Retracted: Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance
2015; Springer Nature; Volume: 7; Issue: 8 Linguagem: Inglês
10.15252/emmm.201505088
ISSN1757-4684
AutoresChris Earl, Teh Wooi Keong, Shi‐qi An, Sarah Murdoch, Yvonne McCarthy, Junkal Garmendia, Joseph Ward, J. Maxwell Dow, Liang Yang, George A. O’Toole, Robert P. Ryan,
Tópico(s)Respiratory and Cough-Related Research
ResumoTHIS ARTICLE HAS BEEN RETRACTED20 May 2015Open Access Retracted: Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance Chris S Earl Chris S Earl Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Teh Wooi Keong Teh Wooi Keong Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Shi-qi An Shi-qi An Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Sarah Murdoch Sarah Murdoch Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Yvonne McCarthy Yvonne McCarthy School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland Search for more papers by this author Junkal Garmendia Junkal Garmendia Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain Search for more papers by this author Joseph Ward Joseph Ward Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author J Maxwell Dow J Maxwell Dow School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland Search for more papers by this author Liang Yang Liang Yang Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore Search for more papers by this author George A O'Toole George A O'Toole Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Robert P Ryan Corresponding Author Robert P Ryan Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Chris S Earl Chris S Earl Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Teh Wooi Keong Teh Wooi Keong Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Shi-qi An Shi-qi An Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Sarah Murdoch Sarah Murdoch Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Yvonne McCarthy Yvonne McCarthy School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland Search for more papers by this author Junkal Garmendia Junkal Garmendia Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain Search for more papers by this author Joseph Ward Joseph Ward Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author J Maxwell Dow J Maxwell Dow School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland Search for more papers by this author Liang Yang Liang Yang Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore Search for more papers by this author George A O'Toole George A O'Toole Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Robert P Ryan Corresponding Author Robert P Ryan Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Author Information Chris S Earl1, Teh Wooi Keong2, Shi-qi An1, Sarah Murdoch1, Yvonne McCarthy3, Junkal Garmendia4,5, Joseph Ward6, J Maxwell Dow3, Liang Yang2, George A O'Toole7 and Robert P Ryan 1 1Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK 2Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore 3School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland 4Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain 5Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain 6Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, UK 7Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA *Corresponding author. Tel: +44 1382 384272; E-mail: [email protected] EMBO Mol Med (2015)7:1018-1033https://doi.org/10.15252/emmm.201505088 Retraction(s) for this article Retraction: "Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance"12 March 2018 See also: J Reidl & E Monsó (August 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Glucocorticosteroids are used as a main treatment to reduce airway inflammation in people with asthma who suffer from neutrophilic airway inflammation, a condition frequently associated with Haemophilus influenzae colonization. Here we show that glucocorticosteroids have a direct influence on the behavior of H. influenzae that may account for associated difficulties with therapy. Using a mouse model of infection, we show that corticosteroid treatment promotes H. influenzae persistence. Transcriptomic analysis of bacteria either isolated from infected mouse airway or grown in laboratory medium identified a number of genes encoding regulatory factors whose expression responded to the presence of glucocorticosteroids. Importantly, a number of these corticosteroid-responsive genes also showed elevated expression in H. influenzae within sputum from asthma patients undergoing steroid treatment. Addition of corticosteroid to H. influenzae led to alteration in biofilm formation and enhanced resistance to azithromycin, and promoted azithromycin resistance in an animal model of respiratory infection. Taken together, these data strongly suggest that H. influenzae can respond directly to corticosteroid treatment in the airway potentially influencing biofilm formation, persistence and the efficacy of antibiotic treatment. Synopsis Transcriptional and phenotypic analyses show a direct effect of glucocorticosteroids on the behavior of Haemophilus influenzae increasing bacterial persistence and antimicrobial resistance during respiratory infection. The glucocorticosteroid beclomethasone promotes H. influenzae persistence without influencing the host inflammatory response in a mouse model of acute infection. Beclomethasone induces specific alteration in the expression of H. influenzae genes implicated in biofilm formation, host colonization and survival in vivo. Examination of sputum samples from asthma patients undertaking steroid treatment showed many of the same alterations in H. influenzae gene expression as those induced by beclomethasone. Mutation analysis and transcriptome profiling identified the alternate sigma factor RpoE as an element in the glucocortico-steroid response. Mutation of rpoE or addition of glucocorticosteroid to H. influenzae led to alteration in biofilm formation and enhanced resistance to antibiotics in models of respiratory infection. Introduction Asthma is a chronic inflammatory condition of the airways, frequently distinguished by abnormal immune responses to environmental antigens and microbes, which leads to recurrent episodes of cough, wheezing and breathlessness (Wenzel, 2006, 2012). An estimated 300 million people worldwide suffer from asthma. Up to 30% of these patients suffer from neutrophilic asthma, which is characterized by substantial increases in airway neutrophils. Chronic colonization by bacteria is evident in the airways of patients with neutrophilic asthma, with Haemophilus influenzae being the species most frequently isolated. Inhaled glucocorticosteroids, through their potent anti-inflammatory action, are the foundation of asthma therapy (Ito et al, 2006). However, in a very high proportion of cases, neutrophilic asthmatics respond poorly to glucocorticosteroid treatment (Essilfie et al, 2011, 2012). Chronic bacterial infection has been associated with steroid-resistant neutrophilic asthma, although the mechanisms producing treatment resistance in such infections are poorly understood (Beigelman et al, 2014). The occurrence of Haemophilus spp., Streptococcus spp. or Moraxella catarrhalis in the neutrophilic asthmatic airway has been positively correlated with sputum neutrophilia and lower FEV1. The presence of H. influenzae in particular has been associated with the activation of airway inflammation pathways in those asthmatics with relative steroid resistance (Green et al, 2014). H. influenzae infection has been shown to contribute in part to allergic airways disease through alterations in IL-17 (Simpson et al, 2006; Berry et al, 2007). Furthermore, a strong relationship between chronic H. influenzae infection and the development of steroid-resistant neutrophilic asthma has been suggested using murine models of ovalbumin (OVA)-induced allergic airway disease (Essilfie et al, 2011, 2012). In this model, the combination of infection and allergic airways disease promotes bacterial persistence leading to the development of a phenotype similar to steroid-resistant neutrophilic asthma. These findings indicate that targeting bacterial infection in steroid-resistant asthma may have a therapeutic benefit. One aspect of the pathogenesis and therapy of neutrophilic asthma that has not received consideration is the potential direct influence of glucocorticosteroids on the behavior of H. influenzae. There are no reports of a direct antibacterial action of glucocorticosteroids; nevertheless, an impact on bacterial functions that promote persistence and tolerance to antibiotics might impinge on the efficacy of therapy. In this context, it is noteworthy that the severity of asthma appears to be increased in patients with bacterial infections who have received prolonged therapy with glucocorticosteroids. Here we have examined the impact of glucocorticosteroid treatment on H. influenzae during respiratory infection and neutrophilic asthma, as well as investigating impacts of this therapeutic in vitro on H. influenzae biology. Using a mouse model of acute infection, we show that the presence of glucocorticosteroids promotes H. influenzae persistence without influencing the host inflammatory response. Comparative transcriptional analysis of H. influenzae grown under standard laboratory conditions in the absence and presence of beclomethasone revealed that this glucocorticosteroid induces altered expression of genes implicated in biofilm formation and host colonization. Furthermore, the majority of these genes showed similar alterations in expression when transcriptomes of bacteria grown in medium without glucocorticosteroid were compared with those of bacteria isolated from the airway of infected mice. Importantly, many of the H. influenzae genes with expression that was responsive to corticosteroid in vitro also had altered expression in bacteria within sputum samples from asthma patients undertaking inhaled steroid treatment. In parallel, we examined the influence of glucocorticosteroids on H. influenzae behavior by addressing the effects on biofilm formation and resistance to azithromycin, a frontline drug prescribed to asthma patients. Addition of glucocorticosteroids changed the architecture and increased the tolerance to azithromycin of H. influenzae biofilms developed in flow cells. Finally, we developed a mutant screen that allowed the identification of a potential role for the RpoE-MclA sigma factor–anti-sigma factor system (HI0628–HI0629) as a mediator of this glucocorticosteroid response. Taken together, these findings indicate that H. influenzae can respond directly to glucocorticosteroid treatment in the lung where it may influence biofilm formation, persistence and the efficacy of antibiotic therapy. Results Glucocorticosteroids promote H. influenzae persistence in a mouse model Mouse models of H. influenzae have been rarely reported since it is difficult for this bacterium to infect mice (Vallee et al, 1992; Wu et al, 1997). Here, we pursued a new mouse model of lung infection in order to determine whether glucocorticosteroids have the ability to modulate H. influenzae infection. We used 4-week-old C57BL/6 mice to assess the effects of supplementation of H. influenzae with beclomethasone, a model glucocorticosteroid used widely in the clinic (van den Berge et al, 2011), on the bacterial load in the lung. Generally, young immature mice are more susceptible to bacterial infection than fully grown mice. In addition, the mice were pretreated intraperitoneally with cyclophosphamide to induce granulocytopenia. When cyclophosphamide-pretreated mice were intranasally inoculated with 1 × 109 CFU of H. influenzae, the bacteria resided in the lungs of the mice at more than 105 CFU for up to 3–4 days post-inoculation. Next, the cyclophosphamide-treated C57BL/6 mice were infected with H. influenzae and treated by inhaling PBS with or without 50 μM beclomethasone, a clinically relevant dose based on the weight of the mice used (Daley-Yates et al, 2001). The lung and spleen bacterial load was determined at 1, 3, 5 or 7 days post-infection. C57BL/6 mice infected with H. influenzae with PBS cleared almost all bacteria from the lungs within 5 days (Fig 1A). However, with the addition of beclomethasone, bacteria were still present in substantial numbers at 7 days (Fig 1A), with ~1,000-fold more bacteria present in the beclomethasone-treated mice versus control. Similar patterns were seen in the bacterial loads in the spleen, where after 7 days the numbers of bacteria in beclomethasone-treated mice were significantly higher (~1.5 log higher) than in untreated mice (Fig 1B). To extend this study, we also examined the influence of mometasone and prednisolone, two other glucocorticosteroids in clinical use, on H. influenzae survival in the cyclophosphamide-mouse model. Treatment with prednisolone (50 μM) gave a similar increased persistence phenotype as was observed for beclomethasone treatment (Supplementary Fig S1). Mometasone (50 μM) also appeared to promote H. influenzae persistence, although its influence was considerably less than the other two steroids tested. Figure 1. Administration of corticosteroids promotes the persistence of H. influenzae in mice A–D. C57BL/6 mice were infected with H. influenzae in PBS with or without supplementation with beclomethasone. Animals were killed at 1, 3, 5 and 7 days after infection, and 10-fold serial dilutions of the lung homogenates (A) and spleen homogenates (B) were plated to determine the bacterial load. Airway inflammation represented by total and differential cell counts in bronchoalveolar lavage fluid (BALF). The number of total leukocytes and in particular of neutrophils, monocytes and lymphocytes recruited in the airways was analyzed in BALF after day 3 (C) and day 7 (D) of infection. Data information: Values represent the mean ± standard deviation (SD). The data are pooled from three independent experiments. Statistical significance by two-tailed Student's t-test is indicated: *P < 0.05, **P < 0.01. Download figure Download PowerPoint The effects of beclomethasone on H. influenzae persistence were also examined in C57BL/6 mice that did not receive the cyclophosphamide treatment. The same trend was observed where beclomethasone treatment enhanced H. influenzae persistence in the airway, although in this case, no dissemination to the spleen was seen (Supplementary Fig S2). Taken together, these data indicate that treatment with beclomethasone enhanced H. influenzae persistence in the airway and systemically in this model of infection. Numerous previous studies have demonstrated H. influenzae infection of the murine airway induces the recruitment of leukocytes and other inflammation markers, which mirrors in part what is seen in a clinical setting (Hansen et al, 1988; Essilfie et al, 2011, 2012). In the current study, the inflammatory response of challenged mice was examined in terms of total leukocyte recruitment in the airways (see 4). In the first set of experiments, mice were pre-treated with cyclophosphamide. At 3 days post-infection, the cell numbers that influx into the airways (including neutrophils, lymphocyte and eosinophils) were similar in beclomethasone-treated mice and mice that were treated with PBS only (Fig 1C). By 7 days post-infection, however, neutrophil numbers in the mice treated with beclomethasone remained at the level seen at 3 days and were significantly higher than those seen in PBS alone (Fig 1D). In contrast, overall lymphocyte numbers were similar in mice treated with beclomethasone compared with mice treated with PBS alone (Fig 1D). The effects of beclomethasone on the inflammatory response induced by H. influenzae in C57BL/6 mice that did not receive the cyclophosphamide treatment were also examined. In this case, more neutrophils were recruited on day 3 as with the cyclophosphamide-treated mice (Supplementary Fig S2), but this elevation returned to normal levels by day 4. The observed continued recruitment of neutrophils at day 7 in the cyclophosphamide-mouse model of H. influenzae infection was unexpected. However, this observation is not unprecedented in the clinical setting; examination of neutrophilic asthma patients showed neutrophil numbers increase in those patients undergoing inhaled corticosteroid treatment (see Simpson et al, 2006). Overall, our results suggest that the presence of beclomethasone increases the capacity of H. influenzae to persist in mice but these findings do not establish whether this is a direct effect of the glucocorticosteroid on the bacteria or whether this is an indirect effect, mediated through an influence on the host, including recruitment of immune cells. Glucocorticosteroids induce specific changes in H. influenzae gene expression in culture As an approach to understanding the possible direct influence of glucocorticosteroids on H. influenzae, we examined the impact of beclomethasone on gene expression of bacteria using high-throughput cDNA sequencing techniques. For these experiments, bacteria were grown in complex sBHI medium, which is brain–heart infusion medium supplemented with hemin and NAD (see 4). RNA was derived from bacteria grown in the presence and absence of beclomethasone (50 μM) and, following rRNA depletion, the remaining RNA was fragmented, reverse-transcribed into cDNA and sequenced using the Illumina platform. Between 20 and 30 million reads were derived from each sample, which were aligned to non-rRNA sequences in the H. influenzae genome, comprising the 1,788 annotated ORFs (Supplementary Table S1). The correlation of technical replicates was very high, suggesting that variations introduced during library construction and sequencing do not significantly contribute to differences in gene expression between samples. A total of 61 genes were significantly differentially expressed by at least threefold (Padj < 1 × 10−5) during growth of H. influenzae in sBHI in the presence or absence of beclomethasone (see 4). Of the 61 genes regulated by beclomethasone, 29 genes were up-regulated and 32 genes were down-regulated (Fig 2A and B and Supplementary Table S2). Figure 2. H. influenzae genes differentially regulated during growth in complex media and during lung infection in the presence of corticosteroids A. Venn diagram showing the overlap of H. influenzae genes whose expression is up-regulated or down-regulated by the presence of corticosteroids in sBHI medium (in vitro) or in the mouse model of infection (in vivo). Genes that are divergently regulated in these conditions are not depicted in this Venn diagram but can be found in the complete RNA-Seq dataset detailed in Supplementary Table S2. B. COG map detailing significantly enriched or scarce functional categories expressed in vitro and in vivo during the presence of corticosteroids. C, D. Comparison of relative fold changes between RNA-seq and qRT–PCR results in vitro (C) and in vivo (D) in the presence of corticosteroids. All qRT–PCR results were normalized using the Cts obtained for the 16S rRNA amplifications run in the same plate. The relative levels of gene transcripts are determined from standard curves. Values given are the mean and standard deviation of triplicate measurements (three biological and three technical replicates). Download figure Download PowerPoint Those genes whose expression was increased by beclomethasone treatment included those with protein products involved in adaptive responses such as iron uptake (HI0994), biofilm formation (HI1344), stress response (HI0628) and antimicrobial resistance (HI1275) as well as the yfe operon (HI0359–HI0362), which encodes an ABC transporter putatively involved in metal ion uptake and previously implicated in H. influenzae virulence regulation, adherence to host cells and host immune evasion (Whitby et al, 2006; Jalalvand et al, 2013; Su et al, 2013). The effect of beclomethasone on the level of transcript was validated by quantitative reverse-transcription polymerase chain reaction (qRT–PCR). The genes selected for these analyses represented those from diverse functional classes with some previously implicated in virulence and biofilm regulation in H. influenzae with a fold change of three or higher (Fig 2C). Importantly, this subset of those genes that showed a response to beclomethasone showed a similar alteration in expression when H. influenzae was cultured in the presence of prednisolone, another glucocorticosteroid taken by asthmatics (Supplementary Figs S3 and S4). In contrast, many of these genes showed no alteration in expression in response to dehydroepiandrosterone, an unrelated steroid that is found abundantly in human tissue (Supplementary Fig S3). Genes showing specific changes in expression during H. influenzae infection include a subset that is influenced by glucocorticosteroids in culture The effects of glucocorticosteroids on gene expression of H. influenzae in culture raised the issue of whether similar alterations in gene expression occur in the mouse model treated with glucocorticosteroids. As with other opportunistic pathogens, H. influenzae modulates its gene expression upon colonization of its mammalian host. To gain understanding of this adaptation of H. influenzae to the host environment (in the presence of beclomethasone treatment), we systematically cataloged the transcriptomes of bacteria grown in vivo, using high-throughput cDNA sequencing techniques. RNA was derived from organisms isolated directly from the lung homogenates of infected mice and, following rRNA depletion, the remaining RNA was fragmented, reverse-transcribed into cDNA and sequenced. Illumina-based RNA-seq was used for the characterization of bacteria within the lung tissue, which reach densities of 105 CFU/ml. We chose to profile the infection 3 days post-inoculation because this represents the midpoint of the trajectory of the infection. A total of 35–40 million reads were derived from murine samples and were aligned to non-rRNA sequences in the H. influenzae genome (Supplementary Table S1). To identify genes that are differentially expressed during murine infection and in sBHI medium with or without glucocorticosteroid, we compared the RNA-seq data for these conditions using DESeq, a differential expression analysis package for RNA-seq data that presumes that abundance of reads can be modeled by a negative binomial distribution (An et al, 2013). These analyses revealed that expression of 129 (7.2%) of the 1,788 annotated H. influenzae ORFs was significantly altered (> 3-fold, Padj < 1 × 10−5) in the mouse model of infection by comparison with bacteria grown in culture without glucocorticosteroids; expression of 58 genes (3.2%) was up-regulated, whereas expression of 71 genes (4.0%) was down-regulated (Fig 2A and B and Supplementary Table S2). Comparison of these alterations in the transcriptome with those induced by the presence of beclomethasone in culture revealed a significant overlap (Fig 2A and Supplementary Table S2). Nevertheless, a large number of genes (n = 82) whose expression is altered in bacteria in the host are not regulated by glucocorticosteroids in culture, consistent with the contention that these are responses of H. influenzae to other aspects of the host environment. While the majority of these differentially expressed genes were annotated as hypothetical proteins, several have recently been shown to encode factors required by H. influenzae for growth and/or colonization of the host. These included HI0174 (mnmA), HI0274 (gltX), HI0479 (atpD), HI0629 (mclA), HI1547 (aroG) and HI1548 (lolE) (Gawronski et al, 2009). Additional regulated genes encoded factors that have been previously reported to contribute to H. influenzae virulence: HI1544 which encodes a NAD(P)H oxidoreductase and HI1707 which encodes the sensor kinase, FirS (Steele et al, 2012). To assess the reliability of RNA-seq data in determining the relative abundances of individual transcripts in the presence of beclomethasone, we used the same total RNA samples determined by qRT–PCR: the mRNA levels of five up-regulated genes (HI0075, HI0095, HI0628, HI1275 and HI1677) and four down-regulated genes (HI0127, HI0811, HI1177 and HI1431) (Fig 2D). The ratios of the transcripts from samples determined by RNA-seq to those obtained by qRT–PCR were in concordance. Transcript levels of glucocorticosteroid-responsive H. influenzae genes in sputum from asthma patients The above findings suggest that glucocorticosteroids may influence the expression of specific H. influenzae genes in asthma patients. In order to test this hypothesis, the transcript level of three corticosteroid-responsive genes (HI0359, HI0628 and HI0994) was established by qRT–PCR on sputum samples collected from twenty-four asthma patients. A comparison of the demographics and asthma characteristics of these patients are shown in Table 1. There were no significant differences in gender, smoking history (defined as % current and past smoker), obesity (defined as BMI ≥ 30 kg/m2), nasal disease, atopy and serum IgE. All patients were taking standard inhaled glucocorticosteroid therapy using beclomethasone or mometasone routinely. Table 1. Summary of bacteria identified by culturing and the detection of bacterial gene transcription in the sputum from asthma patients Sample no. Age (y) and sex FEV1% Inhaled steroid treatmenta Detected bacterial strainsb mRNA levelc HI0359 HI0628 HI0994 S1 27 (M) 26 Bec Hi, Sp 24.9 (0.4) 4.4 (0.9) 7.1 (0.2) S2 26 (F) 32 Bec Hi, Pa, Sa, MRSA, Ko, Sm 18.9 (0.4) 4.3 (0.6) 6.9 (0.6) S3 26 (F) 29 Bec Hi, Pa, Sa, Ko, Sm 12.8 (0.1) 4.4 (0.2) 6.7 (0.3) S5 24 (M) 33 Bec Hi, Sm, Mc 9.3 (0.9) 4.6 (0.8) 6.4 (0.2) S8 22 (F) 23 Bec Hi, Pa, Sa, Ko, Sm 8.6 (0.2) 7.8 (0.2) 5.2 (0.2) S9 26 (M) 24 Bec Hi, Pa, Sa, Ko, Sm 8.5 (0.1) 4.7 (0.8) 5.1 (0.1) S10 25 (M) 33 Bec Hi, Pa, Sm, Mc 8.2 (0.3) 5.1 (0.4) 5.0 (0.7) S12 26 (F) 31 Bec Hi, Pa, Sa, Ko, Sm 7.2 (0.3) 4.2 (0.6) 4.7 (0.4) S13 26 (F) 30 Bec Hi, Pa, Sa, MRSA, Ko, Sm 6.3 (0.1) 4.0 (0.6) 4.2 (0.2) S14 26 (F) 27 Bec Hi, Pa, Sa, Ko, Sm, Sp 6.2 (0.3) 3.0 (0.7) 4.1 (0.5) S15 25 (F) 26 Bec Hi, Ko, Sm 6.0 (0.2) 3.3 (0.4) 3.9 (0.8) S16 23 (M) 29 Bec Hi, Pa, Sa, Ko, Sm 5.2 (0.3) 2.9 (0.7) 3.5 (0.6) S18 25 (M) 32 Bec Hi, Pa, Sa, Ko, Sm 4.7 (0.7) 2.5 (0.6) 3.4 (0.8) S22 26 (M) 26 Bec Hi, Pa, Sa, Ko, Sm 4.5 (0.9) 2.3 (0.6) 3.4 (0.7) S24 27 (M) 28 Bec Hi, Pa, Sa, Ko, Sm 3.9 (0.4) 2.2 (0.4) 3.3 (0.9) S6 23 (M) 30 Mom Hi, Pa, Sa, Ko, Sm 3.7 (0.8) 2.9 (0.1) 2.9 (0.3) S7 22 (M) 27 Mom Hi, Ko, Sm, Sp 2.5 (0.2) 2.3 (0.6) 2.6 (0.2) S17 26 (F) 31 Mom Hi, Ko, Sm 2.5 (0.1) 2.8 (0.2) 2.5 (0.2) S19 26 (F) 30 Mom Hi, Pa, Sa, Ko, Sm 2.5 (0.5) 2.7 (0.5) 2.2 (0.6) S23 25 (F) 31 Mom Hi, Ko, Sm 2.4 (0.6) 2.1 (0.5) 2.0 (0.9) S11 26 (M) 30 Bec Pa, Sa, Ko, Sm N/A N/A N/A S20 27 (F) 30 Bec Pa, Sa, Ko, Sm N/A 3.1 (0.5) N/A S21 26 (F) 29 Mom Pa, Sa, Ko, Sm 2.0 (0.4) N/A N/A S4 26 (M) 30 N/A Pa, Sa, MRSA, Ko, Sm N/A N/A N/A a Current steroid treatment, Bec (beclomethasone) or Mom (mometasone). b Abbreviations of microbes identified by culture-based methods: Hi (H. influenzae), Pa (Pseudomonas aeruginosa), Sa (Staphylococcus aureus), Ko (Klebsiella oxytoca), Sm (Stenotrophomonas maltophilia), MRSA (methicillin-resistant S. aureus), Sp (Streptococcus pneumoniae) and Mc (Moraxella catarrhalis). c Fold change relative to 16S RNA gene expression (standard deviation). Inhaled beclomethasone is prescribed much more frequently (in 90% of cases) than mometasone, probably because it is effective but of lower cost
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