Publications by authors named "Pat G Casey"

47 Publications

Autochthonous faecal viral transfer (FVT) impacts the murine microbiome after antibiotic perturbation.

BMC Biol 2020 11 20;18(1):173. Epub 2020 Nov 20.

APC Microbiome Ireland, University College Cork, Cork, Ireland.

Background: It has become increasingly accepted that establishing and maintaining a complex and diverse gut microbiota is fundamental to human health. There are growing efforts to identify means of modulating and influencing the microbiota, especially in individuals who have experienced a disruption in their native microbiota. Faecal microbiota transplantation (FMT) is one method that restores diversity to the microbiota of an individual by introducing microbes from a healthy donor. FMT introduces the total microbial load into the recipient, including the bacteria, archaea, yeasts, protists and viruses. In this study, we investigated whether an autochthonous faecal viral transfer (FVT), in the form of a sterile faecal filtrate, could impact the recovery of a bacteriome disrupted by antibiotic treatment.

Results: Following antibiotic disruption of the bacteriome, test mice received an FVT harvested prior to antibiotic treatment, while control mice received a heat- and nuclease-treated FVT. In both groups of mice, the perturbed microbiome reverted over time to one more similar to the pre-treatment one. However, the bacteriomes of mice that received an FVT, in which bacteriophages predominate, separated from those of the control mice as determined by principal co-ordinate analysis (PCoA). Moreover, analysis of the differentially abundant taxa indicated a closer resemblance to the pre-treatment bacteriome in the test mice that had received an FVT. Similarly, metagenomic sequencing of the virome confirmed that faecal bacteriophages of FVT and control mice differed over time in both abundance and diversity, with the phages constituting the FVT persisting in mice that received them.

Conclusions: An autochthonous virome transfer reshaped the bacteriomes of mice post-antibiotic treatment such that they more closely resembled the pre-antibiotic microbiota profile compared to mice that received non-viable phages. Thus, FVT may have a role in addressing antibiotic-associated microbiota alterations and potentially prevent the establishment of post-antibiotic infection. Given that bacteriophages are biologically inert in the absence of their host bacteria, they could form a safe and effective alternative to whole microbiota transplants that could be delivered during/following perturbation of the gut flora.
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http://dx.doi.org/10.1186/s12915-020-00906-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679995PMC
November 2020

Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection.

Microbiome 2019 01 18;7(1). Epub 2019 Jan 18.

APC Microbiome Ireland, University College Cork, Cork, Ireland.

Background: A westernized diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here, we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice.

Results: We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen, in the gut and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low fat (LF)- or chow-fed animals. Prior to Listeria infection, short-term consumption of HF diet elevated levels of Firmicutes including Coprococcus, Butyricicoccus, Turicibacter and Clostridium XIVa species. During active infection with L. monocytogenes, microbiota changes were further exaggerated but host inflammatory responses were significantly downregulated relative to Listeria-infected LF- or chow-fed groups, suggestive of a profound tempering of the host response influenced by infection in the context of a HF diet. The effects of diet were seen beyond the gut, as a HF diet also increased the sensitivity of mice to systemic infection and altered gene expression profiles in the liver.

Conclusions: We adopted a systems approach to identify the effects of HF diet upon L. monocytogenes infection through analysis of host responses and microbiota changes (both pre- and post-infection). Overall, the results indicate that short-term consumption of a westernized diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.
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http://dx.doi.org/10.1186/s40168-019-0621-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339339PMC
January 2019

A long and abundant non-coding RNA in .

Microb Genom 2017 09 17;3(9):e000126. Epub 2017 Jul 17.

2APC Microbiome Institute, University College Cork, Cork, Ireland.

, found in the intestinal microbiota of humans and animals, is studied as an example of the sub-dominant intestinal commensals that may impart benefits upon their host. Strains typically harbour at least one megaplasmid that encodes functions contributing to contingency metabolism and environmental adaptation. RNA sequencing (RNA-seq)transcriptomic analysis of strain UCC118 identified the presence of a novel unusually abundant long non-coding RNA (lncRNA) encoded by the megaplasmid, and which represented more than 75 % of the total RNA-seq reads after depletion of rRNA species. The expression level of this 520 nt lncRNA in UCC118 exceeded that of the 16S rRNA, it accumulated during growth, was very stable over time and was also expressed during intestinal transit in a mouse. This lncRNA sequence is specific to the species; however, among 45 genomes analysed, not all (only 34) harboured the sequence for the lncRNA. This lncRNA was produced in 27 tested strains, but at strain-specific expression levels. High-level lncRNA expression correlated with high megaplasmid copy number. Transcriptome analysis of a deletion mutant lacking this lncRNA identified altered expression levels of genes in a number of pathways, but a definitive function of this new lncRNA was not identified. This lncRNA presents distinctive and unique properties, and suggests potential basic and applied scientific developments of this phenomenon.
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http://dx.doi.org/10.1099/mgen.0.000126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643018PMC
September 2017

Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of : Analysis in Cell Culture and Murine Infection Models.

Front Microbiol 2017 26;8:1797. Epub 2017 Sep 26.

APC Microbiome Institute, University College Cork, Cork, Ireland.

is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-) based vector (pPL2CBR) expressing codon optimized CBR- under the control of a highly expressed Listerial promoter (P) for and have compared this to a -based system expressing bacterial luciferase for BLI of the pathogen using growth experiments and models. The CBR- plasmid stably integrates into the chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR- labeled emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, -labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR- labeled bacteria was more efficient than the signal from -labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR- is suited to BLI approaches and demonstrated enhanced sensitivity relative to in the context of infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to and may have significant applications in the analysis of pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2017.01797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622934PMC
September 2017

Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation.

Front Microbiol 2017 31;8:964. Epub 2017 May 31.

APC Microbiome Institute and School of Microbiology, University College CorkCork, Ireland.

Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, and , were further characterised, indicating that is involved in ferrous iron transport, while the -encoded transport system imports both ferrous and ferric iron. Transcription studies showed that and constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the cluster does not affect UCC2003's ability to colonise the gut of a murine model.
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http://dx.doi.org/10.3389/fmicb.2017.00964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449479PMC
May 2017

Listeria monocytogenes mutants defective in gallbladder replication represent safety-enhanced vaccine delivery platforms.

Hum Vaccin Immunother 2016 08 23;12(8):2059-2063. Epub 2016 Feb 23.

a APC Microbiome Institute, University College Cork , Cork , Ireland.

The Gram positive intracellular pathogen Listeria monocytogenes represents a promising vaccine or therapeutic DNA delivery vector that has been successfully administered to humans in clinical trials. However in generating Listeria mutants with therapeutic potential it is important to balance safety attenuation with efficacy. Here we show that L. monocytogenes mutants with a reduced capacity for murine gallbladder replication are capable of stimulating T cell responses in mice and protecting vaccinated animals from secondary challenge. Mutation of L. monocytogenes genes lmo2566 or lmo0598 resulted in significant attenuation in the murine model yet mutants retained a capacity for intracellular growth and stimulation of T cell responses against key Listeria epitopes (LLO and P60). Importantly the mutants showed a reduced capacity for growth in the gallbladders of vaccinated mice as well as significantly reduced faecal shedding indicating that this approach generates live Listeria-based vector delivery systems with a reduced capacity for the spread of live genetically modified microorganisms into the natural environment.
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http://dx.doi.org/10.1080/21645515.2016.1154248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4994736PMC
August 2016

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S: roles in virulence and acid tolerance.

PLoS One 2014 11;9(11):e112649. Epub 2014 Nov 11.

Food Biosciences, University of Reading, Reading, United Kingdom.

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227838PMC
March 2016

Exopolysaccharide-producing probiotic Lactobacilli reduce serum cholesterol and modify enteric microbiota in ApoE-deficient mice.

J Nutr 2014 Dec 15;144(12):1956-62. Epub 2014 Oct 15.

Alimentary Pharmabiotic Centre, Teagasc, Food Research Centre Moorepark, Fermoy, Cork, Ireland

Background: Probiotic bacteria have been associated with a reduction in cardiovascular disease risk, a leading cause of death and disability.

Objectives: The aim of this study was to assess the impact of dietary administration of exopolysaccharide-producing probiotic Lactobacillus cultures on lipid metabolism and gut microbiota in apolipoprotein E (apoE)-deficient mice.

Methods: First, we examined lipid metabolism in response to dietary supplementation with recombinant β-glucan-producing Lactobacillus paracasei National Food Biotechnology Centre (NFBC) 338 expressing the glycosyltransferase (Gtf) gene from Pediococcus parvulus 2.6 (GTF), and naturally exopolysaccharide-producing Lactobacillus mucosae Dairy Product Culture Collection (DPC) 6426 (DPC 6426) compared with the non-β-glucan-producing isogenic control strain Lactobacillus paracasei NFBC 338 (PNZ) and placebo (15% wt:vol trehalose). Second, we examined the effects on the gut microbiota of dietary administration of DPC 6426 compared with placebo. Probiotic Lactobacillus strains at 1 × 10(9) colony-forming units/d per animal were administered to apoE(-/-) mice fed a high-fat (60% fat)/high-cholesterol (2% wt:wt) diet for 12 wk. At the end of the study, aortic plaque development and serum, liver, and fecal variables involved in lipid metabolism were analyzed, and culture-independent microbial analyses of cecal content were performed.

Results: Total cholesterol was reduced in serum (P < 0.001; ∼33-50%) and liver (P < 0.05; ∼30%) and serum triglyceride concentrations were reduced (P < 0.05; ∼15-25%) in mice supplemented with GTF or DPC 6426 compared with the PNZ or placebo group, respectively. In addition, dietary intervention with GTF led to increased amounts of fecal cholesterol excretion (P < 0.05) compared with all other groups. Compositional sequencing of the gut microbiota revealed a greater prevalence of Porphyromonadaceae (P = 0.001) and Prevotellaceae (P = 0.001) in the DPC 6426 group and lower proportions of Clostridiaceae (P < 0.05), Peptococcaceae (P < 0.001), and Staphylococcaceae (P < 0.01) compared with the placebo group.

Conclusion: Ingestion of exopolysaccharide-producing lactobacilli resulted in seemingly favorable improvements in lipid metabolism, which were associated with changes in the gut microbiota of mice.
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http://dx.doi.org/10.3945/jn.114.191627DOI Listing
December 2014

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

PLoS One 2014 28;9(5):e98111. Epub 2014 May 28.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098111PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037206PMC
June 2015

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract.

Microbiology (Reading) 2014 Feb 28;160(Pt 2):439-445. Epub 2013 Nov 28.

Microbiology Department, University College Cork, Ireland.

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-β, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 10(8)-2×10(8) per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-β was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.
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http://dx.doi.org/10.1099/mic.0.068767-0DOI Listing
February 2014

Two-tiered biological containment strategy for Lactococcus lactis-based vaccine or immunotherapy vectors.

Hum Vaccin Immunother 2014 6;10(2):333-7. Epub 2013 Nov 6.

Alimentary Pharmabiotic Centre; University College Cork; Cork, Ireland; Department of Microbiology; University College Cork; Cork, Ireland.

The concept of biological containment was developed as a strategy to prevent environmental dissemination of engineered live vaccine or drug delivery vehicles. A mutation in the gene encoding thymidylate synthase (thyA), a key enzyme in the pyrimidine biosynthetic pathway, has previously been shown to limit growth of L. lactis vectors under restrictive conditions. We hypothesized that further mutations in the pyrimidine biosynthetic pathway might enhance the stability and safety of live L. lactis vectors. We show that a double mutation in the genes encoding ThyA and CTP synthase (PyrG) in L. lactis confers double auxotrophy for both thymidine and cytidine. However, the combination of two mutations failed to enhance the biological containment phenotype of the engineered strain. In the absence of thymine/thymidine, the thyA mutant exhibited a strong bactericidal phenotype. However, creation of the double mutant caused the loss of this phenotype, though survival in the mouse GI tract was enhanced. The implications for biological containment of live L. lactis based delivery vectors are discussed.
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http://dx.doi.org/10.4161/hv.26954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185911PMC
January 2015

A mariner transposon-based signature-tagged mutagenesis system for the analysis of oral infection by Listeria monocytogenes.

PLoS One 2013 12;8(9):e75437. Epub 2013 Sep 12.

Department of Microbiology, University College Cork, Cork, Ireland.

Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM) system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610), lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842), lmOh7858_2579 (encoding the HupDGC hemin transport system) and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system). We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771922PMC
June 2014

In vivo assessment of growth and virulence gene expression during commensal and pathogenic lifestyles of luxABCDE-tagged Enterococcus faecalis strains in murine gastrointestinal and intravenous infection models.

Appl Environ Microbiol 2013 Jul 19;79(13):3986-97. Epub 2013 Apr 19.

Department of Chemistry, Biotechnology and Food Science, Laboratory of Microbial Gene Technology and Food Microbiology, The Norwegian University of Life Sciences, Ås, Norway.

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R(2) > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10(9) CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ~400- and ~900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.
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http://dx.doi.org/10.1128/AEM.00831-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697570PMC
July 2013

Virulence aspects of Listeria monocytogenes LO28 high pressure-resistant variants.

Microb Pathog 2013 Jun-Jul;59-60:48-51. Epub 2013 Apr 17.

Top Institute Food and Nutrition (TIFN), P.O. Box 557, 6700 AN Wageningen, The Netherlands.

High pressure treatment is a novel food processing approach for reducing pathogens in foods and food ingredients. However, relatively little is known about the pathogenic potential of organisms that survive the treatment. Twelve previously isolated and characterized variants of Listeria monocytogenes LO28 obtained after a high pressure treatment were assessed for their virulence potential and antibiotic susceptibility. Ten variants showed attenuated virulence while two variants retained full virulence in a mouse model of infection. Seven of the attenuated variants demonstrated a reduction in virulence factor activity. Compared to the wild type, all variants exhibited similar or increased susceptibility to multiple antibiotics commonly used in listeriosis treatment.
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http://dx.doi.org/10.1016/j.micpath.2013.04.007DOI Listing
November 2013

In vivo activity of nisin A and nisin V against Listeria monocytogenes in mice.

BMC Microbiol 2013 Feb 1;13:23. Epub 2013 Feb 1.

Department of Microbiology, University College Cork, Cork, Ireland.

Background: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo.

Results: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model.More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 10(5) cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen.

Conclusion: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.
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http://dx.doi.org/10.1186/1471-2180-13-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616995PMC
February 2013

A mutant in the Listeria monocytogenes Fur-regulated virulence locus (frvA) induces cellular immunity and confers protection against listeriosis in mice.

J Med Microbiol 2013 Feb 25;62(Pt 2):185-190. Epub 2012 Oct 25.

School of Pharmacy, University College Cork, Cork, Ireland.

Listeria monocytogenes is a Gram-positive intracellular pathogen that is responsible for listeriosis, a potentially fatal, food-borne illness. Due to its cytoplasmic location during infection, this pathogen can mediate a long-lasting cellular immune response, which makes attenuated strains strong candidates for vaccine development. Recently, our group identified and characterized frvA (Fur-regulated virulence factor A), and deletion of this gene resulted in disruption of iron homeostasis and a strong attenuation in virulence. Despite significant attenuation in the mouse infection model, the frvA mutant was capable of intracellular growth in antigen-presenting cells. Indeed, mice immunized with L. monocytogenes ΔfrvA were able to effectively stimulate specific CD8(+) T cells to the listerial epitopes LLO(91-99) and P60(217-225) at levels comparable with L. monocytogenes strain EGDe. Most notably, mice immunized with ΔfrvA then subsequently challenged with the wild-type strain were completely protected from listerial infection. On the basis of these results, we advocate the use of ΔfrvA as a live attenuated listerial vaccine, and propose that this mutant may serve as a platform for the development of a future vaccine delivery vehicle.
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http://dx.doi.org/10.1099/jmm.0.049114-0DOI Listing
February 2013

Residual antibiotics disrupt meat fermentation and increase risk of infection.

mBio 2012 28;3(5):e00190-12. Epub 2012 Aug 28.

Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.

Unlabelled: Fermented sausages, although presumed safe for consumption, sometimes cause serious bacterial infections in humans that may be deadly. Not much is known about why and when this is the case. We tested the hypothesis that residual veterinary antibiotics in meat can disrupt the fermentation process, giving pathogenic bacteria a chance to survive and multiply. We found that six commercially available starter cultures were susceptible to commonly used antibiotics, namely, oxytetracycline, penicillin, and erythromycin. In meat, statutorily tolerable levels of oxytetracycline and erythromycin inhibited fermentation performance of three and five of the six starter cultures, respectively. In model sausages, the disruption of meat fermentation enhanced survival of the pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium compared to successful fermentations. Our work reveals an overlooked risk associated with the presence of veterinary drugs in meat.

Importance: Antibiotics have for a long time been used as growth promoters in farm animals, and while they are banned as such in Europe, their clinical use in farm animals still accounts for the majority of consumption. Here, we examined how acceptable levels of antibiotics in meat influence fermentation. Our results show that commonly used bacterial starter cultures are sensitive to residual antibiotics at or near statutorily tolerable levels, and as a result, processed sausages may indeed contain high levels of pathogens. Our findings provide a possible explanation for outbreaks and disease cases associated with consumption of fermented sausages and offer yet another argument for limiting the use of antimicrobials in farm animals.
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http://dx.doi.org/10.1128/mBio.00190-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445968PMC
January 2013

H5N1 moratorium: Missing the point.

Authors:
Pat G Casey

Bioeng Bugs 2012 May-Jun;3(3):144. Epub 2012 May 1.

The recent moratorium on research using engineered H5N1 influenza viruses is a move which cannot achieve its aims as it ignores the prevalence of molecular biology.
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http://dx.doi.org/10.4161/bbug.20004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370931PMC
October 2012

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe.

Microbiology (Reading) 2012 Jul 13;158(Pt 7):1684-1693. Epub 2012 Apr 13.

School of Pharmacy, University College Cork, Cork, Ireland.

Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, Listeria monocytogenes is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, gcpE, lytB (encoding proteins in the MEP pathway) and hmgR (encoding a protein in the mevalonate pathway). We demonstrate that the hmgR gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact hmgR gene was supplied in trans using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate hmgR mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in L. monocytogenes EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.
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http://dx.doi.org/10.1099/mic.0.056069-0DOI Listing
July 2012

Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells.

mBio 2012 6;3(2):e00029-12. Epub 2012 Mar 6.

TEAGASC Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.

Unlabelled: Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (φNH-4) and a podovirus (φMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 10(7) to 2 × 10(7) P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients.

Importance: Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.
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http://dx.doi.org/10.1128/mBio.00029-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302570PMC
June 2012

A putative P-type ATPase required for virulence and resistance to haem toxicity in Listeria monocytogenes.

PLoS One 2012 21;7(2):e30928. Epub 2012 Feb 21.

Alimentary Pharmabiotic Centre, Department of Microbiology, University College Cork, Cork, Ireland.

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030928PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3283593PMC
June 2012

The Lantibiotic Lacticin 3147 Prevents Systemic Spread of Staphylococcus aureus in a Murine Infection Model.

Int J Microbiol 2012 12;2012:806230. Epub 2012 Jan 12.

Department of Microbiology, University College Cork, College Road, Cork, Ireland.

The objective of this study was to investigate the in vivo activity of the lantibiotic lacticin 3147 against the luminescent Staphylococcus aureus strain Xen 29 using a murine model. Female BALB/c mice (7 weeks old, 17 g) were divided into groups (n = 5) and infected with the Xen 29 strain via the intraperitoneal route at a dose of 1 × 10(6) cfu/animal. After 1.5 hr, the animals were treated subcutaneously with doses of phosphate-buffered saline (PBS; negative control) or lacticin 3147. Luminescent imaging was carried 3 and 5 hours postinfection. Mice were then sacrificed, and the levels of S. aureus Xen 29 in the liver, spleen, and kidneys were quantified. Notably, photoluminescence and culture-based analysis both revealed that lacticin 3147 successfully controlled the systemic spread of S. aureus in mice thus indicating that lacticin 3147 has potential as a chemotherapeutic agent for in vivo applications.
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http://dx.doi.org/10.1155/2012/806230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265090PMC
August 2012

A novel host-responsive sensor mediates virulence and type III secretion during Pseudomonas aeruginosa-host cell interactions.

Microbiology (Reading) 2012 Apr 19;158(Pt 4):1057-1070. Epub 2012 Jan 19.

BIOMERIT Research Centre, Department of Microbiology, University College Cork, Cork, Ireland.

Sensitive sensory mechanisms are instrumental in affording Pseudomonas aeruginosa the capacity to establish diverse yet severe human infections, which can manifest themselves in long-term untreatable disease. The ability of P. aeruginosa to tightly regulate gene expression and virulence factor production, in response to activation of these sensory components, enables the pathogen to sustain infection despite the host immune response and aggressive antibiotic treatment. Although a number of factors are recognized as playing a role in early infection, very little is known regarding the sensors involved in this process. In this study, we identified P. aeruginosa PA3191 as a novel host-responsive sensor that plays a key role during P. aeruginosa-host interactions and is required for optimum colonization and dissemination in a mouse model of infection. We demonstrated that PA3191 contributed to modulation of the type III secretion system (T3SS) in response to host cells and T3SS-inducing conditions in vitro. PA3191 (designated GtrS) acted in concert with the response regulator GltR to regulate the OprB transport system and subsequently carbon metabolism. Through this signal transduction pathway, T3SS activation was mediated via the RsmAYZ regulatory cascade and involved the global anaerobic response regulator Anr.
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http://dx.doi.org/10.1099/mic.0.056127-0DOI Listing
April 2012

Investigation of the role of ZurR in the physiology and pathogenesis of Listeria monocytogenes.

FEMS Microbiol Lett 2012 Feb 20;327(2):118-25. Epub 2011 Dec 20.

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.

Listeria monocytogenes is a Gram positive pathogen that is ubiquitous in the environment. It is a facultative anaerobic rod that causes listeriosis, a disease with potentially lethal consequences for susceptible individuals. During infection, the pathogen is capable of sequestering metal ions to act as vital biocatalysts in cellular processes. The zinc uptake regulator (ZurR) is predicted to coordinate uptake of zinc from the external environment. An in-frame deletion of the zurR gene resulted in a mutant exhibiting a small colony phenotype and a smaller cell size. The zurR mutant was unaffected under conditions of zinc limitation but demonstrated increased sensitivity to toxic levels of zinc. The mutant also demonstrated a significant (1-log) reduction in virulence potential in the murine model of infection. Using a bioinformatic approach, we identified a number of potentially Zur-regulated genes in the genome of L. monocytogenes. Quantitative RT-PCR demonstrated significant de-repression of zurA, lmo0153, and lmo1671 in the zurR mutant background indicating that these putative transporters are ZurR regulated.
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http://dx.doi.org/10.1111/j.1574-6968.2011.02472.xDOI Listing
February 2012

E. coli O104:H4: social media and the characterization of an emerging pathogen.

Bioeng Bugs 2011 Jul-Aug;2(4):189-93. Epub 2011 Jul 1.

Alimentary Pharmabiotic Centre and Microbiology Department, University College Cork, Ireland.

The 2011 German E. coli O104:H4 outbreak resulted in thousands of cases of enterohaemorrhagic illness, with approximately 25% of these progressing to develop haemolytic uraemic syndrome (HUS). This high rate of progression to HUS was the first indicator that the bacterial cause of illness was not a typical enterohaemorrhagic E. coli (EHEC) strain. Collaborative bioinformatic analysis while the outbreak was still in progress indicated that the O104:H4 strain was in fact an enteroaggregative E. coli (EAEC) strain which had acquired genes for the production of Shiga - like toxin.
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http://dx.doi.org/10.4161/bbug.2.4.16961DOI Listing
March 2012

Factors affecting survival of Listeria monocytogenes and Listeria innocua in soil samples.

Arch Microbiol 2011 Nov 25;193(11):775-85. Epub 2011 May 25.

Department of Microbiology, University College Cork, Cork, Ireland.

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.
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http://dx.doi.org/10.1007/s00203-011-0716-7DOI Listing
November 2011

The truncated phage lysin CHAP(k) eliminates Staphylococcus aureus in the nares of mice.

Bioeng Bugs 2010 Nov-Dec;1(6):404-7

Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland.

The endolysin LysK derived from staphylococcal phage K has previously been shown to have two enzymatic domains, one of which is an N-acetylmuramoyl-L-alanine amidase and the other a cysteine/histidine-dependant amidohydrolase/peptidase designated CHAP(k). The latter, when cloned as a single-domain truncated enzyme, is conveniently overexpressed in a highly-soluble form. This enzyme was shown to be highly active in vitro against live cell suspensions of S. aureus. In the current study, the IVIS imaging system was used to demonstrate the effective elimination of a lux labeled S. aureus from the nares of BALB/c mice.
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http://dx.doi.org/10.4161/bbug.1.6.13422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056090PMC
October 2011

Efficacy of a Lactococcus lactis ΔpyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes.

Bioeng Bugs 2010 Jan-Feb;1(1):66-74

School of Pharmacy, University College Cork, Cork, Ireland.

Listeria monocytogenes is a significant food-borne pathogen and the causative agent of listeriosis, a disease which manifests as meningitis in immunocompromised adults or infection of the fetus and miscarriage in pregnant women. We have previously used Lactococcus lactis, a GRAS (Generally Regarded As Safe) organism, as a vaccine vector against listeriosis by engineering plasmid-mediated expression of the immunodominant antigen from L. monocytogenes, listeriolysin O (LLO). However, the environmental release of an engineered vaccine vector carrying a replicating plasmid during clinical usage may raise safety concerns. Here we describe the integration of the LLO gene (hly) into the L. lactis chromosome through homologous double crossover to allow stable expression, in order to avoid the use of antibiotic selection markers and to eliminate the requirement for a plasmid-based system. The approach was designed to simultaneously eliminate the pyrG gene encoding the CTP synthase which is responsible for converting UTP to CTP in a unique step in the de novo pyrimidine synthesis in L. lactis. This gene was targeted in order to restrict bacterial replication outside of the host (biological containment). The resulting cytidine auxotroph was able to secrete LLO constitutively and could elicit LLO(91-99)-specific CD8(+) T lymphocytes in the murine infection model. Moreover, protection against lethal challenge with L. monocytogenes was accomplished after intraperitoneal (IP) vaccination with the constructed strain. The implications for the use of cytidine auxotropy in biological containment are discussed.
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http://dx.doi.org/10.4161/bbug.1.1.10284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035148PMC
October 2011

Fate and efficacy of lacticin 3147-producing Lactococcus lactis in the mammalian gastrointestinal tract.

FEMS Microbiol Ecol 2011 Jun 7;76(3):602-14. Epub 2011 Mar 7.

Teagasc, Food Research Centre Moorepark, Fermoy, Co. Cork, Ireland.

Gastrointestinal survival of the bacteriocin-producing strain, Lactococcus lactis DPC6520, was evaluated systematically in vitro and in vivo with a view to using this strain to deliver biologically active lacticin 3147, a broad-spectrum bacteriocin, to the gut. The activity of the lacticin 3147 producer was also evaluated against two clinically relevant pathogens: Clostridium difficile and Listeria monocytogenes. When suspended in an appropriate matrix, the lactococcal strain is capable of surviving simulated gastrointestinal juices similar to the porcine probiotic, Lactobacillus salivarius DPC6005. Upon administration of L. lactis DPC6520 to pigs (n=4), excretion rates of ∼10(2) -10(5) CFU g(-1) faeces were observed by day 5. Although passage through the gastrointestinal tract (GIT) did not affect lacticin 3147 production by L. lactis DPC6520 isolates, activity was undetectable in faecal samples by an agar well diffusion assay. Furthermore, L. lactis DPC6520 had no inhibitory effect on C. difficile or other bacterial populations in a human distal colon model, while lactococcal counts declined 10,000-fold over 24 h. The lacticin 3147 producer failed to prevent L. monocytogenes infection in a mouse model, even though a mean L. lactis DPC6520 count of 4.7 × 10(4) CFU g(-1) faeces was obtained over the 5-day administration period. These data demonstrate that L. lactis DPC6520 is capable of surviving transit through the GIT, and yet lacks antimicrobial efficacy in the models of infection used.
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http://dx.doi.org/10.1111/j.1574-6941.2011.01069.xDOI Listing
June 2011