R Latham

Pseudomonas aeruginosa Uses Multiple Pathways To Acquire Iron during Chronic Infection in Cystic Fibrosis Lungs

ABSTRACT Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription–quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF.

Platelet-activating factor receptor (PAFr) is upregulated in small airways and alveoli of smokers and COPD patients

PAFr is a cell adhesion site for specific bacteria, notably non‐typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. We previously published that PAFr expression is significantly upregulated in the large airways of smokers, especially in COPD. We have now investigated PAFr expression in the epithelium and Rbm of small airways and in the alveolar compartment in smokers and patients with both COPD and small airway disease.

Molecular analysis of changes in Pseudomonas aeruginosa load during treatment of a pulmonary exacerbation in cystic fibrosis

BACKGROUND Intravenous antibiotics for pulmonary exacerbations (PEs) of cystic fibrosis (CF) usually target Pseudomonas aeruginosa. Insights into the CF lung microbiome have questioned this approach. We used RT-qPCR to determine whether intravenous antibiotics reduced P. aeruginosa numbers and whether this correlated with improved lung function. We also investigated antibiotic effects on other common respiratory pathogens in CF. METHODS Sputa were collected from patients when stable and again during a PE. Sputa were expectorated into a RNA preservation buffer for RNA extraction and preparation of cDNA. qPCR was used to enumerate viable P. aeruginosa as well as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Burkholderia cepacia complex and Aspergillus fumigatus. RESULTS Fifteen CF patients were followed through 21 PEs. A complete set of serial sputum samples was unavailable for two patients (three separate PEs). P. aeruginosa numbers did not increase immediately prior to a PE, but numbers during intravenous antibiotic treatment were reduced ≥4-log in 6/18 and ≥1-log in 4/18 PEs. In 7/18 PEs, P. aeruginosa numbers changed very little with intravenous antibiotics and one patient demonstrated a ≥2-log increase in P. aeruginosa load. H. influenzae and S. pneumoniae were detected in ten and five PEs respectively, but with antibiotic treatment these bacteria rapidly became undetectable in 6/10 and 4/5 PEs, respectively. There was a negative correlation between P. aeruginosa numbers and FEV1 during stable phase (r(s)=0.75, p<0.05), and reductions in P. aeruginosa load with intravenous antibiotic treatment correlated with improved FEV1 (r(s)=0.52, p<0.05). CONCLUSIONS Exacerbations are not due to increased P. aeruginosa numbers in CF adults. However, lung function improvements correlate with reduced P. aeruginosa burden suggesting that current antibiotic treatment strategies remain appropriate in most patients. Improved understanding of PE characterised by unchanged P. aeruginosa numbers and minimal lung function improvement following treatment may allow better targeted therapies.

An antagonist of the platelet-activating factor receptor inhibits adherence of both nontypeable Haemophilus influenzae and Streptococcus pneumoniae to cultured human bronchial epithelial cells exposed to cigarette smoke

Background COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells. Objective To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae. Methods Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken. Results PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure. Conclusion WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD.

Toll-like receptor signaling is functional in immune cells of the endangered Tasmanian devil

Devil facial tumour disease (DFTD) is a fatally transmissible cancer that threatens the Tasmanian devil population. As Tasmanian devils do not produce an immune response against DFTD cells, an effective vaccine will require a strong adjuvant. Activation of innate immune system cells through toll-like receptors (TLRs) could provide this stimulation. It is unknown whether marsupials, including Tasmanian devils, express functional TLRs. We isolated RNA from peripheral blood mononuclear cells and, with PCR, detected transcripts for TLRs 2, 3, 4, 5, 6, 7, 8, 9, 10 and 13. Stimulation of the mononuclear cells with agonists to these TLRs increased the expression of downstream TLR signaling products (IL1α, IL6, IL12A and IFNβ). Our data provide the first evidence that TLR signaling is functional in the mononuclear cells of the Tasmanian devil. Future DFTD vaccination trials will incorporate TLR agonists to enhance the immune response against DFTD.

The main rhinovirus respiratory tract adhesion site (ICAM-1) is upregulated in smokers and patients with chronic airflow limitation (CAL)

BackgroundICAM-1 is a major receptor for ~60% of human rhinoviruses, and non-typeable Haemophilus influenzae, two major pathogens in COPD. Increased cell-surface expression of ICAM-1 in response to tobacco smoke exposure has been suggested. We have investigated epithelial ICAM-1 expression in both the large and small airways, and lung parenchyma in smoking-related chronic airflow limitation (CAL) patients.MethodsWe evaluated epithelial ICAM-1 expression in resected lung tissue: 8 smokers with normal spirometry (NLFS); 29 CAL patients (10 small-airway disease; 9 COPD-smokers; 10 COPD ex-smokers); Controls (NC): 15 normal airway/lung tissues. Immunostaining with anti-ICAM-1 monoclonal antibody was quantified with computerized image analysis. The percent and type of cells expressing ICAM-1 in large and small airway epithelium and parenchyma were enumerated, plus percentage of epithelial goblet and submucosal glands positive for ICAM- 1.ResultsA major increase in ICAM-1 expression in epithelial cells was found in both large (p < 0.006) and small airways (p < 0.004) of CAL subjects compared to NC, with NLFS being intermediate. In the CAL group, both basal and luminal areas stained heavily for ICAM-1, so did goblet cells and sub-mucosal glands, however in either NC or NLFS subjects, only epithelial cell luminal surfaces stained. ICAM-1 expression on alveolar pneumocytes (mainly type II) was slightly increased in CAL and NLFS (p < 0.01). Pack-years of smoking correlated with ICAM-1 expression (r = 0.49; p < 0.03).ConclusionAirway ICAM-1 expression is markedly upregulated in CAL group, which could be crucial in rhinoviral and NTHi infections. The parenchymal ICAM-1 is affected by smoking, with no further enhancement in CAL subjects.

Filtration of crushed tablet suspensions has potential to reduce infection incidence in people who inject drugs

INTRODUCTION AND AIMS The medical complications of injecting preparations from crushed tablets can be severe, and most can be attributed to the injection of insoluble particles and micro-organisms. Previously we have shown that most of the particles can be removed by filtration, but it was not known whether bacteria could also be filtered in the presence of a high particle load. This study aims to determine the feasibility of filtration to remove bacteria from injections prepared from tablets. DESIGN AND METHODS Injections were prepared from crushed slow-release morphine tablets, in mixed bacterial suspensions of Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa. The injection suspensions were passed through syringe filters of porosity 0.45 or 0.20 μm, or combined 0.8 then 0.2 μm, and the bacterial load was counted. RESULTS Bacterial concentrations in unfiltered injections were 2.5-4.3 × 10(6) colony forming units mL(-1) . Both the 0.20 and 0.45 μm filters blocked unless a prefilter (cigarette filter) was used first. The 0.2 μm filter and the combined 0.8/0.2 μm filter reduced the bacteria to the limit of detection (10 colony forming units mL(-1) ) or below. Filtration through a 0.45 μm filter was slightly less effective. DISCUSSION AND CONCLUSIONS Use of a 0.2 μm filter, together with other injection hygiene measures, offers the prospect of greatly reducing the medical complications of injecting crushed tablets and should be considered as a highly effective harm reduction method. It is very likely that these benefits would also apply to other illicit drug injections, although validation studies are needed.

Identifying Haemophilus haemolyticus and Haemophilus influenzae by SYBR Green real-time PCR

SYBR Green real time PCR assays for protein D (hpd), fuculose kinase (fucK) and [Cu, Zn]-superoxide dismutase (sodC) were designed for use in an algorithm for the identification of Haemophilus influenzae and H. haemolyticus. When tested on 127 H. influenzae and 60 H. haemolyticus all isolates were identified correctly.

Molecular detection of Haemophilus influenzae in COPD sputum is superior to conventional culturing methods

School of Medicine and Menzies Research Institute, University of Tasmania, Hobart, Australia.