Mucoid morphotype variation of Burkholderia multivorans during chronic cystic fibrosis lung infection is correlated with changes in metabolism, motility, biofilm formation and virulence

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens infecting hosts such as cystic fibrosis (CF) patients. Long-term Bcc infection of CF patients' airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates displaying different morphotypes from a chronically infected CF patient to evaluate trait development during lung infection. Expression profiling of mucoid D2095 and non-mucoid D2214 isolates revealed decreased expression of genes encoding products related to virulence-associated traits and metabolism in D2214. Furthermore, D2214 showed no exopolysaccharide production, lower motility and chemotaxis, and more biofilm formation, particularly under microaerophilic conditions, than the clonal mucoid isolate D2095. When Galleria mellonella was used as acute infection model, D2214 at a cell number of approximately 7×10(6) c.f.u. caused a higher survival rate than D2095, although 6 days post-infection most of the larvae were dead. Infection with the same number of cells by mucoid D2095 caused larval death by day 4. The decreased expression of genes involved in carbon and nitrogen metabolism may reflect lower metabolic needs of D2214 caused by lack of exopolysaccharide, but also by the attenuation of pathways not required for survival. As a result, D2214 showed higher survival than D2095 in minimal medium for 28 days under aerobic conditions. Overall, adaptation during Bcc chronic lung infections gave rise to genotypic and phenotypic variation among isolates, contributing to their fitness while maintaining their capacity for survival in this opportunistic human niche

Pseudomonas aeruginosa: Role in the pathogenesis of the CF lung lesion

Lung disease is the leading cause of morbidity and mortality in individuals with cystic fibrosis (CF), with P. aeruginosa the main pulmonary infectious agent. Although CF patients can become infected with other microorganisms (such as Burkholderia cepacia complex, Staphylococcus aureus, Haemophilus influenzae, and atypical mycobacteria), P. aeruginosa predominates, eventually infecting ∼80% of patients. Once established, P. aeruginosa infection usually persists until death. The interaction between the CF host and this opportunistic pathogen is unique and most likely directly contributes to the classical end-stage pathology of CF lung disease. However, the extent to which this constitutes success by the pathogen or failure by the host, or both, is yet to be determined. Many important questions remain regarding host susceptibility, the role of both innate and adaptive immune defenses, bacterial infectivity and transmission, and pathogen virulence factors. Here, we discuss some recent advances toward understanding this complex interaction between host and pathogen and how the interplay influences the CF lung lesion

Pharmacodynamics of ciprofloxacin against Pseudomonas aeruginosa planktonic and biofilm‐derived cells

© 2019 The Society for Applied Microbiology The influence of growth phase and state on the survival and recovery of Pseudomonas aeruginosa exposed to ciprofloxacin was investigated using batch culture grown planktonic cells and disaggregated biofilm populations. Biofilms were either nonantibiotic exposed or previously exposed to ciprofloxacin before disaggregation and subsequent challenge with ciprofloxacin. Viable counts showed that late stationary phase cells were tolerant to ciprofloxacin over 24h exposure, while all other populations presented a biphasic killing pattern. In contrast, the metabolic activity of planktonic and biofilm-derived cells remained similar to controls during the initial 6h of ciprofloxacin exposure, despite a significant reduction in viable cell numbers. A similar effect was observed when assessing the postantibiotic effect of 1h ciprofloxacin exposure. Thus, although cell reduction occurred, the metabolic status of the cells remained unchanged. The recovery of disaggregated biofilm cells previously exposed to ciprofloxacin was significantly quicker than naïve biofilm cells, and this latter population's recovery was significantly slower than all planktonic populations. Results from this work have implications for our understanding of biofilm-related infections and their resilience to antimicrobial treatment. Significance and Impact of the Study: Removal of biofilms from surfaces and infection sites via disaggregation and induction of dispersion may reverse their antibiotic tolerant state. However, little is known of the recovery of the cells upon disaggregation from biofilms. Driven by this gap in knowledge we quantified the effect of ciprofloxacin on disaggregated biofilms of Pseudomonas aeruginosa, including those previously exposed to ciprofloxacin. Our results provide further insight into bacterial resilience, regrowth, and antimicrobial efficacy, as reduction in cell viability does not directly correlate with the metabolic activity of bacteria at the time of the exposure to antimicrobials. Thus, despite a perceived reduction in viability, the potential for cell persistence and regrowth remains and recovery is quicker upon subsequent exposure to antimicrobial, supporting the increase in resilience and recurrence of infections

Differential post-transcriptional activation of human phagocytes by different Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa is a pulmonary pathogen in individuals with impaired mucocillary clearance such as cystic fibrosis or mechanical ventilation. Non-opsonic phagocytosis of P. aeruginosa can be mediated by either CR3 or CD14 and different strains appear to have a bias towards one or the other receptor. Strain Fc808 is ingested through CD14 whereas P1 (Fc194) uses CR3. In an in vitro culture system, the inflammatory response of macrophages to these two different strains of P. aeruginosa was divergent at the protein level, with higher IL-6 and tumour necrosis factor (TNF)-α production generated in response to strain P1 and higher IL-1β production in response to strain Fc808. Interaction of macrophages with these two bacterial strains induced distinct gene expression patterns as detected by gene array analysis, with prominence of genes encoding pro-inflammatory cytokines, surface receptors, transcription factors and proteins involved in phagocytosis. However, comparison of gene expression data and cytokine response data with the two bacterial strains indicated that production of IL-1β, IL-6 and TNF-α was under differential post-transcriptional control. Interestingly, this effect did not correlate with receptor bias but instead was related to the different LPSs of the two strains. The use of specific mitogen-activated protein kinase (MAPK) inhibitors suggested a role for extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the differential cytokine production by strains P1 and Fc808. These results indicate that strains of the same species of bacteria may induce differential macrophage phagocytic and inflammatory responses with likely consequence for bacterial clearance and host injury

Primary Immunodeficiency to pneumococcal infection due to a defect in Toll-like receptor signaling

Objective: The role of human Toll-like receptors (TLRs) in initiating protective immune responses in vivo is not well understood. We investigated the role of TLR signaling in defense against infection in a 3-year-old boy with a severe defect resulting in recurrent Streptococcus pneumoniae bacteremia. Methods: After classic immunodeficiencies were ruled out, the patient's mononuclear cells, macrophages, and dendritic cells (DCs) were studied. TLR signaling responses to a range of TLR- and interleukin-1 receptor (IL-1R)-specific agonists were investigated pre- and posttranscriptionally by measuring NF-κB translocation and cytokine mRNA and protein expression. Results: The patient's monocytic cells were profoundly deficient in cytokine production in response to a range of microbial-derived TLR agonists and to recombinant IL-1β or IL-18. Lipopolysaccharide (LPS)-induced translocation of NF-κB p50 and p65 and the kinetics of LPS-induced cytokine mRNA transcription were normal except for IL-6 and IL-12p40, which were poorly transcribed. Despite deficient responses to TLR agonists by the patient's DCs and B cells, CD40L responses were normal. Conclusions: We describe a patient with deficient TLR-mediated cytokine production with intact interleukin receptor-associated kinase (IRAK)-4 expression, NF-κB translocation, and enhanced susceptibility to infection. This patient demonstrates that TLR signaling, in the presence of intact antibody responses, may be a nonredundant requirement for defense against pyogenic infections

The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization

Dendritic cells (DC) are instrumental in orchestrating an appropriately polarized Th cell response to pathogens. DC exhibit considerable phenotypic and functional plasticity, influenced by lineage, Ag engagement, and the environment in which they develop and mature. In this study, we identify the human cationic peptide LL-37, found in abundance at sites of inflammation, as a potent modifier of DC differentiation, bridging innate and adaptive immune responses. LL-37-derived DC displayed significantly up-regulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated costimulatory molecule expression, enhanced secretion of Th-1 inducing cytokines, and promoted Th1 responses in vitro. LL-37 may be an attractive therapeutic candidate for manipulating T cell polarization by DC

Surgical Masks Reduce Airborne Spread of Pseudomonas aeruginosa in Colonized Patients with Cystic Fibrosis

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IRAK-4 mutation (Q293X): Rapid detection and characterization of defective post-transcriptional TLR/IL-1R responses in human myeloid and non-myeloid cells

Innate immunodeficiency has recently been reported as resulting from the Q293X IRAK-4 mutation with consequent defective TLR/IL-1R signaling. In this study we report a method for the rapid allele-specific detection of this mutation and demonstrate both cell type specificity and ligand specificity in defective IL-1R-associated kinase (IRAK)-4-deficient cellular responses, indicating differential roles for this protein in human PBMCs and primary dermal fibroblasts and in LPS, IL-1β, and TNF-α signaling. We demonstrate transcriptional and post-transcriptional defects despite NF-κB signaling and intact MyD88-independent signaling and propose that dysfunctional complex 1 (IRAK1/TRAF6/TAK1) signaling, as a consequence of IRAK-4 deficiency, generates specific defects in MAPK activation that could underpin this patient's innate immunodeficiency. These studies demonstrate the importance of studying primary human cells bearing a clinically relevant mutation; they underscore the complexity of innate immune signaling and illuminate novel roles for IRAK-4 and the fundamental importance of accessory proinflammatory signaling to normal human innate immune responses and immunodeficiencies

Primary immune deficiencies presenting in adults: seven years of experience from Iran.

Primary immunodeficiencies (PIDs) are not solely diseases of childhood. We describe the clinical presentation and outcome for 55 adult patients with previously unrecognized PIDs. This series provides unique data regarding PIDs presenting in adulthood, and serves as a timely reminder that physicians must consider the diagnosis of PIDs in their adult patients. Using the experience gained from these patients, we outline key "warning signs" suggestive of an underlying PID. Only through increased physician awareness will patients with PIDs receive timely diagnosis and optimal management