Philip Murphy

Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis.

Intractable formation of biofilm by and infection with the opportunistic pathogen Pseudomonas aeruginosa are hallmarks of cystic fibrosis (CF). Lactoferrin, an innate immunity protein, has recently been shown to inhibit the formation of P. aeruginosa biofilm. Partial cleavage of lactoferrin by the proteases neutrophil elastase and Pseudomonas elastase has previously been described in CF. Here, we show that cathepsins in CF secretions are responsible for complete and rapid cleavage of lactoferrin. We demonstrate that levels of lactoferrin in P. aeruginosa-positive sputum samples are decreased when corrected for inflammatory burden and that P. aeruginosa-positive sputum samples have significantly higher cathepsin activity and significantly reduced ability to inhibit formation of biofilm, compared with P. aeruginosa-negative sputum samples. We also show that cleavage of lactoferrin by cathepsin results in loss of both its microbicidal and antibiofilm activity. Loss of such a vital innate immunity protein clearly has important implications for the pathogenesis of chronic P. aeruginosa lung infection in patients with CF.

LL-37 complexation with glycosaminoglycans in cystic fibrosis lungs inhibits antimicrobial activity, which can be restored by hypertonic saline.

There is an abundance of antimicrobial peptides in cystic fibrosis (CF) lungs. Despite this, individuals with CF are susceptible to microbial colonization and infection. In this study, we investigated the antimicrobial response within the CF lung, focusing on the human cathelicidin LL-37. We demonstrate the presence of the LL-37 precursor, human cathelicidin precursor protein designated 18-kDa cationic antimicrobial protein, in the CF lung along with evidence that it is processed to active LL-37 by proteinase-3. We demonstrate that despite supranormal levels of LL-37, the lung fluid from CF patients exhibits no demonstrable antimicrobial activity. Furthermore Pseudomonas killing by physiological concentrations of exogenous LL-37 is inhibited by CF bronchoalveolar lavage (BAL) fluid due to proteolytic degradation of LL-37 by neutrophil elastase and cathepsin D. The endogenous LL-37 in CF BAL fluid is protected from this proteolysis by interactions with glycosaminoglycans, but while this protects LL-37 from proteolysis it results in inactivation of LL-37 antimicrobial activity. By digesting glycosaminoglycans in CF BAL fluid, endogenous LL-37 is liberated and the antimicrobial properties of CF BAL fluid restored. High sodium concentrations also liberate LL-37 in CF BAL fluid in vitro. This is also seen in vivo in CF sputum where LL-37 is complexed to glycosaminoglycans but is liberated following nebulized hypertonic saline resulting in increased antimicrobial effect. These data suggest glycosaminoglycan–LL-37 complexes to be potential therapeutic targets. Factors that disrupt glycosaminoglycan–LL-37 aggregates promote the antimicrobial effects of LL-37 with the caveat that concomitant administration of antiproteases may be needed to protect the now liberated LL-37 from proteolytic cleavage.

Comparison of antibiotic susceptibility of Burkholderia cepacia complex organisms when grown planktonically or as biofilm in vitro

This study determined the antibiotic susceptibility of planktonic and biofilm cultures of Burkholderia cepacia complex organisms, a group of highly problematic pathogens associated with cystic fibrosis patients. The biofilm inhibitory concentrations were considerably higher than the corresponding minimum inhibitory concentrations for meropenem and piperacillin-tazobactam. However, tobramycin and amikacin were efficacious against both biofilm and planktonic cultures. Overall this study showed that biofilm susceptibility testing might be more clinically appropriate for determining antibiotic therapy for Burkholderia cepacia complex infections in cystic fibrosis patients.

An outbreak of colonization with linezolid-resistant Staphylococcus epidermidis in an intensive therapy unit

OBJECTIVES To report an outbreak of colonization with linezolid-resistant Staphylococcus epidermidis in an intensive therapy unit (ITU). METHODS An outbreak of colonization with linezolid-resistant S. epidermidis affecting 16 patients in an ITU was investigated using PFGE. Environmental and staff screening was carried out as part of the investigation. Usage of linezolid in the hospital and in the ITU was reviewed. Resistant strains were screened for the presence of the G2576T mutation using PCR-RFLP genotyping. The interventions made to control the outbreak were restriction of linezolid prescription and specific infection control measures, including isolation of colonized patients and increased environmental cleaning. RESULTS Linezolid-resistant S. epidermidis strains from the 16 colonized patients were genetically related. The same strain was also cultured from environmental samples in the ITU. An increase in linezolid usage in the hospital and in the ITU occurred in the 6 months prior to the emergence of the resistant strain. Infection control measures and restriction of linezolid prescription controlled the outbreak. All resistant isolates contained the G2576T mutation. CONCLUSIONS An outbreak of colonization with linezolid-resistant S. epidermidis occurred in the ITU in our institution. The resistant strain colonized the environment and probably spread from patient to patient. The outbreak was associated with an increase in the linezolid usage in the ITU and in the institution as a whole. Restriction of linezolid usage and infection control measures were introduced to control the outbreak. The emergence of linezolid resistance in S. epidermidis has implications for the use of linezolid as a therapeutic agent.

Early detection of Pseudomonas aeruginosa - comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF)

BackgroundPseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum.MethodsAdult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (opr L) gene locus and (ii) the exotoxin A (ETA) gene locus.ResultsBy sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The opr L locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the opr L target negative. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months.ConclusionsThis study indicates that molecular detection of PA in sputum employing the opr L gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.

Multilocus Sequence Typing Reveals that the Population Structure of Candida dubliniensis Is Significantly Less Divergent than That of Candida albicans

ABSTRACT The pathogenic yeast Candida dubliniensis is phylogenetically very closely related to Candida albicans, and both species share many phenotypic and genetic characteristics. DNA fingerprinting using the species-specific probe Cd25 and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal gene cluster previously showed that C. dubliniensis is comprised of three major clades comprising four distinct ITS genotypes. Multilocus sequence typing (MLST) has been shown to be very useful for investigating the epidemiology and population biology of C. albicans and has identified many distinct major and minor clades. In the present study, we used MLST to investigate the population structure of C. dubliniensis for the first time. Combinations of 10 loci previously tested for MLST analysis of C. albicans were assessed for their discriminatory ability with 50 epidemiologically unrelated C. dubliniensis isolates from diverse geographic locations, including representative isolates from the previously identified three Cd25-defined major clades and the four ITS genotypes. Dendrograms created by using the unweighted pair group method with arithmetic averages that were generated using the data from all 10 loci revealed a population structure which supports that previously suggested by DNA fingerprinting and ITS genotyping. The MLST data revealed significantly less divergence within the C. dubliniensis population examined than within the C. albicans population. These findings show that MLST can be used as an informative alternative strategy for investigating the population structure of C. dubliniensis. On the basis of the highest number of genotypes per variable base, we recommend the following eight loci for MLST analysis of C. dubliniensis: CdAAT1b, CdACC1, CdADP1, CdMPIb, CdRPN2, CdSYA1, exCdVPS13, and exCdZWF1b, where “Cd” indicates C. dubliniensis and “ex” indicates extended sequence.

Cognitive predictors of problem drinking and AUDIT scores among college students

Evidence from a number of substance abuse populations suggests that substance abuse is associated with a cluster of differences in cognitive processes. However, investigations of this kind in non-clinical samples are relatively few. The present study examined the ability of alcohol-attentional bias (an alcohol Stroop task), impulsive decision-making (a delay discounting task), and impaired inhibitory control (a GO-NOGO task) to: (a) discriminate problem from non-problem drinkers among a sample of college students; (b) predict scores on the Alcohol Use Disorders Identification Test (AUDIT; a measure of alcohol consumption, drinking behaviour, and alcohol-related problems) across all of the student drinkers; (c) predict AUDIT scores within the subgroups of problem and non-problem student drinkers. In logistic regression controlling for gender and age, student drinkers with elevated alcohol-attentional bias and impulsive decision-making were over twice as likely to be a problem than a non-problem drinker. Multiple regression analysis of the entire sample revealed that all three cognitive measures were significant predictors of AUDIT scores after gender and age had been controlled; the cognitive variables together accounted for 48% of the variance. Moreover, subsequent multiple regressions revealed that impaired inhibitory control was the only significant predictor of AUDIT scores for the group of non-problem drinkers, and alcohol-attentional bias and impulsive decision-making were the only significant predictors of AUDIT scores for the group of problem drinkers. Finally, both impulsive decision-making and impaired inhibitory control were significantly correlated with alcohol-attentional bias across the whole sample. Implications are discussed relating to the development of problematic drinking.

The Effect of Aspergillus fumigatus Infection on Vitamin D Receptor Expression in Cystic Fibrosis

RATIONALE Aspergillus fumigatus (A. fumigatus) in cystic fibrosis (CF) is increasingly recognized. Although allergic bronchopulmonary aspergillosis (ABPA) leads to deterioration of pulmonary function, the effect of A. fumigatus colonization in the absence of ABPA remains unclear. OBJECTIVES To address this, we examined individuals with CF with A. fumigatus who were ABPA negative to identify the effects of itraconazole therapy on Aspergillus-induced lung inflammation. METHODS The effect of A. fumigatus on nuclear vitamin D receptor (VDR) expression was investigated using qRT-PCR and Western blotting. IL-5 and IL-13 levels were quantified by ELISA. The effect of itraconazole was assessed by a combination of high-resolution computed tomography, lung function test, and microbiological analysis. MEASUREMENTS AND MAIN RESULTS We demonstrate that A. fumigatus down-regulates VDR in macrophages and airway epithelial cells and that the fungal metabolite gliotoxin (Gt) is the main causative agent. Gt overcame the positive effect of 1,25-OH vitamin D(3) on VDR expression in vitro, resulting in increased IL-5 and IL-13 production. In vivo, A. fumigatus positivity was associated with increased Gt in CF bronchoalveolar lavage fluid and increased bronchoalveolar lavage fluid levels of IL-5 and IL-13. After airway eradication of A. fumigatus with itraconazole, we observed decreased Gt, IL-5 and IL-13, improved respiratory symptoms, and diminished high-resolution computed tomography mosaic pattern consistent with sustained pulmonary function. CONCLUSIONS This study provides a rationale for the therapeutic effect of itraconazole and implied that the therapeutic potential of vitamin D supplementation in preventing ABPA is only feasible with concurrent elimination of A. fumigatus to permit VDR expression and its positive functional consequences.

Prevalence of thermophilic Campylobacter spp. in ready-to-eat foods and raw poultry in Northern Ireland.

Although there have been numerous studies investigating the prevalence of campylobacters in animals and raw meats, there are limited data on the persistence of these organisms in ready-to-eat (RTE) foodstuffs. Although poultry is now well established as a major reservoir of thermophilic campylobacters, it is widely assumed that hazard analysis critical control point (HACCP) controls in commercial and industrial settings are effective in eliminating this hazard through thorough cooking of RTE products. Therefore, it was the primary aim of this study to investigate the effectiveness of HACCP controls in eliminating campylobacters in such cooked RTE foods by attempting to isolate viable organisms from product. Concurrently, the results of this study demonstrate that local poultry is highly contaminated with campylobacters. Commercially available RTE foodstuffs (n = 2,030) consisting of 1,061 poultry-related cooked products and 969 other products were analyzed and were not found to contain thermophilic Campylobacter spp. In addition, 107 raw chickens (63 fresh birds and 44 frozen birds) were sampled, and 94% of the fresh birds and 77% of the frozen birds examined were demonstrated to be contaminated with campylobacters, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari accounting for 69, 30, and 1% of the contaminating organisms, respectively. In general, commercially available RTE foodstuffs, including cooked poultry, are not commonly contaminated with campylobacters and thus do not appear to represent a significant cause of clinical infection of Campylobacter spp. in Northern Ireland. However, raw poultry produce, including fresh and frozen chicken, frequently tested positive for campylobacters. Implementation of HACCP systems by food processors will help to minimize and/or eliminate the risk posed by this organism to the consumer.

The changing pattern of antimicrobial resistance within 42,033 Escherichia coli isolates from nosocomial, community and urology patient-specific urinary tract infections, Dublin, 1999-2009.

Study Type – Therapy (practice patterns cohort)