Membrane-bound nitrate reductase is required for anaerobic growth in cystic fibrosis sputum

The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.     

The microorganisms in chronically infected end-stage and non-end-stage cystic fibrosis patients

Patients suffering from cystic fibrosis (CF) develop chronic lung infections because of highly viscous mucus, where bacteria can form biofilms. In this study, we investigated the microorganisms present in the lungs of end-stage and non-end-stage patients using standard culturing techniques and molecular methods. Tissue and sputum samples (n?=?34) from explanted lungs of five end-stage patients were examined along with routine expectorates (n?=?15) from 13 patients with non-end-stage CF, representing earlier stages of chronic lung infections. Previously, using peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH), we have shown that Pseudomonas aeruginosa was the sole pathogen in end-stage CF lungs (Pediatr Pulmonol 2009, 44: 547). In this study, this tendency was supported by the results of real-time PCR, confirming previous results obtained by standard culturing and 16S rRNA gene analysis (J Clin Microbiol 2011, 49: 4352). Conversely, the non-end-stage patients were found to harbor several species by culturing. PNA FISH confirmed heterogeneous microbiota and showed that the bacteria were located in monospecies aggregates with no apparent physical interaction between the different microcolonies. In conclusion, standard culturing identifies the dominating pathogens, which seem to reside in monospecies microcolonies. The possibility of signaling between the distinct microcolonies still has to be verified and elucidated.     

Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.     

Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients

The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gram-negatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.     

Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum

The sputum (mucus) layer of the cystic fibrosis (CF) lung is a complex substrate that provides Pseudomonas aeruginosa with carbon and energy to support high-density growth during chronic colonization. Unfortunately, the CF lung sputum layer has been difficult to mimic in animal models of CF disease, and mechanistic studies of P. aeruginosa physiology during growth in CF sputum are hampered by its complexity. In this study, we performed chromatographic and enzymatic analyses of CF sputum to develop a defined, synthetic CF sputum medium (SCFM) that mimics the nutritional composition of CF sputum. Importantly, P. aeruginosa displays similar phenotypes during growth in CF sputum and in SCFM, including similar growth rates, gene expression profiles, carbon substrate preferences, and cell-cell signaling profiles. Using SCFM, we provide evidence that aromatic amino acids serve as nutritional cues that influence cell-cell signaling and antimicrobial activity of P. aeruginosa during growth in CF sputum.     

Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens

The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities≥10(2) rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.     

Multiple roles of TorD-like chaperones in the biogenesis of molybdoenzymes

Studies on cultured cells and in infection models have shown that cell density-dependent quorum-sensing (QS) controls many of the known virulence factors of Pseudomonas aeruginosa . However, it is less clear what role QS plays in chronic human lung infections associated with cystic fibrosis (CF). The involvement of QS in biofilm development, crucial to the establishment of long-term infections, suggests a role in the early stages of infection. However, the accumulation of QS mutants during chronic CF infections has been taken to indicate that any role diminishes thereafter. Here, we discuss the evidence for a continuing role for QS in P. aeruginosa CF infections, including QS activity in CF sputa and CF-relevant effects of QS-regulated products, such as pyocyanin. Bacterial population behaviour in CF is complex, and the exact roles of QS remains unclear. Therapeutic strategies directed against QS suggest that a greater understanding of bacterial populations during infection would be a valuable research goal from a clinical perspective.     

Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.     

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.     

Evolution and adaptation in Pseudomonas aeruginosa biofilms driven by mismatch repair system-deficient mutators

Pseudomonas aeruginosa is an important opportunistic pathogen causing chronic airway infections, especially in cystic fibrosis (CF) patients. The majority of the CF patients acquire P. aeruginosa during early childhood, and most of them develop chronic infections resulting in severe lung disease, which are rarely eradicated despite intensive antibiotic therapy. Current knowledge indicates that three major adaptive strategies, biofilm development, phenotypic diversification, and mutator phenotypes [driven by a defective mismatch repair system (MRS)], play important roles in P. aeruginosa chronic infections, but the relationship between these strategies is still poorly understood. We have used the flow-cell biofilm model system to investigate the impact of the mutS associated mutator phenotype on development, dynamics, diversification and adaptation of P. aeruginosa biofilms. Through competition experiments we demonstrate for the first time that P. aeruginosa MRS-deficient mutators had enhanced adaptability over wild-type strains when grown in structured biofilms but not as planktonic cells. This advantage was associated with enhanced micro-colony development and increased rates of phenotypic diversification, evidenced by biofilm architecture features and by a wider range and proportion of morphotypic colony variants, respectively. Additionally, morphotypic variants generated in mutator biofilms showed increased competitiveness, providing further evidence for mutator-driven adaptive evolution in the biofilm mode of growth. This work helps to understand the basis for the specific high proportion and role of mutators in chronic infections, where P. aeruginosa develops in biofilm communities.     

Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.     

Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.     

Molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients

Background

There is strong evidence that culture-based methods detect only a small proportion of bacteria present in the respiratory tracts of cystic fibrosis (CF) patients.

Methodology/Principal Findings

Standard microbiological culture and phenotypic identification of bacteria in sputa from CF patients have been compared to molecular methods by the use of 16S rDNA amplification, cloning and sequencing. Twenty-five sputa from CF patients were cultured that yield 33 isolates (13 species) known to be pathogens during CF. For molecular cloning, 760 clones were sequenced (7.2±3.9 species/sputum), and 53 different bacterial species were identified including 16 species of anaerobes (30%). Discrepancies between culture and molecular data were numerous and demonstrate that accurate identification remains challenging. New or emerging bacteria not or rarely reported in CF patients were detected including Dolosigranulum pigrum, Dialister pneumosintes, and Inquilinus limosus.

Conclusions/Significance

Our results demonstrate the complex microbial community in sputa from CF patients, especially anaerobic bacteria that are probably an underestimated cause of CF lung pathology. Metagenomic analysis is urgently needed to better understand those complex communities in CF pulmonary infections.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.     

Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis

In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.     

IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice

Patients with cystic fibrosis (CF) develop chronic Pseudomonas aeruginosa lung infection with mucoid strains of P. aeruginosa; these infections cause significant morbidity. The immunological response in these infections is characterized by an influx of neutrophils to the lung and subsequent lung damage over time; however, the underlying mediators to this response are not well understood. We recently reported that IL-23 and IL-17 were elevated in the sputum of patients with CF who were actively infected with P. aeruginosa; however, the importance of IL-23 and IL-17 in mediating this inflammation was unclear. To understand the role that IL-23 plays in initiating airway inflammation in response to P. aeruginosa, IL-23p19(-/-) (IL-23 deficient) and wild-type (WT) mice were challenged with agarose beads containing a clinical, mucoid isolate of P. aeruginosa. Levels of proinflammatory cytokines, chemokines, bacterial dissemination, and inflammatory infiltrates were measured. IL-23-deficient mice had significantly lower induction of IL-17, keratinocyte-derived chemokine (KC), and IL-6, decreased bronchoalveolar lavage (BAL) neutrophils, metalloproteinase-9 (MMP-9), and reduced airway inflammation than WT mice. Despite the reduced level of inflammation in IL-23p19(-/-) mice, there were no differences in the induction of TNF and interferon-gamma or in bacterial dissemination between the two groups. This study demonstrates that IL-23 plays a critical role in generating airway inflammation observed in mucoid P. aeruginosa infection and suggests that IL-23 could be a potential target for immunotherapy to treat airway inflammation in CF.     

RISA-HPLC analysis of lung bacterial colonizers of cystic fibrosis children

Microbiological analysis of sputum samples, from children affected by cystic fibrosis (CF) and showing signs of acute or chronic infections, is routinely performed by culture-dependent approaches involving selective media and biochemical tests. These identification schemes are time-consuming, and may lead to false negative results. The aim of this work was to evaluate the efficacy of a Ribosomal Intergenic Spacer Analysis (RISA) coupled to high performance liquid chromatography (HPLC) for the detection and monitoring of CF lung microbial colonizers including Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, the Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans. These RISA-HPLC analyses were performed over a 10-months period on infants (below 18 months) and children that were or were not yet known to be colonised by P. aeruginosa. The RISA-HPLC profiles were found specific of the patients' microbial communities. A specific P. aeruginosa RISA-HPLC peak corresponding to 550 bp PCR products was recorded, and used to investigate P. aeruginosa persistence through time and after various therapeutic treatments. The RISA-HPLC profiles showed the CF children to be colonized by few bacterial species, and sometimes revealed peaks corresponding to bacterial species that were not detected by the selective plating approaches. Significant RISA-HPLC infra-specific variations were observed for most bacterial colonizers of CF lungs except P. aeruginosa. These species could yield as much as 5 distinct RISA-HPLC peaks, with some of these profiles being strain-specific. RISA-HPLC shows a great potential for revealing colonization by novel emerging pathogens, and for evaluating the efficacy of therapeutic treatments on the global bacterial community of CF lungs.