Infections In Cystic Fibrosis Patients Research Articles

Hypermutator Pseudomonas aeruginosa Exploits Multiple Genetic Pathways To Develop Multidrug Resistance during Long-Term Infections in the Airways of Cystic Fibrosis Patients.

Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI, encoding the β-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying β-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.

Antibacterial and Antivirulence Activity of Glucocorticoid PYED-1 against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia, an environmental Gram-negative bacterium, is an emerging nosocomial opportunistic pathogen that causes life-threatening infections in immunocompromised patients and chronic pulmonary infections in cystic fibrosis patients. Due to increasing resistance to multiple classes of antibiotics, S. maltophilia infections are difficult to treat successfully. This makes the search for new antimicrobial strategies mandatory. In this study, the antibacterial activity of the heterocyclic corticosteroid deflazacort and several of its synthetic precursors was tested against S. maltophilia. All compounds were not active against standard strain S. maltophilia K279a. The compound PYED-1 (pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1) showed a weak effect against some S. maltophilia clinical isolates, but exhibited a synergistic effect with aminoglycosides. PYED-1 at sub-inhibitory concentrations decreased S. maltophilia biofilm formation. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of biofilm- and virulence- associated genes (StmPr1, StmPr3, sphB, smeZ, bfmA, fsnR) was significantly suppressed after PYED-1 treatment. Interestingly, PYED-1 also repressed the expression of the genes aph (3′)-IIc, aac (6′)-Iz, and smeZ, involved in the resistance to aminoglycosides.

The origin of extracellular DNA in bacterial biofilm infections in vivo.

ABSTRACTExtracellular DNA (eDNA) plays an important role in both the aggregation of bacteria and in the interaction of the resulting biofilms with polymorphonuclear leukocytes (PMNs) during an inflammatory response. Here, transmission electron and confocal scanning laser microscopy were used to examine the interaction between biofilms of Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of PMN DNA with a thymidine analogue and immunohistochemistry were applied to localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), neutrophil elastase (NE) and citrullinated H3 (citH3). Host-derived eDNA was observed surrounding bacterial biofilms but not within the biofilms. H3 localized to the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for neutrophil extracellular traps (NETs) was detected only sporadically indicating that most host-derived eDNA in vivo was not a result of NETosis. Together these observations show that, in these in vivo biofilm infections with P. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host.

Pseudomonas aeruginosa antibody response in cystic fibrosis decreases rapidly following lung transplantation

BackgroundSpecific Pseudomonas aeruginosa (PA) precipitating immunoglobulin G antibodies in serum are correlated with PA biofilm infection and are used as diagnostic and prognostic markers in cystic fibrosis (CF). The aim of this study was to examine the change of PA antibody response in CF patients after bilateral sequential lung transplantation (LTx). MethodsPA antibodies and airway bacteriology were retrospectively evaluated in 20 chronically infected CF patients, who underwent LTx between 2001 and 2016 at Rigshospitalet, Copenhagen. Yearly precipitin counts from one year before LTx and up to five years after LTx were compared. Monthly airway cultures were examined in the five-year period after LTx. In addition, crossed immunoelectrophoresis (CIE) were analysed for each patient for antigenic similarities from time of infection, pre-LTx and post-LTx. ResultsAll patients experienced a significant drop in PA antibodies from one year pre-LTx to one year post-LTx (p < 0.0001). The PA antibody level did not differ between those, who became reinfected immediately after LTx, and those, who did not. No patients regained the high pre-LTx precipitin levels in the following five years. The antigenic specificities of the sera post-LTx were in each patient similar to the antigenic specificities at the beginning of infection indicating a decades long memory of their immune response like an “immunological fingerprint”. ConclusionsAfter LTx a significant and continuous reduction in PA antibodies was observed. The reduction was independent of immediate reinfection after LTx. A novel three-factor explanatory model is presented.

Antibody response against Pseudomonas aeruginosa and its relationship with immune mediators in the upper and lower airways of cystic fibrosis patients.

The upper airways (UAW) are a niche and a reservoir of Pseudomonas aeruginosa strains that cause chronic infection of the lower airways (LAW) in cystic fibrosis (CF). Here, we assessed the role of anti-P. aeruginosa immunoglobulin A (IgA) and IgG antibodies in upper and lower airway infections in cystic fibrosis patients. Nasal lavage fluid and induced sputum samples of 40 CF patients were microbiologically cultured. We searched for correlations between anti-P. aeruginosa IgA and IgG levels, measured by enzyme-linked immunosorbent assay (optical density), and unspecific immune mediators in both specimens. Anti-P. aeruginosa IgA (median optical density: 0.953 vs 0.298) and IgG (0.120 vs 0.059) were significantly higher in nasal lavage than in sputum, but not significantly different between patients with and without chronic P. aeruginosa infection in UAW. Matrix metallopeptidase-9 (MMP-9) in nasal lavage and neutrophil elastase (NE) in sputum were predictors of IgA in nasal lavage and IgA in sputum, respectively. IgA was a predictor of myeloperoxidase (MPO) in nasal lavage. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was a predictor of IgG in sputum. IgG, TIMP-1, and NE in sputum were predictors of IgG in nasal lavage. The anti-P. aeruginosa IgA response was more prominent in CF patients' UAW, indicating a lower degree of inflammatory responses. Proteases may play a role in the anti-P. aeruginosa humoral response in the upper and LAW, and anti-P. aeruginosa IgG may be involved in the crosstalk between upper and lower airways in cystic fibrosis patients.

Pseudomonas aeruginosa Modulates the Antiviral Response of Bronchial Epithelial Cells

Cystic fibrosis (CF) patients frequently acquire Pseudomonas aeruginosa infections that have been associated with a bad prognosis and an increased rate of pulmonary exacerbations. Respiratory viruses can cause exacerbations in chronic pulmonary diseases including COPD or asthma and have been suggested to contribute to exacerbations also in CF. In this study we investigated a possible link between P. aeruginosa infection and susceptibility to respiratory viruses. We show that P. aeruginosa is able to block the antiviral response of airway epithelial cells thereby promoting virus infection and spread. Mechanistically, P. aeruginosa secretes the protease AprA in a LasR dependent manner, which is able of directly degrading epithelial-derived IFNλ resulting in inhibition of IFN signaling. In addition, we correlate the virus infection status of CF patients with the ability of patients' P. aeruginosa isolates to degrade IFNλ. In line with this, the infection status of CF patients correlated significantly with the amount of respiratory viruses in sputum. Our data suggest that the interplay between P. aeruginosa and respiratory virus infections might partially explain the association of increased rates of pulmonary exacerbations and P. aeruginosa infections in CF patients.

Discovery of Ubonodin, an Antimicrobial Lasso Peptide Active against Members of the Burkholderia cepacia Complex.

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.

Imaging of the bacterial interactions in lung co-infection in cystic fibrosis patients

Objective. To identify bacterial interactions at the site of infection in cystic fibrosis patients and to assess their possible effects on the course of infection. Materials and Methods. The following strains were used in this study: Alcaligenes faecalis LGBP strain, isolated from the environment; clinical isolates of Pseudomonas aeruginosa; Achromobacter xylosoxidans, Acinetobacter baumannii, Alcaligenes faecalis, and Bacillus subtilis strains; the standard laboratory P. aeruginosa PAO1 strain and its lysogens by temperate bacteriophages of various species, and its phageresistant mutants. Imaging and evaluation of the effects of bacterial interaction was performed in an in vitro co-infection with A. faecalis LGBP and the tested strains. Results. The bacteria of A. faecalis which are often involved in the lung co-infection in cystic fibrosis have been shown to stimulate the growth of most of the tested P. aeruginosa strains, as well as bacteria of some other species (for example, B. subtilis). The interspecies interactions pattern depends primarily on the strain of A. faecalis and physiological features of the infecting P. aeruginosa strains. When growing concurrently, the contacts between bacteria may change both the physical properties of the contacting bacteria surface (propagation rate) and the course of biochemical reactions in the contacting bacteria (occurrence of pigmentation, change in auto-plaquing pattern, reduction in alginate production). Conclusions. The results suggest that visually recognizable interactions are similar to the interactions of A. faecalis LGBP, exhibited in vitro with clinical isolates of P. aeruginosa, may influence on the course of chronic infections and their treatment results. Expanding of model studies of bacterial interspecies interactions may contribute to better understanding of their molecular mechanism that may be useful for optimizing therapy.

IV antibiotics no more effective than oral for eradicating infection in cystic fibrosis patients, study suggests

IV antibiotics no more effective than oral for eradicating infection in cystic fibrosis patients, study suggests

Bacterial pulmonary colonization with Pseudomonas aeruginosa in cystic fibrosis patients

Introduction : Cystic fibrosis (CF) is the most common life-limiting autosomal recessive condition in Caucasians. Lung damage is the main determinant of morbidity and mortality. The most important factor for chronic lung infection is the colonization with Pseudomonas aeruginosa . Aim : Understanding the nature of the infection with Pseudomonas aeruginosa and its factors of virulence and compiling statistics on age-related bacterial pulmonary colonization in patients with cystic fibrosis in Expert Center-Varna. Methods : Review of the medical literature and microbiological examination (sputum specimen, oropharyngeal swabs, BAL) of patients with cystic fibrosis at the Expert Center-Varna. Results : With increasing age of the patient with cystic fibrosis there has also been an increase in the rate of pulmonary colonization with Pseudomonas aeruginosa - up to 85% in patients over 18 years of age. Conclusion : Pseudomonas aeruginosa is largely responsible for morbidity and mortality in chronically infected cystic fibrosis patients. Therefore, prevention and early diagnosis are vital to the prognosis of these patients.

Multidrug-Resistant and Clinically Relevant Gram-Negative Bacteria Are Present in German Surface Waters.

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored blaVIM–1. Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.

Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity.

Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds, and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP-expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP-expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both nonself P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.IMPORTANCE Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight into how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing.

Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.

Tedizolid is a promising antimicrobial option for the treatment of Staphylococcus aureus infections in cystic fibrosis patients.

Tedizolid is a protein synthesis inhibitor in clinical use for the treatment of Gram-positive infections. Pulmonary MRSA infections are a growing problem in patients with cystic fibrosis (CF) and the efficacy of tedizolid-based therapy in CF pulmonary infections is unknown. To evaluate the in vitro and in vivo activity of tedizolid and predict the likelihood of tedizolid resistance selection in CF-background Staphylococcus aureus strains. A collection of 330 S. aureus strains (from adult and paediatric patients), either of normal or small colony variant (SCV) phenotypes, gathered at three CF centres in the USA was used. Tedizolid activity was assessed by broth microdilution, Etest and time-kill analysis. In vivo tedizolid efficacy was tested in a murine pneumonia model. Tedizolid in vitro mutants were obtained by 40 days of exposure and progressive passages. Whole genome sequencing of clinical S. aureus strains with reduced susceptibility to tedizolid was performed. MRSA strain MIC90s were tedizolid 0.12-0.25 mg/L and linezolid 1-2 mg/L; for MSSA strains, MIC90s were tedizolid 0.12 mg/L and linezolid 1-2 mg/L. Two strains, WIS 441 and Seattle 106, with tedizolid MICs of 2 mg/L and 1 mg/L, respectively, had MICs above the FDA tedizolid breakpoint (0.5 mg/L). Tedizolid at free serum concentrations exhibited a bacteriostatic effect. Mean bacterial burdens in lungs (log10 cfu/g) for WIS 423-infected mice were: control, 11.2±0.5; tedizolid-treated (10 mg/kg), 3.40±1.87; linezolid-treated (40 mg/kg), 4.51±2.1; and vancomycin-treated (30 mg/kg), 5.21±1.93. For WIS 441-infected mice the (log10 cfu/g) values were: control, 9.66±0.8; tedizolid-treated, 3.18±1.35; linezolid-treated 5.94±2.19; and vancomycin-treated, 4.35±1.7. These results suggest that tedizolid represents a promising therapeutic option for the treatment of CF-associated MRSA/MSSA infections, having potent in vivo activity and low resistance potential.

Adapting to the Airways: Metabolic Requirements of Pseudomonas aeruginosa during the Infection of Cystic Fibrosis Patients.

Pseudomonas aeruginosa is one of the major causes of morbidity and mortality of cystic fibrosis patients. During the infection, the bacteria colonize the nutritional rich lung mucus, which is present in the airway secretions in the patients, and they adapt their phenotype accordingly to the lung environment. In the airways, P. aeruginosa undergoes a broad metabolic rewiring as a consequence of the nutritional and stressful complexity of the lungs. However, the role of such metabolic rewiring on the infection outcome is poorly understood. Here, we review the metabolic evolution of clinical strains of P. aeruginosa during a cystic fibrosis lung infection and the metabolic functions operating in vivo under patho-physiological conditions. Finally, we discuss the perspective of modeling the cystic fibrosis environment using genome scale metabolic models of P. aeruginosa. Understanding the physiological changes occurring during the infection may pave the way to a more effective treatment for P. aeruginosa lung infections.

Evaluation of Fraction of Exhaled Nitric Oxide (FeNO) in CF Children and its Association with Sputum Culture

Background: Exhaled nitric oxide (FeNO) in cystic fibrosis (CF) patients is reduced when compared with healthy people, and it has now been found that FeNO has a relative association with airway clearance index. Studies have shown the role of some infections in changing the FeNO level; however, the role of respiratory infections in FeNO has not yet been thoroughly studied. Objectives: The objective of this study was to investigate the possibility of FeNO usage to monitor the infections in CF patients. Methods: This cross-sectional case-control study was conducted on CF patients between the age of five to 18 with positive sputum culture, through simple non-random census method. FeNO levels were measured in 30 healthy children and 30 CF children with positive sputum culture. CF patients were treated by antibiotic therapy for two weeks; in the case offending negative sputum culture, the FeNO level was re-measured. FeNO levels were re-evaluated in 13 patients after four weeks. Results: There was no statistical difference between both groups in terms of age and weight. The level of FeNO in CF patients was significantly lower than in healthy children (22.1 ± 10.1 versus 30.0 ± 11.0 in the control group and P = 0.003). In 27 children, two weeks after administration of antibiotic therapy, sputum culture was negative. The mean of FeNO in these patients was 16.4 ± 5.5 at the time of the negative sputum culture, which was significantly lower than FeNO before starting the treatment. (P: 0.003). The mean FeNO was 13.0 ± 7.41 in 13 patients who were re-measured four weeks after starting the treatment. There was no significant difference between FeNO level two weeks after treatment and four weeks after starting treatment (P: 0.292). Patients with pseudomona sputum culture were not significantly different from those with non-pseudomona sputum culture in terms of primary FeNO and FeNO changes after the treatment (P value: 0.084 and 0.094, respectively). Conclusions: However, in our study, FeNO was decreased after administration of antibiotic treatment in CF patients, but according to the sample size and conflicting or similar results in other studies, currently, FeNO levels cannot be used as a way of monitoring the treatment of infection in CF patients.

The prevalence of viral infections in children with cystic fibrosis in a tertiary care center in Saudi Arabia.

IntroductionStudies have shown that pulmonary exacerbations in cystic fibrosis (CF) patients are associated with respiratory viruses. The most common agent causing viral infections in patients with CF before the age of 3 years is respiratory syncytial virus.ObjectivesTo obtain the prevalence of the different types of viral infection in CF patients and to identify its relation with the type of bacterial infection, (CFTR) mutations and pulmonary function test (PFT).MethodologyA retrospective charts review of 387 patients with CF of all age groups who were screened for the detection of viruses during respiratory exacerbation from the period of January 1, 1984 to June 1, 2016.ResultsA total of 159 CF patients had pulmonary exacerbation and had viral PCR obtained. Fifty-eight patients (36%) had positive viral PCR. Males were more commonly infected in 30/58 patients (52%) compared to females in 28 patients (48%). Forty-five of 58 patients (78%) were alive and 13 patients (22%) died. Rhinovirus was the most frequently reported viral PCR in 33/74 sample (45%). Out of 74 viral PCR, 41 (55.4%) were during the colder seasons (October–February) and 33 (44.5%) during the warmer seasons (March–September). During viral infection and viral recurrence, there was an increase in bacterial colonization specifically of H. influenza and staphylococcus aureus. The most common CFTR mutation for the CF viral infection is: 3120+1G>A in Intron 16 in 11/57 patients (19%). The Eastern Province had the highest viral infection of 24 out of 57 patients (42%). Follow-up PFT post viral infection showed no significant difference in the type and the severity of PFT compared to the initial PFT during the viral illness.ConclusionViral infections contributed to the increase in morbidity and mortality of CF patients in our population, and rhinovirus was the most common causative agent. Viral infections and viral recurrence increased the prevalence of bacterial infection of specific pathogens such as H. influenza and S. aureus. Physicians should be aware to prevent progressive lung damage in CF patients by treating the concomitant viral and bacterial infections. Viral infection may be associated with some common CFTR mutations.

Isolation and identification of Pandoraea\xa0spp. From bronchoalveolar lavage of cystic fibrosis patients in Iran

BackgroundPandoraea species are gram negative, motile, non-spore forming, rod shaped and oxidase positive, obligate aerobes bacteria, and have one polar flagellum. Most of Pandoraea species are associated with lung infections in cystic fibrosis patients. Cystic fibrosis is the most prevalent autosomal recessive hereditary disease in the world that affects various organs of the body. The main important cause of death in these patients is lung involvement. This study was conducted to isolate and identify Pandoraea bacterium from bronchoalveolar lavage and sputum samples of cystic fibrosis patients in Shiraz, Iran.MethodsIn this research 31 samples of bronchoalveolar lavage and sputum were examined by culture and PCR method. Then confirmed isolates were evaluated for susceptibility to different antibiotics and ability to produce biofilm.ResultsThe results of this study after cultivation, purification and DNA extraction led to the isolation of 4 Pandoraea bacterium by PCR using specific primers. Antibiotic susceptibility test were indicated all isolates were resistant to gentamicin, amikacin and imipenem and susceptible to ciprofloxacin, trimethoprim-sulfumethoxazole, piperacillin and tetracycline. Ability to create biofilm was indicated by some of Pandoraea isolates. According to findings of this study, ability to synthesis biofilm by Pandoraea isolates and resistance to some antibiotics are very important.ConclusionsOur study notes the role of P. pnomenusa as an emerging pathogen that can cause chronic lung colonization in CF patients. Identification tools need to be accurate and must be based on molecular techniques. Also our findings should raise awareness about antibiotic resistance in cystic fibrosis patients in Iran and ability of including bacterial agents to produce biofilm is an alarm for public health. Thus clinicians should exercise caution about finding of clinical relevance of this pathogen to the infection and prescribing antibiotics, especially in cases of children infections.

Feedback of airborne bacterial consortia to haze pollution with different PM2.5 levels in typical mountainous terrain of Jinan, China

Polluted air is as harmful as polluted water sources to public health. As air living organisms, the research on microbial consortia under haze stress with different PM2.5 levels in a mountainous environment remains very limited. This study investigated the dynamic changes in bacterial cell counts, apoptosis, human pathogens, consortia characteristics, metabolic pathways, and the biochemical functions under haze conditions with various degrees of pollution (leading pollutant PM2.5) from August to December 2017 in a typical mountainous terrain of Jinan, China. Samples were evaluated with flow cytometry and 16S rRNA gene amplicon sequencing. Results indicated that cell counts ranged from 6.83 × 105 ± 1.27 × 104 (non-polluted air, NP) to 2.32 × 106 ± 3.56 × 104 (heavily polluted air, HP) cell m−3 air. The proportion of viable apoptotic and necrotic cells were positively correlated to PM2.5. Burkholderia cenocepacia (36.6%) was the most abundant human pathogen found in HP; this gram-negative bacterium is associated with potentially lethal respiratory infections in cystic fibrosis patients. The relative abundance of the phylum Proteobacteria (63.8%) in NP first decreased in lightly polluted (LP) (41.3%) and moderately polluted air (MP) (26.3%) then increased in HP (81.0%). Cupriavidus (22.9%) and BTEX-degrading bacteria (0.6%, Pseudomonas) were found in HP. Metabolic pathways with significant differences included cell motility and endocrine and immune diseases that exhibited increasing relative abundance as pollution levels increased. The diversity of biochemical functions was found to be decreased in hazy air.

In silico Analysis Reveals Distribution of Quorum Sensing Genes and Consistent Presence of LuxR Solos in the Pandoraea Species

The most common quorum sensing (QS) system in Gram-negative bacteria consists of signaling molecules called N-acyl-homoserine lactones (AHLs), which are synthesized by an enzyme AHL synthase (LuxI) and detected by a transcriptional regulator (LuxR) that are usually located in close proximity. However, many recent studies have also evidenced the presence of LuxR solos that are LuxR-related proteins in Proteobacteria that are devoid of a cognate LuxI AHL synthase. Pandoraea species are opportunistic pathogens frequently isolated from sputum specimens of cystic fibrosis (CF) patients. We have previously shown that P. pnomenusa strains possess QS activity. In this study, we examined the presence of QS activity in all type strains of Pandoraea species and acquired their complete genome sequences for holistic bioinformatics analyses of QS-related genes. Only four out of nine type strains (P. pnomenusa, P. sputorum, P. oxalativorans, and P. vervacti) showed QS activity, and C8-HSL was the only AHL detected. A total of 10 canonical luxIs with adjacent luxRs were predicted by bioinformatics from the complete genomes of aforementioned species and publicly available Pandoraea genomes. No orphan luxI was identified in any of the genomes. However, genes for two LuxR solos (LuxR2 and LuxR3 solos) were identified in all Pandoraea genomes (except two draft genomes with one LuxR solo gene), and P. thiooxydans was the only species that harbored no QS-related activity and genes. Except the canonical LuxR genes, LuxIs and LuxR solos of Pandoraea species were distantly related to the other well-characterized QS genes based on phylogenetic clustering. LuxR2 and LuxR3 solos might represent two novel evolutionary branches of LuxR system as they were found exclusively only in the genus. As a few luxR solos were located in close proximity with prophage sequence regions in the genomes, we thus postulated that these luxR solos could be transmitted into genus Pandoraea by transduction process mediated by bacteriophage. The bioinformatics approach developed in this study forms the basis for further characterization of closely related species. Overall, our findings improve the current understanding of QS in Pandoraea species, which is a potential pharmacological target in battling Pandoraea infections in CF patients.