet International, Inc.). Expectorated sputum was fixed in disposable vinyl specimen molds (Cryomold Standard Tissue-Tek 4557, Sakura Finetek, Europe) with Tissue-Tek. The fixed samples were snap frozen in −80 ◦ C hexane (Sigma).

Cryosections of sputum samples The snap frozen blocks were cut in 8 ␮m thick slices on a −18 ◦ C cryostat (Tissue-Tek 2000cryo, USA). Slices were fixated to microscope slide.

Whole cell hybridization One drop of PNA-FISH probe specific for S. maltophilia (11912, Advandx) was applied on the fixed S. maltophilia cells on slides with a 22 mm × 22 mm coverslip (no.1.5H) on top. The samples were incubated on a workstation (Advandx) for 90 min at 55 ◦ C before incubation for 30 min at 55 ◦ C in a 4 ml 60× wash solution (CP0022, Advandx) diluted in 240 ml MilliQ H2 O followed by air drying in darkness for 15 min. In addition, for sputum samples 100 ␮l Syto 59 (Invitrogen) diluted 1000× in NaCl was added for staining of all DNA and samples were incubated for 15 min followed by PBS rinsing and air drying in darkness. A drop of Hard Set mounting medium (Vectashield Vector) was added on top of the slide, sealed with a coverslip and air dried for 10 min.

Microscopy and image analysis Mounted sample slides were scanned with a Zeiss Imager.Z2 microscope with a LSM 710 CLSM and the accompanying software Zeiss Zen 2010 v. 6.0. (Zeiss, Germany). Fluorescence images were recorded by laser excitation at 594 nm and an emission of 615 nm with a resolution of 6144 × 6144 pixels at a color depth of 16 bits using a 63×/1.4 oil objective; each pixel was scanned twice. Images were stored in 16 bit TIF format. Quantification of fluorescence from individual cells was performed using the freeware Image J (National Institutes of Health, USA). Discrimination between cells and background signal was done by thresholding using the automated “Multithreshold” macro for Image J (Baler et al., 2009). For quantification, the Image J function “Analyze Particles” was used. For each of the analyzed isolates, we used ∼17,000 mean fluorescence intensity units as the lowest limit of detection separating growth from non-growth.

Statistical methods Statistical significance was evaluated by Kruskal–Wallis test followed by Dunn’s multiple comparison test or ordinary one-way ANOVA test followed by Dunnett’s multiple comparison test. A p value ≤0.05 was considered statistically significant. The tests were performed with Prism 6.1 (GraphPad Software, La Jolla, CA, USA).

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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Fig. 1. N2 O production by isolated pathogens from chronically infected CF patients. N2 O concentrations in isolates from chronically infected CF patients grown in anaerobic LB broth without supplementation or supplemented with either 10 mM NO3 − or 10 mM NO2 − incubated for 1 and 2 days. P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7). Statistical significance was determined using Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

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Results

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N2 O production in isolates from chronically infected CF patients

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for P. aeruginosa isolates, indicating the ability for anaerobic growth on NO2 − . Similar results were observed for the reference strains (online supplement Fig. 2).

A significant increase in N2 O concentration was found for P. aeruginosa, A. xylosoxidans and B. multivorans in anaerobic LB media supplemented with NO3 − when incubated for 1 or 2 days (Fig. 1). S. maltophilia isolates were not capable to increase the N2 O concentration significantly when incubated in anaerobic LB broth supplemented with either NO3 − or NO2 − (Fig. 1), suggesting that S. maltophilia does not contain enzymes for the release of N2 O. In contrast, the corresponding reference strains of the highly pathogenic CF isolates demonstrated low amounts of N2 O production (online supplement Fig. 1), indicating that the pathogenic clinical strains were adapted to anaerobic respiration during lung infection.

Growth of isolates from chronically infected CF patients

Biomass accumulation in isolates from chronically infected CF patients

Extracellular concentrations of NO3 − and NO2 − in isolates from chronically infected CF patients

Accumulation of biomass could be controlled by removal of the O2 and by supplementation of NO3 − or NO2 − as determined by the absorbance (Fig. 2) and by visual inspection (Fig. 3). Anaerobic cultures of all 4 isolated pathogens showed a significant increase in absorbance when supplemented with NO3 − (Fig. 2) demonstrating that all 4 pathogens have the ability to grow anaerobically by NO3 − reduction. However, significantly higher absorbance was seen on day two for P. aeruginosa, A. xylosoxidans and B. multivorans than for S. maltophilia in anaerobic cultures supplemented with NO3 − indicating slower growth by S. maltophilia. Supplementation with NO2 − resulted in significantly increased biomass only

The concentration of NO3 − in anaerobic LB broth supplemented with 10 mM NO3 − was significantly decreased on day 1 and 2 for all 4 pathogens, but S. maltophilia was not capable of consuming the entire amount of NO3 − (Fig. 5A). The ability to grow in the absence of O2 with NO3 − as a terminal electron acceptor may be explained by the demonstration of nar genes encoding the membrane-bound nitrate reductase (NAR) in a clinical S. maltophilia isolate (Crossman et al., 2008). This indicates that NO3 − consumption due to NAR activity was present in all species. Following NO3 − consumption, a significant increase in the concentration of NO2 − was demonstrated for the CF highly pathogenic species P. aeruginosa, A. xylosoxidans,

Supplementation of anaerobic cultures with 10 mM NO3 − resulted in significantly more CFU/ml for the P. aeruginosa isolates on day 1 and 2 (Fig. 4A) and for A. xylosoxidans on day 1 (Fig. 4B). In contrast, supplementation with 10 mM NO3 − did not increase the number of CFU/ml for B. multivorans (Fig. 4C) and S. maltophilia (Fig. 4D) significantly. Only S. maltophilia isolates were affected by supplementation with 10 mM NO2 − , which resulted in a significant decrease in CFU/ml (Fig. 4D). Reference strains demonstrated similar results as the clinical isolates (online supplement Fig. 3).

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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Fig. 2. Bacterial density of isolated pathogens from chronically infected CF patients. Estimation of biomass according to OD600 in isolates from chronically infected CF patients grown in anaerobic LB broth without supplementation or supplemented with either 10 mM NO3 − or 10 mM NO2 − for 2 days. P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7). Statistical significance was determined using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.

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B. multivorans (Fig. 5B). Accordingly, only the highly pathogenic CF strains were able to export NO2 − produced from reduction of NO3 − to the medium. The lack of increased extracellular content of NO2 − during NO3 − consumption in S. maltophilia may, in part, be due to assimilatory denitrification. Apparently, only P. aeruginosa was able to consume NO2 − significantly (Fig. 5C) indicating that this pathogen contains more active nitrite reductase (NIR). In contrast to the fast consumption of NO3 − seen on day 1, the concentration of NO2 − was significantly reduced after 2 days. This resembles recent findings in infected CF sputum (Kolpen et al., 2014b) and suggests that the rate of NO2 − reduction is slower than the rate of NO3 − reduction.

Correlation between specific growth rate and cellular ribosome content in S. maltophilia isolates To establish a correlation between the bacterial growth rate and the cellular content of 16S rRNA, quantitative PNA FISH (specific for 16S rRNA of S. maltophilia ribosomes) was performed on 3 S. maltophilia isolates harvested during growth, where the corresponding

growth rate was calculated (online supplement Fig. 5). The correlation between the growth rate and the intensity of the PNA FISH fluorescence was calculated according to the regression lines, where R2 = 0.533 for isolate I, R2 = 0.789 for isolate II, and R2 = 0.680 for isolate III. These standard curves were then used for estimating the in situ growth rate of S. maltophilia in sputum samples (Table 1). Q4

Detection of non-growing S. maltophilia cells in sputum from chronically infected CF patients by rRNA hybridization Using the correlation between the specific growth rate and cellular ribosome content in S. maltophilia isolates (online supplement Fig. 5), the in situ growth rate of S. maltophilia in sputum was estimated for the first time. Interestingly, the majority of cells analyzed in sputum were found to be non-growing to slow-growing cells based on the reduced numbers of ribosomes (Table 2). None of the cells were approaching the in vitro growth rates measured in aerobic LB broth for cells isolated from the sputum samples. This was confirmed by confocal fluorescence microscopy (Fig. 6). The median specific growth rates were found to be 0 divisions h−1

Table 1 Demographic data of CF patients expressed as numbers of patients in a group or median (range) (age, FEV1%, FVC % and duration of chronic infection. P. aeruginosa Number (males) Age (years) FEV1 (%) predicted FVC (%) Duration of chronic infection (years)

7 (6) 26 (24–43) 70 (43–83) 110 (88–128) 16 (1–30)

A. xylosoxidans

B. multivorans

S. maltophilia

9 (5) 21 (16–27) 69 (22–96) 94 (48–115) 5 (3–11)

9 (4) 29 (18–41) 55 (27–89) 91 (47–109) 8 (3–13)

7 (4) 18 (9–34) 77 (59–96) 108 (81–113) 2 (1–4)

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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ARTICLE IN PRESS M. Kolpen et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx Table 2 Growth of S. maltophilia in sputum from chronically infected CF patients.

Median (MFIU) Range (MFIU) Median (divisions h−1 ) Range (divisions h−1 )

Sputum I

Sputum II

Sputum III

11,185 10,943–17,114 0 0–0.217

11,802 10,896–19,246 0 0–0.194

ND ND ND ND

ND: not detected.

with a range from 0 to 0.217 divisions h−1 for sputum sample I and 0 divisions h−1 with a range from 0 to 0.194 divisions h−1 for sputum sample II, while no growth could be detected in sputum sample III (Table 2). These sputum growth rates were considerably lower than during aerobic culturing, where growth rates exceeded 0.8 divisions h−1 were detected (online supplement Fig. 5) and suggest that S. maltophilia is dormant in the sputum samples. Discussion

Fig. 3. Visual examination of bacterial growth of isolated pathogens from chronically infected CF patients. Examination of growth as visualized in isolates from chronically infected CF patients grown in aerobic or anaerobic LB broth without supplementation or supplemented with either 10 mM NO3 − or 10 mM NO2 − for 2 days. P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7).

Even though several human pathogens possess the genetic setup for denitrification (Philippot, 2002; Zumft, 1997), the clinical significance of denitrification has been questioned (Philippot, 2005). Our data indicate that certain Gram-negative pathogenic species causing severe lung damage during chronic lung infection in CF (2012a; 2011; 2012b; Burns et al., 1998; Drevinek and Mahenthiralingam, 2010; Rønne et al., 2006) are able to perform denitrification, while denitrification is absent in S. maltophilia that lead to less severe decrease of lung function after establishing a chronic infection (Dalbøge et al., 2011; Waters et al., 2013). To demonstrate denitrification, we measured the concentration of N2 O, which is recognized as a bona fide marker for denitrification in anaerobic cultures (Zumft, 1997). We revealed production of N2 O in LB media supplemented with NO3 − by the three highly pathogenic CF species in accordance with the sequenced genes encoding enzymes and the identified enzymatic activities involved in denitrification (Silvestrini et al., 1989; Arai et al., 1990; Kawasaki et al., 1995, 1997; Jakobsen et al., 2013). The detection of N2 O required supplementation with 10 mM of NO3 − , which is higher than the reported concentrations in expectorated sputum (Kolpen et al., 2014a,b; Hassett, 1996). The actual spatiotemporal distribution of NO3 − in the lungs is, however, not known, but the concentrations of involved electron acceptors may vary more than a 100-fold in sputum samples (Kolpen et al., 2014a). For P. aeruginosa, the ability to consume NO2 − and increase the biomass during NO2 − supplementation further confirms that this pathogen can perform denitrification. Furthermore, aiming to kill lung pathogens with NO2 − call for careful monitoring of whether the supplemented NO2 − leads to expansion or decrease of the pathogen population (Yoon et al., 2006; Major et al., 2010). In contrast, S. maltophilia isolates failed to produce N2 O in agreement with the absence of reported genes encoding for known enzymes involved in gas production from nitrogen oxides. All four species, however, demonstrated NAR activity as seen by the increased biomass and consumption of NO3 − during NO3 − supplementation, which corresponds to the active NAR enzyme in environmental isolates of S. maltophilia (Woodard et al., 1990), the nar genes in a clinical isolate of S. maltophilia (Crossman et al., 2008) and the well described genetic setups in the three highly pathogenic strains (Silvestrini et al., 1989; Arai et al., 1990; Kawasaki et al., 1995, 1997). The low anaerobic growth of S. maltophilia may be related to the inability of S. maltophilia to perform anaerobic NO3 − respiration using denitrification as indicated by the failure to deplete NO3 − completely together with the lack of NO2 − accumulation and generation of N2 O. However, the modest consumption of NO3 − and induction of growth during

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Fig. 4. Bacterial growth of isolated pathogens from chronically infected CF patients. Growth estimated as CFU/ml in isolates from chronically infected CF patients grown in anaerobic LB broth without supplementation or supplemented with either 10 mM NO3 − or 10 mM NO2 − for 1 and 2 days. P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7). Statistical significance was determined using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.

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NO3 − supplementation together with the absence of biomarkers for active enzymes downstream of the NAR in the denitrification pathway suggests that S. maltophilia is able to grow by respiration of NO3 − without employing further reduction steps of terminal electron acceptors. Accordingly, decreased expression of the nar genes in S. maltophilia during anaerobic conditions may also provide explanations for the observed slower growth, but awaits further investigations. In addition, we speculate if the anaerobic persistence of S. maltophilia may also be supported by fermentative processes as in P. aeruginosa where anaerobic growth or survival is promoted by arginine (Vander et al., 1984) or pyruvate (Eschbach et al., 2004). Since it may be argued that the observed differences between the isolated species are caused by intra-strain variability we estimated the variability for the 4 laboratory strains from 5 individual experimental set-ups (supplemental online Figs. 1–4). In general, the coefficients of variations were too low to influence on the observed inter-species differences. Formation of biofilm has been demonstrated in clinical isolates of all four pathogens in vitro (Jakobsen et al., 2013; Pompilio et al., 2010; Silva et al., 2011; Aaron et al., 2002). But so far, only P. aeruginosa biofilm aggregates have been demonstrated directly in chronically infected CF lungs (Bjarnsholt et al., 2009). Biofilm formation enables P. aeruginosa to withstand the bactericidal activity of the numerous active PMNs in the endobronchial mucus, where antibiotic treatment is less effective than in the alveoli (Bjarnsholt et al., 2009). It has recently been shown that denitrification is one mechanism, by which P. aeruginosa may achieve energy (Kolpen et al., 2014b) to maintain the observed growth (Yang et al., 2008; Kragh et al., 2014) in the anaerobic parts of the endobronchial

secretions. The present demonstration of denitrification and anaerobic growth of P. aeruginosa isolates during NO3 − supplementation underlines the significance of denitrification for proliferation of P. aeruginosa in the endobronchial CF mucus. The advantage of anaerobic metabolism in the infected CF bronchi is likely to depend on the intense O2 depletion caused by the accumulated activated PMNs and diffusion limitations due to the mucoid matrix of the biofilm aggregates. In fact, NO3 − supplementation restores the growth rate when P. aeruginosa is exposed to O2 depletion by human PMNs (Kragh et al., 2014). Although the amount of endobronchial accumulation of activated PMNs during chronic A. xylosoxidans lung infection in CF has not been quantified and compared to that during chronic P. aeruginosa infection, systemic inflammatory mediators suggest similar PMN accumulation during lung infection with A. xylosoxidans (Hansen et al., 2010; Jensen et al., 2006). Accordingly, intense O2 depletion by the accumulated PMNs is also likely to cause anaerobiosis in endobronchial CF mucus infected by A. xylosoxidans. Therefore, our finding of denitrification and anaerobic growth in A. xylosoxidans isolates indicates that A. xylosoxidans is able to proliferate in the infected endobronchial CF mucus. B. multivorans belongs to the Burkholderia cenocepacia complex (Bcc) and Bcc cells can be detected in CF mucus invading PMNs. This successful invasion is probably a consequence of the anaerobic environment where the amount of available O2 is too low for the PMNs to produce bactericidal amounts of reactive oxygen species by the respiratory burst (Sousa et al., 2007). Our results indicate that B. multivorans may engage denitrification to promote intracellular proliferation in the anaerobic PMNs of infected endobronchial CF mucus.

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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Fig. 5. Consumption of NO3 − and NO2 − by isolated pathogens from chronic infected CF patients. (A) NO3 − concentration in anaerobic LB broth supplemented with 10 mM NO3 − for 1 and 2 days by P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7). (B) NO2 − concentration in anaerobic LB broth supplemented with 10 mM NO3 − for 1 and 2 days by P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7). (C) NO2 − concentration in anaerobic LB broth supplemented with 10 mM NO2 − for 1 and 2 days by P. aeruginosa (n = 12), A. xylosoxidans (n = 9), B. multivorans (n = 9) and S. maltophilia (n = 7). Statistical significance was determined using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Asterisk indicates intervals of statistical significance between day 0 to day 1 and day 2. *Statistical significant increase. **Statistical significant decrease.

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The ability of S. maltophilia to activate PMNs during lung infection in mice (Waters et al., 2007) and the high level of IgG antibodies against these bacteria in CF patients (Dalbøge et al., 2011) indicate that S. maltophilia can also stimulate inflammatory activities of

PMNs including the formation of anoxic conditions in the infected endobronchial mucus by O2 depletion during the respiratory burst, which may induce selection for anaerobic metabolism. Since anaerobic respiration by denitrification is a process where each reduction

Fig. 6. S. maltophilia in CF sputum. Visualization of a representative sputum sample from a CF patient chronically infected with S. maltophilia analyzed by Syto 59 staining combined with PNA FISH using a S. maltophilia specific PNA FISH probe (16S rRNA). The bar in each photograph represents 2 ␮m.

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step may lead to increased growth of the biomass (Strohm et al., 2007; John and Whatley, 1970; Koike and Hattori, 1975a,b; Thauer et al., 1977), we expected that S. maltophilia, which only exhibits NAR activity, had lower anaerobic growth during NO3 − supplementation than the species with more complete denitrification pathways. These in vitro findings may explain our findings of mainly non-growing or slow-growing S. maltophilia in CF sputum as inferred by quantitating the ribosomal content with PNA FISH. We therefore suggest that the low pathogenicity of S. maltophilia is associated with dormancy resulting from the poor ability to grow anaerobically. In particular, dormancy may prevent spreading from the anaerobic bronchial mucus to the lower respiratory tract and induction of lung tissue deterioration. The low pathogenicity of S. maltophilia in cystic fibrosis patients may also partly result from the lack of many conventional key virulence determinants (Crossman et al., 2008). In comparison, the growth advantage provided by denitrification may give rise to the higher pathogenicity of P. aeruginosa, A. xylosoxidans and B. multivorans by enabling proliferation that is sufficient for spreading from the anaerobic bronchial mucus to other parts of the lungs resulting in tissue destruction. On the other hand, the slow growth of S. maltophilia may confer increased tolerance to antibiotics leading to the low success of eradication of this bacterium by antibiotics, which parallels the high resistance of the slow growing small-colony variants of Staphylococcus aureus (Proctor et al., 2006). Association between denitrification and pathogenicity has previously been proposed to be involved in the severe lung function decline in CF patients infected with lasR mutants of P. aeruginosa, as mutations in lasR promote faster growth and enhanced NO production in hypoxic cultures supplemented with NO3 − (Hoffman et al., 2009, 2010). Thus, the present study extends the possible role of denitrification from contributing to the pathogenicity between mutants within one species to affecting the varying pathogenicity between several Gram-negative species causing chronic lung infection in CF patients. Denitrification may, however, not be the only mechanism supporting pathogenicity involving oxygen depletion in infected CF lungs since Burkholderia cenocepacia, which may cause severe acute pulmonary deterioration with bacteraemia (Ciofu et al., 2013), is able to persist during anoxia (Sass et al., 2013) and to grow at micro-oxic conditions without employing denitrification (Pessi et al., 2013). In conclusion, this study separated highly pathogenic CF species P. aeruginosa, A. xylosoxidans and B. multivorans from the low pathogenic S. maltophilia by the ability to grow anaerobically by denitrification. This indicates that denitrification may contribute to the pathogenicity of Gram-negative bacteria during chronic lung infection in CF. Funding

Michael Kühl was supported by a grant the Danish Research Q6 Council for Independent Research | Natural Sciences (FNU). 528 Q5 527

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Competing interests None. Acknowledgement We thank the Danish Cystic Fibrosis Association for support Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm. 2014.07.002.

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References Aaron, S.D., Ferris, W., Ramotar, K., Vandemheen, K., Chan, F., Saginur, R., 2002. Single and combination antibiotic susceptibilities of planktonic, adherent, and biofilmgrown Pseudomonas aeruginosa isolates cultured from sputa of adults with cystic fibrosis. J. Clin. Microbiol. 40, 4172–4179. Andersen, K., Kjær, T., Revsbech, N.P., 2001. An oxygen insensitive microsensor for nitrous oxide. Sens. Actuators B Chem. 81, 42–48. 2011. Cystic Fibrosis Foundation Patient Registry Annual Data Report, 2010. Cystic Fibrosis Foundation, Bethesda, MD. 2012a. ECFS Patient Registry Annual Data Report, 2008–2009 data. European Cystic Fibrosis Society, Karup, Denmark. 2012b. UK CF Registry Annual Data Report, 2010. Cystic Fibrosis Trust, Bromley, Kent, UK. Arai, H., Iiyama, K., 2013. Role of nitric oxide-detoxifying enzymes in the virulence of Pseudomonas aeruginosa against the silkworm, Bombyx mori. Biosci. Biotechnol. Biochem. 77, 198–200. Arai, H., Sanbongi, Y., Igarashi, Y., Kodama, T., 1990. Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa. FEBS Lett. 261, 196–198. Baler, K., Landini, G., Rasband, W., 2009. MultiTresholder. Baltimore, R.S., Christie, C.D., Smith, G.J., 1989. Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis. Implications for the pathogenesis of progressive lung deterioration. Am. Rev. Respir. Dis. 140, 1650–1661. Bjarnsholt, T., Jensen, P.Ø., Fiandaca, M.J., Pedersen, J., Hansen, C.R., Andersen, C.B., Pressler, T., Givskov, M., Høiby, N., 2009. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr. Pulmonol. 44, 547–558. Borriello, G., Werner, E., Roe, F., Kim, A.M., Ehrlich, G.D., Stewart, P.S., 2004. Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob. Agents Chemother. 48, 2659–2664. Boucher, R.C., 2007. Evidence for airway surface dehydration as the initiating event in CF airway disease. J. Intern. Med. 261, 5–16. Burns, J.L., Emerson, J., Stapp, J.R., Yim, D.L., Krzewinski, J., Louden, L., Ramsey, B.W., Clausen, C.R., 1998. Microbiology of sputum from patients at cystic fibrosis centers in the United States. Clin. Infect. Dis. 27, 158–163. Chen, F., Xia, Q., Ju, L.K., 2003. Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD(P)H fluorescence. Appl. Environ. Microbiol. 69, 6715–6722. Ciofu, O., Hansen, C.R., Høiby, N., 2013. Respiratory bacterial infections in cystic fibrosis. Curr. Opin. Pulm. Med. 19, 251–258. Crossman, L.C., Gould, V.C., Dow, J.M., Vernikos, G.S., Okazaki, A., Sebaihia, M., Saunders, D., Arrowsmith, C., Carver, T., Peters, N., Adlem, E., Kerhornou, A., Lord, A., Murphy, L., Seeger, K., Squares, R., Rutter, S., Quail, M.A., Rajandream, M.A., Harris, D., Churcher, C., Bentley, S.D., Parkhill, J., Thomson, N.R., Avison, M.B., 2008. The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants. Genome Biol. 9, R74, http://dx.doi.org/10.1186/gb-2008-9-4-r74. Dalbøge, C.S., Hansen, C.R., Pressler, T., Høiby, N., Johansen, H.K., 2011. Chronic pulmonary infection with Stenotrophomonas maltophilia and lung function in patients with cystic fibrosis. J. Cyst. Fibros. 10, 318–325. Drevinek, P., Mahenthiralingam, E., 2010. Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence. Clin. Microbiol. Infect. 16, 821–830. Eschbach, M., Schreiber, K., Trunk, K., Buer, J., Jahn, D., Schobert, M., 2004. Longterm anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation. J. Bacteriol. 186, 4596–4604. Gaston, B., Ratjen, F., Vaughan, J.W., Malhotra, N.R., Canady, R.G., Snyder, A.H., Hunt, J.F., Gaertig, S., Goldberg, J.B., 2002. Nitrogen redox balance in the cystic fibrosis airway: effects of antipseudomonal therapy. Am. J. Respir. Crit. Care Med. 165, 387–390. Grasemann, H., Ioannidis, I., Tomkiewicz, R.P., de Groot, H., Rubin, B.K., Ratjen, F., 1998. Nitric oxide metabolites in cystic fibrosis lung disease. Arch. Dis. Child. 78, 49–53. Hansen, C.R., Pressler, T., Nielsen, K.G., Jensen, P.Ø., Bjarnsholt, T., Høiby, N., 2010. Inflammation in Achromobacter xylosoxidans infected cystic fibrosis patients. J. Cyst. Fibros. 9, 51–58. Hansen, N., Rasmussen, A.K.I., Fiandaca, M.J., Kragh, K.N., Bjarnsholt, T., Høiby, N., Stender, H., Guardabassi, L., 2014. Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization. New Microbes Q7 Hum. New Resist. Microbes Hum., 1–3. Hassett, D.J., 1996. Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen. J. Bacteriol. 178, 7322–7325. Hoboth, C., Hoffmann, R., Eichner, A., Henke, C., Schmoldt, S., Imhof, A., Heesemann, J., Hogardt, M., 2009. Dynamics of adaptive microevolution of hypermutable Pseudomonas aeruginosa during chronic pulmonary infection in patients with cystic fibrosis. J. Infect. Dis. 200, 118–130. Hoffman, L.R., Kulasekara, H.D., Emerson, J., Houston, L.S., Burns, J.L., Ramsey, B.W., Miller, S.I., 2009. Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression. J. Cyst. Fibros. 8, 66–70. Hoffman, L.R., Richardson, A.R., Houston, L.S., Kulasekara, H.D., Martens-Habbena, W., Klausen, M., Burns, J.L., Stahl, D.A., Hassett, D.J., 2010. Nutrient availability as a mechanism for selection of antibiotic tolerant Pseudomonas aeruginosa within the CF airway. PLoS Pathog. 6, e1000712. Høiby, N., 2000. Microbiology of cystic fibrosis. In: Hodson, G.D., M.E. (Eds.), Cystic Fibrosis. Arnold, London, UK, pp. 83–107.

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Jakobsen, T.H., Hansen, M.A., Jensen, P.Ø., Hansen, L., Riber, L., Cockburn, A., Kolpen, M., Rønne Hansen, C., Ridderberg, W., Eickhardt, S., Hansen, M., Kerpedjiev, P., Alhede, M., Qvortrup, K., Burmølle, M., Moser, C., Kühl, M., Ciofu, O., Givskov, M., Sørensen, S.J., Høiby, N., Bjarnsholt, T., 2013. Complete genome sequence of the cystic fibrosis pathogen Achromobacter xylosoxidans NH44784-1996 complies with important pathogenic phenotypes. PLOS ONE 8, e68484. Jensen, P.Ø., Moser, C., Kharazmi, A., Presler, T., Koch, C., Høiby, N., 2006. Increased serum concentration of G-CSF in cystic fibrosis patients with chronic Pseudomonas aeruginosa pneumonia. J. Cyst. Fibros. 5, 145–151. John, P., Whatley, F.R., 1970. Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans. Biochim. Biophys. Acta 216, 342–352. Jones, K.L., Hegab, A.H., Hillman, B.C., Simpson, K.L., Jinkins, P.A., Grisham, M.B., Owens, M.W., Sato, E., Robbins, R.A., 2000. Elevation of nitrotyrosine and nitrate concentrations in cystic fibrosis sputum. Pediatr. Pulmonol. 30, 79–85. Kakishima, K., Shiratsuchi, A., Taoka, A., Nakanishi, Y., Fukumori, Y., 2007. Participation of nitric oxide reductase in survival of Pseudomonas aeruginosa in LPS-activated macrophages. Biochem. Biophys. Res. Commun. 355, 587–591. Kawasaki, S., Arai, H., Igarashi, Y., Kodama, T., 1995. Sequencing and characterization of the downstream region of the genes encoding nitrite reductase and cytochrome c-551 (nirSM) from Pseudomonas aeruginosa: identification of the gene necessary for biosynthesis of heme d1. Gene 167, 87–91. Kawasaki, S., Arai, H., Kodama, T., Igarashi, Y., 1997. Gene cluster for dissimilatory nitrite reductase (nir) from Pseudomonas aeruginosa: sequencing and identification of a locus for heme d1 biosynthesis. J. Bacteriol. 179, 235–242. Kimura, A., Yuhara, S., Ohtsubo, Y., Nagata, Y., Tsuda, M., 2012. Suppression of pleiotropic phenotypes of a Burkholderia multivorans fur mutant by oxyR mutation. Microbiology 158, 1284–1293. Knowles, M.R., Boucher, R.C., 2002. Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Invest. 109, 571–577. Koike, I., Hattori, A., 1975a. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitriteand oxide-limited conditions. J. Gen. Microbiol. 88, 11–19. Koike, I., Hattori, A., 1975b. Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions. J. Gen. Microbiol. 88, 1–10. Kolpen, M., Hansen, C.R., Bjarnsholt, T., Moser, C., Christensen, L.D., van Gennip, M., Ciofu, O., Mandsberg, L., Kharazmi, A., Döring, G., Givskov, M., Høiby, N., Jensen, P.Ø., 2010. Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis. Thorax 65, 57–62. Kolpen, M., Bjarnsholt, T., Moser, C., Hansen, C.R., Rickelt, L.F., Kühl, M., Hempel, C., Pressler, T., Høiby, N., Jensen, P.Ø., 2014a. Nitric oxide production by polymorphonuclear leukocytes in infected cystic fibrosis sputum consumes oxygen. Clin. Exp. Immunol. 177, 310–319. Kolpen, M., Kuhl, M., Bjarnsholt, T., Moser, C., Hansen, C.R., Liengaard, L., Kharazmi, A., Pressler, T., Høiby, N., Jensen, P.Ø., 2014b. Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection. PLOS ONE 9, e84353. Kragh, K.N., Alhede, M., Jensen, P.Ø., Moser, C., Jacobsen, C.S., Seier, S., Eickhardt, S., Trøstrup, H., Christoffersen, L., Hougen, H., Givskov, M., Kühl, M., Høiby, N., Bjarnsholt, T., 2014. Polymorphonuclear Leukocytes Restrict the Growth of Pseudomonas aeruginosa in Lungs of Cystic Fibrosis Patients (submitted). Linnane, S.J., Keatings, V.M., Costello, C.M., Moynihan, J.B., O’Connor, C.M., Fitzgerald, M.X., McLoughlin, P., 1998. Total sputum nitrate plus nitrite is raised during acute pulmonary infection in cystic fibrosis. Am. J. Respir. Crit. Care Med. 158, 207–212. Major, T.A., Panmanee, W., Mortensen, J.E., Gray, L.D., Hoglen, N., Hassett, D.J., 2010. Sodium nitrite-mediated killing of the major cystic fibrosis pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under anaerobic planktonic and biofilm conditions. Antimicrob. Agents Chemother. 54, 4671–4677. Palmer, K.L., Brown, S.A., Whiteley, M., 2007. Membrane-bound nitrate reductase is required for anaerobic growth in cystic fibrosis sputum. J. Bacteriol. 189, 4449–4455. Pessi, G., Braunwalder, R., Grunau, A., Omasits, U., Ahrens, C.H., Eberl, L., 2013. Response of Burkholderia cenocepacia H111 to micro-oxia. PLOS ONE 8, e72939. Philippot, L., 2002. Denitrifying genes in bacterial and Archaeal genomes. Biochim. Biophys. Acta 1577, 355–376. Philippot, L., 2005. Denitrification in pathogenic bacteria: for better or worst? Trends Microbiol. 13, 191–192. Pompilio, A., Crocetta, V., Confalone, P., Nicoletti, M., Petrucca, A., Guarnieri, S., Fiscarelli, E., Savini, V., Piccolomini, R., Di Bonaventura, G., 2010. Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients. BMC Microbiol. 10, 102.

Proctor, R.A., von Eiff, C., Kahl, B.C., Becker, K., McNamara, P., Herrmann, M., Peters, G., 2006. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat. Rev. Microbiol. 4, 295–305. Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., Drumm, M.L., Iannuzzi, M.C., Collins, F.S., Tsui, L., 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073. Rønne, H.C., Pressler, T., Høiby, N., Gormsen, M., 2006. Chronic infection with Achromobacter xylosoxidans in cystic fibrosis patients; a retrospective case control study. J. Cyst. Fibros. 5, 245–251. Sass, A.M., Schmerk, C., Agnoli, K., Norville, P.J., Eberl, L., Valvano, M.A., Mahenthiralingam, E., 2013. The unexpected discovery of a novel low-oxygen-activated locus for the anoxic persistence of Burkholderia cenocepacia. ISME J. 7, 1568–1581. Silva, I.N., Ferreira, A.S., Becker, J.D., Zlosnik, J.E., Speert, D.P., He, J., Mil-Homens, D., Moreira, L.M., 2011. Mucoid morphotype variation of Burkholderia multivorans during chronic cystic fibrosis lung infection is correlated with changes in metabolism, motility, biofilm formation and virulence. Microbiology 157, 3124–3137. Silvestrini, M.C., Galeotti, C.L., Gervais, M., Schinina, E., Barra, D., Bossa, F., Brunori, M., 1989. Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein. FEBS Lett. 254, 33–38. Sousa, S.A., Ulrich, M., Bragonzi, A., Burke, A., Worlitzsch, D., Leitão, J.H., Meisner, C., Eberl, L., Sá-Correia, I., Döring, G., 2007. Virulence of Burkholderia cepacia complex strains in gp91phox−/− mice. Cell. Microbiol. 9, 2817–2825. Spicuzza, L., Sciuto, C., Vitaliti, G., Di, D.G., Leonardi, S., La, R.M., 2009. Emerging pathogens in cystic fibrosis: ten years of follow-up in a cohort of patients. Eur. J. Clin. Microbiol. Infect. Dis. 28, 191–195. Strohm, T.O., Griffin, B., Zumft, W.G., Schink, B., 2007. Growth yields in bacterial denitrification and nitrate ammonification. Appl. Environ. Microbiol. 73, 1420–1424. Thauer, R.K., Jungermann, K., Decker, K., 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, 100–180. Vander, W.C., Pierard, A., Kley-Raymann, M., Haas, D., 1984. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J. Bacteriol. 160, 928–934. Waters, V., Atenafu, E.G., Lu, A., Yau, Y., Tullis, E., Ratjen, F., 2013. Chronic Stenotrophomonas maltophilia infection and mortality or lung transplantation in cystic fibrosis patients. J. Cyst. Fibros. 12, 482–486. Waters, V.J., Gomez, M.I., Soong, G., Amin, S., Ernst, R.K., Prince, A., 2007. Immunostimulatory properties of the emerging pathogen Stenotrophomonas maltophilia. Infect. Immun. 75, 1698–1703. Weiss, R.F., Price, B.A., 1980. Nitrous oxide solubility in water and seawater. Mar. Chem. 8, 347–359. Williams, D.R., Rowe, J.J., Romero, P., Eagon, R.G., 1978. Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport. Appl. Environ. Microbiol. 36, 257–263. Woodard, L.M., Bielkie, A.R., Eisses, J.F., Ketchum, P.A., 1990. Occurrence of nitrate reductase and molybdopterin in Xanthomonas maltophilia. Appl. Environ. Microbiol. 56, 3766–3771. Worlitzsch, D., Tarran, R., Ulrich, M., Schwab, U., Cekici, A., Meyer, K.C., Birrer, P., Bellon, G., Berger, J., Weiss, T., Botzenhart, K., Yankaskas, J.R., Randell, S., Boucher, R.C., Döring, G., 2002. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J. Clin. Invest. 109, 317–325. Yang, L., Haagensen, J.A., Jelsbak, L., Johansen, H.K., Sternberg, C., Høiby, N., Molin, S., 2008. In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections. J. Bacteriol. 190, 2767–2776. Yoon, S.S., Hennigan, R.F., Hilliard, G.M., Ochsner, U.A., Parvativar, K., Kamani, M.C., Allen, H.L., Dekievit, T.R., Gardner, P.R., Schwab, U., Rowe, J.J., Iglewski, B.H., McDermot, T.R., Mason, R.P., Wozniak, D.J., Hancock, R.E., Parsek, M.R., Noah, T.L., Boucher, R.C., Hassett, D.J., 2002. Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Dev. Cell 3, 593–603. Yoon, S.S., Coakley, R., Lau, G.W., Lymar, S.V., Gaston, B., Karabulut, A.C., Hennigan, R.F., Hwang, S., Buettner, G., Schurr, M.J., Mortensen, J.E., Burns, J.L., Speert, D., Boucher, R.C., Hassett, D.J., 2006. Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions. J. Clin. Invest. 116, 436–446. Yuhara, S., Komatsu, H., Goto, H., Ohtsubo, Y., Nagata, Y., Tsuda, M., 2008. Pleiotropic roles of iron-responsive transcriptional regulator Fur in Burkholderia multivorans. Microbiology 154, 1763–1774. Zumft, W.G., 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616. Zumft, W.G., 2005. Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme-copper oxidase type. J. Inorg. Biochem. 99, 194–215.

Please cite this article in press as: Kolpen, M., et al., Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.002

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