Chronic Respiratory Infections Research Articles

Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human macrophages.

Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.

Short, Medium and Long-Term Cause-Specific Mortality Following First-Ever Heart Failure Hospitalisation in New Zealand

Heart failure (HF) is associated with high mortality, but there are limited reports on the underlying cause of death. This study reports short-, medium- and long-term cause-specific mortality following first-ever HF hospitalisation in New Zealand. First-ever HF hospitalisations were identified from hospital discharge coding between 2010 and 2013. Mortality outcomes were obtained via anonymised linkage to national datasets. Short (0-30 days), medium (31-364 days), and long-term (1-5 years) mortality rates were identified. Cause of death was identified from death certification coding and classified as cardiovascular and non-cardiovascular. Cox regression analysis was performed to adjust for confounding variables. A cohort of 34,264 individuals with first-ever HF hospitalisation were identified. Mean age was 75.8±13 years and 50.5% were male. A total of 21,637 (63.1%) died within 5 years of hospitalisation; 4,122 (12.0%) within the first 30 days, 6,358 (18.6%) between 31-364 days, and 11,157 (32.6%) between 1 and 5 years. Older age, male gender, Māori ethnicity, higher socioeconomic deprivation and increased comorbidity were independent factors associated with higher all-cause mortality. Cardiovascular causes accounted for 51% of total deaths. Cardiovascular mortality was 6.0%, 9.5%, and 16.7% at 30 days, 31-364 days, and 1-5 years, respectively. The most common causes of non-cardiovascular mortality were neoplasms, chronic respiratory diseases and infections, accounting for 14.6%, 11.0%, and 5.5% of total deaths respectively. Comorbidity was associated with higher non-cardiovascular mortality (hazard ratio [HR] 3.35; 95% confidence interval [CI] 3.16-3.55) but not cardiovascular mortality (HR 0.79; 95% CI 0.72-0.86). In New Zealand, mortality following first-ever HF hospitalisation is high. Non-cardiovascular death is common and there are ethnic inequities.

Identification of Mycoplasma pneumoniae proteins interacting with NOD2 and their role in macrophage inflammatory response.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is a cell wall-deficient microorganism known to cause chronic respiratory infections in both children and adults. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor primarily responsible for identifying muramyl dipeptide (MDP) found in bacterial cell walls. Previous experiments have demonstrated that Mycoplasma ovipneumoniae induces macrophage autophagy through NOD2. In this study, we conducted RNA-seq analysis on macrophages infected with M. pneumoniae and observed an up-regulation in the expression of genes associated with the NOD2 signaling pathway. Mechanistic investigations further revealed the involvement of the NOD2 signaling pathway in the inflammatory response of macrophages activated by M. pneumoniae. We utilized GST pull-down technology in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to pinpoint the M. pneumoniae proteins that interact with NOD2. Additionally, co-immunoprecipitation (Co-IP) and immunofluorescence co-localization techniques were used to confirm the interaction between DUF16 protein and NOD2. We found that DUF16 protein can enter macrophages and induce macrophage inflammatory response through the NOD2/RIP2/NF-κB pathway. Notably, the region spanning amino acids 13-90 was identified as a critical region necessary for DUF16-induced inflammation. This research not only broadens our comprehension of the recognition process of the intracellular receptor NOD2, but also deepens our understanding of the development of M. pneumoniae infection.

Transient Complete Asthenozoospermia

ABSTRACT Limited research exists on the mechanisms underlying asthenozoospermia associated with acquired ciliary dyskinesia. Primary ciliary dyskinesia links respiratory pathology with infertility and provides a basis for a potential mechanism. The aetiology of asthenozoospermia is often unclear and may be secondary to direct or indirect effects on sperm motility. Here, we report a case – with a brief clinical review – of recovering sperm motility after diagnosis of complete asthenozoospermia coinciding with resolution of chronic respiratory infections. The patient is a 36-year-old male, with initial semen analysis demonstrating 100% immotile sperm. Following the resolution of chronic respiratory infection, subsequent analysis demonstrated functional improvement with 76 million sperm/mL, 8% progressive motility and 4% strict morphology. Our case reinforces a potentially underappreciated role of environmental risk factors in infertility, with a focus on the patient’s history of infections and other risk factors for acquired ciliary dyskinesia, which should be kept in mind when treating patients with asthenozoospermia.

Surveillance and phylogenetic analysis of a pathogenic bacterium candidate in nasal discharge from children.

"The infectious organism lurking in human airways (IOLA)" is a candidate pathogenic bacterium detected in bronchoalveolar lavage fluid specimens derived from adult patients with chronic lower respiratory tract infections. Genomic analyses of IOLA have revealed that it possesses the smallest and most AT-rich genome among human-derived bacteria. However, its biological properties remain unclear because no culture method has been established for IOLA. Here, we conducted a large-scale IOLA surveillance study of nasal discharge specimens from children in Japan and investigated the correlation between IOLA detection frequency and patient characteristics. We detected IOLA in 5.4% (103 of 1,920) of pediatric nasal discharge samples. No significant differences were observed in the frequency of detection based on the patient's background. However, with respect to age, the frequency of detection tended to be significantly high at 2-3 and 6 years old. Phylogenetic analysis revealed five phylotypes in the IOLA 16S rRNA gene sequences, and the sequences detected in adult patients with respiratory infections in a previous study belonged to one of the five phylotypes. The involvement of IOLA in the symptoms is not clear, but IOLA is detected at a relatively high frequency in pediatric nasal discharge. Many subjects with detected IOLA were not always IOLA positive, and IOLA was detected transiently. Our findings suggest that IOLA is horizontally transmitted through groups in nursery and elementary schools, and there are differences in biological characteristics among the IOLA phylotypes.IMPORTANCE"The infectious organism lurking in human airways (IOLA)" is a candidate pathogenic bacterium strongly suspected to be infectious to the respiratory tracts of humans and animals. However, a culture method for IOLA has not been established yet, and its properties remain unclear. In this study, IOLA was detected at a relatively high frequency in the nasal discharge of children, and five phylotypes of IOLA were identified. One of these phylotypes was found in the bronchoalveolar lavage fluid from adult patients, suggesting lineage-specific differences in the pathogenicity of IOLA. Moreover, it was suggested that IOLA is horizontally transmitted when children gather in groups such as nursery and elementary schools. These findings strongly indicate that IOLAs have been clinically undetected so far but are spreading among children, with one lineage being involved in respiratory diseases in adults. Examining the presence of IOLA in clinical specimens may help to understand the etiology of respiratory diseases with unknown causes.

Phage Therapy for Respiratory Infections: Opportunities and Challenges.

We are entering the post-antibiotic era. Antimicrobial resistance (AMR) is a critical problem in chronic lung infections resulting in progressive respiratory failure and increased mortality. In the absence of emerging novel antibiotics to counter AMR infections, bacteriophages (phages), viruses that infect bacteria, have become a promising option for chronic respiratory infections. However, while personalized phage therapy is associated with improved outcomes in individual cases, clinical trials demonstrating treatment efficacy are lacking, limiting the therapeutic potential of this approach for respiratory infections. In this review, we address the current state of phage therapy for managing chronic respiratory diseases. We then discuss how phage therapy may address major microbiologic obstacles which hinder disease resolution of chronic lung infections with current antibiotic-based treatment practices. Finally, we highlight the challenges that must be addressed for successful phage therapy clinical trials. Through this discussion, we hope to expand on the potential of phages as an adjuvant therapy in chronic lung infections, as well as the microbiologic challenges that need to be addressed for phage therapy to expand beyond personalized salvage therapy.

Evaluation of Antibacterial Efficacy of Centella asiatica-Mediated Selenium Oxide Nanoparticles Against Multidrug-Resistant Upper Respiratory Isolates.

Background The evolution of new respiratory diseases, especially upper respiratory tract infections and resistance of pathogens to various antibiotic treatments, needs an alternative way of medication. Chronic respiratory infections in both adults and infants are the major cause of morbidity and mortality, particularly in developing countries. The widespread application of nanomaterials in the field of medicine and the incorporation of nanoparticles in drugs are taken into account. These nanomaterials are involved along with the biosynthesis of plant extract. In this study, selenium oxide nanoparticles (SeO-NPs), known as a significant trace element for human health, were synthesized in an eco-friendly manner. Methodology Green synthesis of Centella asiatica-mediated SeO-NPs was proceeded by titration method and nanoparticles were synthesized. The color intensity, morphological characters, functional properties, and involvement ofphytochemical compounds were studied by using UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDX) analysis. Results The synthesized extract showed a color change from brown to ruby red. Results obtained by characterization and biological assays depicted that the Centella asiatica-mediated SeO-NPs showed absorbance at the peak level 320 nm by UV-Vis spectroscopy, several phytochemical compounds, and O-H functional groups by FT-IR which may be involved in the reduction of the selenium oxide nanoparticles. The XRD showed 57.1% crystalline and 42.6% amorphous nature. The SEM images showed that agglomerated spherical shapes were involved in biological activities. The EDX analysis showed the presence of Se, C, and O compounds. Further, the antibacterial activity of the synthesized nanoparticles showed significant activity in the multidrug-resistant respiratory pathogens. Conclusions Based on the characterization studies and biomedical assays, it can be concluded that the incorporation of SeO-NPs along with the plant extract serves as the best remedy and organic treatment for upper respiratory tract infections. We plan to conduct further in-vivo, toxicity-level studies, and clinical trials.

Bronchiectasis-associated infections and outcomes in a large, geographically diverse electronic health record cohort in the United States

BackgroundBronchiectasis is a pulmonary disease characterized by irreversible dilation of the bronchi and recurring respiratory infections. Few studies have described the microbiology and prevalence of infections in large patient populations outside of specialized tertiary care centers.MethodsWe used the Cerner HealthFacts Electronic Health Record database to characterize the nature, burden, and frequency of pulmonary infections among persons with bronchiectasis. Chronic infections were defined based on organism-specific guidelines.ResultsWe identified 7,749 patients who met our incident bronchiectasis case definition. In this study population, the organisms with the highest rates of isolate prevalence were Pseudomonas aeruginosa with 937 (12%) individuals, Staphylococcus aureus with 502 (6%), Mycobacterium avium complex (MAC) with 336 (4%), and Aspergillus sp. with 288 (4%). Among persons with at least one isolate of each respective pathogen, 219 (23%) met criteria for chronic P. aeruginosa colonization, 74 (15%) met criteria for S. aureus chronic colonization, 101 (30%) met criteria for MAC chronic infection, and 50 (17%) met criteria for Aspergillus sp. chronic infection. Of 5,795 persons with at least two years of observation, 1,860 (32%) had a bronchiectasis exacerbation and 3,462 (60%) were hospitalized within two years of bronchiectasis diagnoses. Among patients with chronic respiratory infections, the two-year occurrence of exacerbations was 53% and for hospitalizations was 82%.ConclusionsPatients with bronchiectasis experiencing chronic respiratory infections have high rates of hospitalization.

Tracking the fate of bacteria-derived site-specific immunomodulators by positron emission tomography

IntroductionSite-specific immunomodulators (SSIs) are a novel class of therapeutics made from inactivated bacterial species designed to regulate the innate immune system in targeted organs. QBECO is a gut-targeted SSI that is being advanced clinically to treat and/or prevent inflammatory bowel disease, cancer, and serious infections of the gastrointestinal (GI) tract and proximal organs, and QBKPN is a lung-targeted SSI that is in clinical development for the treatment and/or prevention of chronic inflammatory lung disease, lung cancers and respiratory tract infections. While these SSIs have demonstrated both safety and proof-of-concept in preclinical and clinical studies, detailed understanding of their trafficking and biodistribution is yet to be fully characterized. MethodsQBECO and QBKPN were radiolabeled with [89Zr] and injected subcutaneously into healthy mice. The mice underwent Positron Emission Tomography (PET) imaging every day for eight days to track biodistribution of the SSIs. Tissue from the site of injection was collected and immunohistologically probed for immune cell infiltration. ResultsDifferential biodistribution of the two SSIs was seen, adhering to their site-specific targeting. QBKPN appeared to migrate from the site of injection (abdomen) to the cervical lymph nodes which are nearer to the respiratory tract and lungs. QBECO remained in the abdominal region, with lymphatic trafficking to the inguinal lymph nodes, which are nearer to GI-proximal tissues/organs. Immune infiltration at the site of injection comprised of neutrophils for both SSIs, and macrophages for only QBKPN. ConclusionRadiolabeling of SSIs allows for longitudinal in vivo imaging of biodistribution and trafficking. PET imaging revealed differential biodistribution of the SSIs based on the organotropism of the bacteria from which the SSI is derived. Trafficking from the site of injection to the targeted site is in part mediated via the lymphatics and involves macrophages and neutrophils.

Co-spray dried inhalable composite powders of ciprofloxacin and alginate oligosaccharide as anti-biofilm therapy

The treatment of chronic respiratory infections caused by biofilm formation are extremely challenging owing to poor drug penetration into the complex biofilm structure and high drug resistance. Local delivery of an antibiotic together with a non-antibiotic adjuvant to the lungs could often enhance the therapeutic responses by targeting different bacterial growth pathways and minimizing drug resistance. In this study, we designed new inhalable dry powders containing ciprofloxacin (CIP) and OligoG (Oli, a low-molecular-weight alginate oligosaccharide impairing the mucoid biofilms by interacting with their cationic ions) to combat respiratory bacterial biofilm infections. The resulting powders were characterized with respect to their morphology, solid-state property, surface chemistry, moisture sorption behavior, and dissolution rate. The aerosol performance and storage stability of the dry powders were also evaluated. The results showed that inhalable dry powders composed of CIP and Oli could be readily accomplished via the wet milling and spray drying process. Upon the storage under 20 ± 2 °C/20 ± 2 % relative humidity (RH) for one month, there was no significant change in the in vitro aerosol performances of the dry powders. In contrast, the dry powders became non-inhalable following the storage at 20 ± 2 °C/53 ± 2 % RH for one month due to the hygroscopic nature of Oli, which could be largely prevented by incorporation of leucine. Collectively, this study suggests that the newly developed co-spray-dried powders composed of CIP and Oli might represent a promising and alternative treatment strategy against respiratory bacterial biofilm infections.

Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms.

People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.

A Polyclonal Antibody against a Burkholderia cenocepacia OmpA-like Protein Strongly Impairs Pseudomonas aeruginosa and B. multivorans Virulence.

Despite advances in therapies, bacterial chronic respiratory infections persist as life-threatening to patients suffering from cystic fibrosis (CF). Pseudomonas aeruginosa and bacteria of the Burkholderia cepacia complex are among the most difficult of these infections to treat, due to factors like their resistance to multiple antibiotics and ability to form biofilms. The lack of effective antimicrobial strategies prompted our search for alternative immunotherapies that can effectively control and reduce those infections among CF patients. Previous work from our group showed that the anti-BCAL2645 goat polyclonal antibody strongly inhibited Burkholderia cenocepacia to adhere and invade cultured epithelial cells. In this work, we showed that the polyclonal antibody anti-BCAL2645 also strongly inhibited the ability of P. aeruginosa to form biofilms, and to adhere and invade the human bronchial epithelial cell line CFBE41o-. The polyclonal antibody also inhibited, to a lesser extent, the ability of B. multivorans to adhere and invade the human bronchial epithelial cell line CFBE41o. We also show that the ability of B. cenocepacia, P. aeruginosa and B. multivorans to kill larvae of the Galleria mellonella model of infection was impaired when bacteria were incubated with the anti-BCAL2645 antibody prior to the infection. Our findings show that an antibody against BCAL2645 possesses a significant potential for the development of new immunotherapies against these three important bacterial species capable of causing devastating and often lethal infections among CF patients.

Bronchiectasis in renal transplant patients: a cross-sectional study

BackgroundBronchiectasis is a chronic airway disease characterized by permanent and irreversible abnormal dilatation of bronchi. Several studies have reported the development of bronchiectasis after renal transplantation (RT), but no prospective study specifically assessed bronchiectasis in this population. This study aimed to compare features of patients with bronchiectasis associated with RT to those with idiopathic bronchiectasis.MethodsNineteen patients with bronchiectasis associated with RT (RT-B group) and 23 patients with idiopathic bronchiectasis (IB group) were prospectively included in this monocentric cross-sectional study. All patients underwent clinical, functional, laboratory, and CT scan assessments. Sputum was collected from 25 patients (n = 11 with RT-B and n = 14 with IB) and airway microbiota was analyzed using an extended microbiological culture.ResultsDyspnea (≥ 2 on mMRC scale), number of exacerbations, pulmonary function tests, total bronchiectasis score, severity and prognosis scores (FACED and E-FACED), and quality of life scores (SGRQ and MOS SF-36) were similar in the RT-B and IB groups. By contrast, chronic cough was less frequent in the RT-B group than in the IB group (68% vs. 96%, p = 0.03). The prevalence and diversity of the airway microbiota in sputum were similar in the two groups.ConclusionClinical, functional, thoracic CT scan, and microbiological characteristics of bronchiectasis are overall similar in patients with IB and RT-B. These results highlight that in RT patients, chronic respiratory symptoms and/or airway infections should lead to consider the diagnosis of bronchiectasis. Further studies are required to better characterize the pathophysiology of RT-B including airway microbiota, its incidence, and impact on therapeutic management.

Inhaled dry powder liposomal azithromycin for treatment of chronic lower respiratory tract infection

A dry powder inhaled liposomal azithromycin formulation was developed for the treatment of chronic respiratory diseases such as cystic fibrosis and bronchiectasis. Key properties including liposome size, charge and encapsulation efficiency powder size, shape, glass transition temperature (Tg), water content and in vitro respiratory deposition were determined. Antimicrobial activity against cystic fibrosis (CF) respiratory pathogens was determined by MIC, MBC and biofilm assays. Cytotoxicity and cellular uptake studies were performed using A549 cells. The average liposome size was 105 nm, charge was 55 mV and encapsulation efficiency was 75 %. The mean powder particle size d[v,50] of 4.54 µm and Mass Median Aerodynamic Diameter (MMAD) was 5.23 µm with a mean Tg of 76˚C and water content of 2.1 %. These excellent physicochemical characteristics were maintained over one year. Liposomal loaded azithromycin demonstrated enhanced activity against P. aeruginosa clinical isolates grown in biofilm. The formulation was rapidly delivered into bacterial cells with > 75 % uptake in 1 h. Rapid uptake into A549 cells via a cholesterol-dependent endocytosis pathway with no cytotoxic effects apparent. These data demonstrate that this formulation could offer benefits over current treatment regimens for people with chronic respiratory infection.

DERMATITIS ARTEFACTA WITH Proteus mirabilis INFECTION IN COINCIDENCE WITH PYODERMA GANGRENOSUM IN SCHIZOPHRENIC PATIENT

Dermatitis artefacta (DA) is a psychiatric disorder with secondary manifestations on the skin. One of the most common skin lesions in DA is chronic ulcer which also can be caused by pyoderma gangrenosum (PG). There are some bacteria found in chronic ulcer such as Proteus mirabilis. A forty-one years old female with chronic wounds on her chest and left leg. Based on history taking of present illness, physical examinations, laboratory tests and histopathology of the skin lesions, the patient was diagnosed with DA and PG. She was also diagnosed with schizophrenia catatonic type. Chest x-ray showed pneumonia while the swab culture showed growth of Proteus mirbabilis. She was given an oral cefadroxile 500 mg three times daily, cetirizine 10 mg once daily, wound dressing and gentamycin ointment twice daily. She also received oral risperidone 0.5 mg twice daily combined with behavioral therapy from Psychiatric Outpatientclinic. Dermatitis artefacta is a psychocutaneous disorder with uncommon morphology. The most common psychological condition related to DA is schicophrenia. There are some specific histopathology features of DA such as dominant destruction of epidermal layer, multinucleated epidermal keratinocytes or destruction of keratinocytes. Chronic ulcers in DA might be mimicking with PG, which is a chronic recidive neutrophilic dermatoses. Proteus mirabilis is one of the most common causes of urinary tract infection, meanwhile it can also cause chronic ulcers and respiratory tract infection as pneumonia.

Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

The use of a train-the-trainer model to educate non-dental healthcare professionals for oral care

Background: Associations between oral health and systemic conditions have been well established in recent literature. Improvements in oral hygiene have consistently shown to significantly reduce the incidence of respiratory infections, which has high morbidity and mortality rates in the frail elderly. However, the simple low-cost intervention of good oral care remains overlooked in healthcare institutions and nursing homes, due to limited awareness and knowledge. Previous efforts for oral care training of non-dental healthcare professionals have been largely sporadic, with little impact and reach to the medical fraternity. Purpose: To overcome these challenges, we utilized a train-the-trainer model, where seven senior nurses from the long-term stay wards of a public community hospital were educated on the importance of good oral health and trained to administer a comprehensive oral care protocol in patients requiring assistance. The Oral Health Education Programme (OHEP) consisted of a full-day workshop on Common Dental Conditions, Oral Health Assessment Technique (OHAT), and oral hygiene instruction for normal and assisted tooth brushing in independent and mechanically ventilated patients respectively. Results: Majority of the nurses felt that the training was effective and applicable to their work. The number of patients with chronic respiratory infections in the wards were reduced after commencement of OHEP. Conclusion: This interprofessional education collaboration using the train-the-trainer model provides a standardized curriculum, while allowing autonomy for the trainers to customize modules relevant to their respective institutions. This partnership model between medical nurses and oral health professionals allows for better sustainability of the programme.

The Role of the NRF2 Pathway in the Pathogenesis of Viral Respiratory Infections.

In humans, acute and chronic respiratory infections caused by viruses are associated with considerable morbidity and mortality. Respiratory viruses infect airway epithelial cells and induce oxidative stress, yet the exact pathogenesis remains unclear. Oxidative stress activates the transcription factor NRF2, which plays a key role in alleviating redox-induced cellular injury. The transcriptional activation of NRF2 has been reported to affect both viral replication and associated inflammation pathways. There is complex bidirectional crosstalk between virus replication and the NRF2 pathway because virus replication directly or indirectly regulates NRF2 expression, and NRF2 activation can reversely hamper viral replication and viral spread across cells and tissues. In this review, we discuss the complex role of the NRF2 pathway in the regulation of the pathogenesis of the main respiratory viruses, including coronaviruses, influenza viruses, respiratory syncytial virus (RSV), and rhinoviruses. We also summarize the scientific evidence regarding the effects of the known NRF2 agonists that can be utilized to alter the NRF2 pathway.

Imipenem heteroresistance but not tolerance in Haemophilus influenzae during chronic lung infection associated with chronic obstructive pulmonary disease.

Antibiotic resistance is a major Public Health challenge worldwide. Mechanisms other than resistance are described as contributors to therapeutic failure. These include heteroresistance and tolerance, which escape the standardized procedures used for antibiotic treatment decision-making as they do not involve changes in minimal inhibitory concentration (MIC). Haemophilus influenzae causes chronic respiratory infection and is associated with exacerbations suffered by chronic obstructive pulmonary disease (COPD) patients. Although resistance to imipenem is rare in this bacterial species, heteroresistance has been reported, and antibiotic tolerance cannot be excluded. Moreover, development of antibiotic heteroresistance or tolerance during within-host H. influenzae pathoadaptive evolution is currently unknown. In this study, we assessed imipenem resistance, heteroresistance and tolerance in a previously sequenced longitudinal collection of H. influenzae COPD respiratory isolates. The use of Etest, disc diffusion, population analysis profiling, tolerance disc (TD)-test methods, and susceptibility breakpoint criteria when available, showed a significant proportion of imipenem heteroresistance with differences in terms of degree among strains, absence of imipenem tolerance, and no specific trends among serial and clonally related strains could be established. Analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes rendered a panel of polymorphisms only found in heteroresistant strains, but gene expression and genome-wide analyses did not show clear genetic traits linked to heteroresistance. In summary, a significant proportion of imipenem heteroresistance was observed among H. influenzae strains isolated from COPD respiratory samples over time. These data should be useful for making more accurate clinical recommendations to COPD patients.

Azithromycin regulates bacterial virulence and immune response in a murine model of ceftazidime-treated Pseudomonas aeruginosa acute pneumonia.

Pseudomonas aeruginosa (PA) remains one of the leading causes of nosocomial acute pneumonia. The array of virulence factors expressed by PA and the intense immune response associated with PA pneumonia play a major role in the severity of these infections. New therapeutic approaches are needed to overcome the high resistance of PA to antibiotics and to reduce the direct damage to host tissues. Through its immunomodulatory and anti-virulence effects, azithromycin (AZM) has demonstrated clinical benefits in patients with chronic PA respiratory infections. However, there is relatively little evidence in PA acute pneumonia. We investigated the effects of AZM, as an adjunctive therapy combined with ceftazidime (CAZ), in a murine model of PA acute pneumonia. We observed that the combined therapy (i) reduces the weight loss of mice 24 h post-infection (hpi), (ii) decreases neutrophil influx into the lungs at 6 and 24 hpi, while this effect is absent in a LPS-induced pneumonia or when PA is pretreated with antibiotics and mice do not receive any antibiotics, and that (iii) AZM, alone or with CAZ, modulates the expression of PA quorum sensing regulators and virulence factors (LasI, LasA, PqsE, PhzM, ExoS). Our findings support beneficial effects of AZM with CAZ on PA acute pneumonia by both bacterial virulence and immune response modulations. Further investigations are needed to clarify the exact underlying mechanisms responsible for the reduction of the neutrophils influx and to better discriminate between direct immunomodulatory properties of AZM, and indirect effects on neutrophilia resulting from bacterial virulence modulation.