K. Schaffer

Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.

ABSTRACT Antibiotic resistance is a significant and developing problem in general medical practice and a common clinical complication in cystic fibrosis patients infected with Pseudomonas aeruginosa. Such infections occur within hypoxic mucous deposits in the cystic fibrosis lung; however, little is known about how the hypoxic microenvironment influences pathogen behavior. Here we investigated the impact of hypoxia on antibiotic resistance in P. aeruginosa. The MICs of a selection of antibiotics were determined for P. aeruginosa grown under either normoxic or hypoxic conditions. The expression of mRNAs for resistance-nodulation-cell division (RND) multidrug efflux pump linker proteins was determined by real-time PCR, and multidrug efflux pump activity was inhibited using Phe-Arg β-naphthylamide dihydrochloride. The MIC values of a subset of clinically important P. aeruginosa antibiotics were higher for bacteria incubated under hypoxia than under normoxia. Furthermore, hypoxia altered the stoichiometry of multidrug efflux pump linker protein subtype expression, and pharmacologic inhibition of these pumps reversed hypoxia-induced antibiotic resistance. We hypothesize that hypoxia increases multidrug resistance in P. aeruginosa by shifting multidrug efflux pump linker protein expression toward a dominance of MexEF-OprN. Thus, microenvironmental hypoxia may contribute significantly to the development of antibiotic resistance in P. aeruginosa infecting cystic fibrosis patients.

Hypoxia, innate immunity and infection in the lung.

The mucosal surface of the lung is the key interface between the external atmosphere and the bloodstream. Normally, this well oxygenated tissue is maintained in state of sterility by a number of innate immune processes. These include a physical and dynamic mucus barrier, the production of microbiocidal peptides and the expression of specific pattern recognition receptors on alveolar epithelial cells and resident macrophages and dendritic cells which recognise microbial structures and initiate innate immune responses which promote the clearance of potentially infectious agents. In a range of diseases, the mucosal surface of the lung experiences decreased oxygen tension leading to localised areas of prominent hypoxia which can impact upon innate immune and subsequent infectious and inflammatory processes. Under these conditions, the lung is generally more susceptible to infection and subsequent inflammation. In the current review, we will discuss recent data pertaining to the role of hypoxia in regulating both host and pathogen in the lung during pulmonary disease and how this contributes to innate immunity, infection and inflammation.

Virulence of an emerging respiratory pathogen, genus Pandoraea, in vivo and its interactions with lung epithelial cells

Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species among Irish CF patients. The objective of this study was to investigate the pathogenicity and potential mechanisms of virulence of Irish P. pulmonicola isolates and strains from other Pandoraea species. Three patients from whom the P. pulmonicola isolates were isolated have since died. The in vivo virulence of these and other Pandoraea strains was examined by determining the ability to kill Galleria mellonella larvae. The P. pulmonicola strains generally were the most virulent of the species tested, with three showing a comparable or greater level of virulence in vivo relative to another CF pathogen, Burkholderia cenocepacia, whilst strains from two other species, Pandoraea apista and Pandoraea pnomenusa, were considerably less virulent. For all Pandoraea species, whole cells were required for larval killing, as cell-free supernatants had little effect on larval survival. Overall, invasive Pandoraea strains showed comparable invasion of two independent lung epithelial cell lines, irrespective of whether they had a CF phenotype. Pandoraea strains were also capable of translocation across polarized lung epithelial cell monolayers. Although protease secretion was a common characteristic across the genus, it is unlikely to be involved in pathogenesis. In conclusion, whilst multiple mechanisms of pathogenicity may exist across the genus Pandoraea, it appears that lung cell invasion and translocation contribute to the virulence of P. pulmonicola strains.

Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen commonly associated with lung and wound infections. Hypoxia is a frequent feature of the microenvironment of infected tissues which induces the expression of genes associated with innate immunity and inflammation in host cells primarily through the activation of the hypoxia-inducible factor (HIF) and Nuclear factor kappaB (NF-κB) pathways which are regulated by oxygen-dependent prolyl-hydroxylases. Hypoxia also affects virulence and antibiotic resistance in bacterial pathogens. However, less is known about the impact of hypoxia on host-pathogen interactions such as bacterial adhesion and infection. In the current study, we demonstrate that hypoxia decreases the internalization of P. aeruginosa into cultured epithelial cells resulting in decreased host cell death. This response can also be elicited by the hydroxylase inhibitor Dimethyloxallyl Glycine (DMOG). Reducing HIF-2α expression or Rho kinase activity diminished the effects of hypoxia on P. aeruginosa infection. Furthermore, in an in vivo pneumonia infection model, application of DMOG 48 h before infection with P. aeruginosa significantly reduced mortality. Thus, hypoxia reduces P. aeruginosa internalization into epithelial cells and pharmacologic manipulation of the host pathways involved may represent new therapeutic targets in the treatment of P. aeruginosa infection.

A low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens.

Abstract Acute respiratory tract infections are a major cause of morbidity and mortality worldwide and exert a considerable economic burden on healthcare systems. Acute respiratory tract infections of the upper and lower respiratory tract are caused by a wide variety of viral and bacterial pathogens, which require comprehensive laboratory investigations. Conventional serological and immunofluorescence-based diagnostic methods for acute respiratory tract infections lack sensitivity when compared to polymerase chain reaction (PCR)-based approaches and the development of new diagnostic methodologies is required, to provide accurate, sensitive and rapid diagnoses. In the present study, a PCR-based low density oligonucleotide microarray was developed for the detection of 16 viral and two atypical bacterial pathogens. The performance of this DNA microarray-based analysis exhibited comparable sensitivities and specificities to multiplex real-time reverse transcription polymerase chain reactions (rtPCRs) confirming the potential diagnostic utility of the method. In contrast to routine multiplex PCR, the microarray incorporates an intrinsic redundancy as multiple and non-identical probes per target on the array allow direct intra-assay confirmation of positives. This study demonstrates that microarray technology provides a viable alternative to conventional serological-based approaches and multiplex PCR for pathogen identification in acute respiratory tract infections.

The impact of hypoxia on bacterial infection

Tissue hypoxia is a common microenvironmental feature during inflammation associated with bacterial infection. Hypoxia has recently been shown to play an important role in both innate and adaptive host immunity through the regulation of transcription factors, including hypoxia‐inducible factor and nuclear factor‐κB, in both infiltrating immunocytes and inflamed resident cells. Recent studies have suggested that, by regulating these important immune effector pathways in host tissues, hypoxia can significantly alter the process of bacterial infection and subsequent disease progression. Although hypoxia is often beneficial in terms of reducing the development of infection, its net effect depends on a number of factors, including the nature of the pathogen and the characteristics of the infection encountered. In this minireview, we will discuss the impact of local tissue hypoxia and the resulting activation of hypoxia‐sensitive pathways on bacterial infection by a range of pathogens. Furthermore, we will review how this knowledge may be used to develop new approaches to anti‐infective therapeutics.

JC virus in the Irish population: Significant increase of genotype 2 in immunocompromised individuals

The human polyomavirus JC virus (JCV) is ubiquitous and can be shed in the urine of more than 40% of the healthy population. Amplification and sequencing of JCV from urine has allowed a distinctive map of the distribution of JCV genotypes worldwide. To define the frequency of JCV urinary excretion and genotype distribution in Ireland, urines from 121 healthy individuals and from 94 immunocompromised individuals (human immunodeficiency virus [HIV]-positive patients and rheumatoid arthritis patients) were collected. JCV DNA was detected by polymerase-chain reaction (PCR) with subsequent nucleotide sequencing of a fragment of the major capsid protein (VP1). JCV was detected in 20.7% of healthy individuals and was found significantly more often in the urine of HIV-positive patients (54.2%; P < .001) and rheumatoid arthritis patients (54.4%; P < .001). In healthy Irish individuals genotype 1 was the predominant genotype in 62.5%, followed by genotype 4 in 16.7% and genotype 2 in 12.5%. In contrast, genotype 2 was significantly more often isolated from the urine of both HIV-positive patients (60%) and rheumatoid arthritis patients (54.4%; P < .01). The pattern of genotype distribution among healthy Irish individuals is in agreement with data reported from other European countries, whereas the overall level of JCV urinary excretion is lower. Previous studies have found genotype 2 significantly more often in cerebrospinal (CSF) samples of patients with progressive multifocal leukoencephalopathy (PML). Here the authors report an increased frequency of genotype 2 in urine samples of immunocompromised non-PML patients. This finding further underlines the hypothesis that there could be biologic differences between JCV genotypes.

Immunoproteomic Analysis of Proteins Expressed by Two Related Pathogens, Burkholderia multivorans and Burkholderia cenocepacia, during Human Infection.

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.

Surveillance of epstein‐barr virus loads in adult liver transplantation: Associations with age, sex, posttransplant times, and transplant indications

Epstein‐Barr virus (EBV)–associated posttransplant lymphoproliferative disorder (PTLD) is a life‐threatening complication after adult orthotopic liver transplantation (AOLT). Besides EBV and immunosuppression, relatively little is known about the pretransplant clinical parameters associated with the risk of PTLD, and the benefit of using EBV surveillance to predict EBV‐associated disease in AOLT patients is uncertain. The aims of this single‐center study were to monitor EBV viral loads (VLs) in AOLT patients and to investigate any associations with age, sex, cytomegalovirus (CMV) serostatus, posttransplant times, and indications for transplantation. 1275 blood samples that were collected from 197 AOLT patients 1 day to more than 15 years after transplantation were investigated with quantitative polymerase chain reaction for EBV and CMV DNA. Seventy‐two percent of the patients had EBV DNAemia less than 100 days after transplantation without clinical manifestations. No association was observed between the EBV copy numbers and the time since transplantation. EBV DNAemia was weakly associated with male sex but was not associated with age, CMV serostatus, or indications for AOLT. The highest EBV VL levels were observed in patients who presented with congenital liver diseases, whereas patients with viral hepatitis maintained high EBV VLs after transplantation. None of the patients developed PTLD during the study period; however, 3 patients presented with EBV‐associated diseases. In conclusion, EBV DNAemia is common in AOLT patients, and routine EBV surveillance has limited value for predicting EBV‐associated morbidity or mortality. Liver Transpl, 2011.

First outbreak of linezolid-resistant vancomycin-resistant Enterococcus faecium in an Irish hospital, February to September 2014

An outbreak of linezolid-resistant vancomycin-resistant Enterococcus faecium (LRVREfm) occurred in the hepatology ward of a tertiary referral hospital in Ireland between February and September 2014. LRVREfm was isolated from 15 patients; pulsed-field gel electrophoresis confirmed spread of a single clone. This is the first report of an outbreak of linezolid-resistant vancomycin-resistant enterococcus in Ireland.