Key publications
Mitchelmore PJ, Withers NJ, Sheldon CD, Scotton CJ, Brown AR (2021). Culture-independent multilocus sequence typing of Pseudomonas aeruginosa for cross-infection screening. Diagnostic Microbiology and Infectious Disease, 100(1), 115315-115315.
Mitchelmore PJ, Randall J, Bull MJ, Moore KA, O'Neill PA, Paszkiewicz K, Mahenthiralingam E, Scotton CJ, Sheldon CD, Withers NJ, et al (2018). Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.
Thorax,
73(7), 677-679.
Abstract:
Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort
While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.
Abstract.
Francis V, Waters EM, Finton-James SE, Gori A, Kadioglu A, Brown A, Porter SL (2018). Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa. Nature Communications, 9, 2219-2219.
McLean CJ, Marles-Wright J, Custodio R, Lowther J, Kennedy AJ, Pollock J, Clarke DJ, Brown AR, Campopiano DJ (2017). Characterization of homologous sphingosine-1- phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.
Journal of Lipid Research,
58(1), 137-150.
Abstract:
Characterization of homologous sphingosine-1- phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei
Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5?-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzymecoupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.-McLean, C. J. Marles-Wright, R. Custodio, J. Lowther, A. J. Kennedy, J. Pollock, D. J. Clarke, A. R. Brown, and D. J. Campopiano. Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.
Abstract.
Custódio R, McLean CJ, Scott AE, Lowther J, Kennedy A, Clarke DJ, Campopiano DJ, Sarkar-Tyson M, Brown AR (2016). Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei.
Molecular Microbiology,
102(6), 1004-1019.
Abstract:
Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei
Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.
Abstract.
Denman CC, Robinson MT, Sass AM, Mahenthiralingam E, Brown AR (2014). Growth on mannitol-rich media elicits a genomewide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner.
Microbiology (United Kingdom),
160(PART 1), 187-197.
Abstract:
Growth on mannitol-rich media elicits a genomewide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner
In common with other members of the Burkholderia cepacia complex (BCC), Burkholderia multivorans is capable of producing exopolysaccharide (EPS) when grown on certain mannitolrich media. The significance of the resulting mucoid phenotype and the genome-wide response to mannitol has never been characterized despite its clinical relevance following the approval of a dried-powder preparation of mannitol as an inhaled osmolyte therapy for cystic fibrosis (CF) patients. In the present study we defined the transcriptional response of B. multivorans ATCC 17616, a model genome-sequenced strain of environmental origin, to growth on mannitol-rich yeast extract media (MYEM). EPS-dependent and -independent impact of MYEM on virulenceassociated traits was assessed in both strain ATCC 17616 and the CF isolate B. multivorans C1576. Our studies revealed a significant transcriptional response to MYEM encompassing approximately 23% of predicted genes within the genome. Strikingly, this transcriptional response identified that EPS induction occurs in ATCC 17616 without the upregulation of the bce-I and bce-II EPS gene clusters, despite their pivotal role in EPS biosynthesis. of approximately 20 differentially expressed putative virulence factors, 16 exhibited upregulation including flagella, ornibactin, oxidative stress proteins and phospholipases. MYEM-grown B. multivorans also exhibited enhanced motility, biofilm formation and epithelial cell invasion. In contrast to these potential virulence enhancements, MYEM-grown B. multivorans C1576 showed attenuated virulence in the Galleria mellonella infection model. All of the observed phenotypic responses occurred independently of EPS production, highlighting the profound impact that mannitol-based growth has on the physiology and virulence of B. multivorans. © 2014 SGM.
Abstract.
Publications by year
2021
Mitchelmore PJ, Withers NJ, Sheldon CD, Scotton CJ, Brown AR (2021). Culture-independent multilocus sequence typing of Pseudomonas aeruginosa for cross-infection screening. Diagnostic Microbiology and Infectious Disease, 100(1), 115315-115315.
2018
Mitchelmore PJ, Randall J, Bull MJ, Moore KA, O'Neill PA, Paszkiewicz K, Mahenthiralingam E, Scotton CJ, Sheldon CD, Withers NJ, et al (2018). Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.
Thorax,
73(7), 677-679.
Abstract:
Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort
While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.
Abstract.
Francis V, Waters EM, Finton-James SE, Gori A, Kadioglu A, Brown A, Porter SL (2018). Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa. Nature Communications, 9, 2219-2219.
2017
McLean CJ, Marles-Wright J, Custodio R, Lowther J, Kennedy AJ, Pollock J, Clarke DJ, Brown AR, Campopiano DJ (2017). Characterization of homologous sphingosine-1- phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.
Journal of Lipid Research,
58(1), 137-150.
Abstract:
Characterization of homologous sphingosine-1- phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei
Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5?-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzymecoupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.-McLean, C. J. Marles-Wright, R. Custodio, J. Lowther, A. J. Kennedy, J. Pollock, D. J. Clarke, A. R. Brown, and D. J. Campopiano. Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.
Abstract.
2016
Custódio R, McLean CJ, Scott AE, Lowther J, Kennedy A, Clarke DJ, Campopiano DJ, Sarkar-Tyson M, Brown AR (2016). Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei.
Molecular Microbiology,
102(6), 1004-1019.
Abstract:
Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of Burkholderia pseudomallei
Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.
Abstract.
2014
Denman CC, Robinson MT, Sass AM, Mahenthiralingam E, Brown AR (2014). Growth on mannitol-rich media elicits a genomewide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner.
Microbiology (United Kingdom),
160(PART 1), 187-197.
Abstract:
Growth on mannitol-rich media elicits a genomewide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner
In common with other members of the Burkholderia cepacia complex (BCC), Burkholderia multivorans is capable of producing exopolysaccharide (EPS) when grown on certain mannitolrich media. The significance of the resulting mucoid phenotype and the genome-wide response to mannitol has never been characterized despite its clinical relevance following the approval of a dried-powder preparation of mannitol as an inhaled osmolyte therapy for cystic fibrosis (CF) patients. In the present study we defined the transcriptional response of B. multivorans ATCC 17616, a model genome-sequenced strain of environmental origin, to growth on mannitol-rich yeast extract media (MYEM). EPS-dependent and -independent impact of MYEM on virulenceassociated traits was assessed in both strain ATCC 17616 and the CF isolate B. multivorans C1576. Our studies revealed a significant transcriptional response to MYEM encompassing approximately 23% of predicted genes within the genome. Strikingly, this transcriptional response identified that EPS induction occurs in ATCC 17616 without the upregulation of the bce-I and bce-II EPS gene clusters, despite their pivotal role in EPS biosynthesis. of approximately 20 differentially expressed putative virulence factors, 16 exhibited upregulation including flagella, ornibactin, oxidative stress proteins and phospholipases. MYEM-grown B. multivorans also exhibited enhanced motility, biofilm formation and epithelial cell invasion. In contrast to these potential virulence enhancements, MYEM-grown B. multivorans C1576 showed attenuated virulence in the Galleria mellonella infection model. All of the observed phenotypic responses occurred independently of EPS production, highlighting the profound impact that mannitol-based growth has on the physiology and virulence of B. multivorans. © 2014 SGM.
Abstract.
2013
Denman CC, Brown AR (2013). Mannitol promotes adherence of an outbreak strain of Burkholderia multivorans via an exopolysaccharide-independent mechanism that is associated with upregulation of newly identified fimbrial and afimbrial adhesins.
Microbiology (United Kingdom),
159(4), 771-781.
Abstract:
Mannitol promotes adherence of an outbreak strain of Burkholderia multivorans via an exopolysaccharide-independent mechanism that is associated with upregulation of newly identified fimbrial and afimbrial adhesins
Burkholderia multivorans, a member of the Burkholderia cepacia complex (Bcc), is an important pathogen of the cystic fibrosis (CF) lung. Mannitol, approved as an inhaled osmolyte therapy for use in CF patients, promotes exopolysaccharide (EPS) production by the Bcc. In the present study, we investigated the role of mannitol-induced EPS in the adherence of B. multivorans. We report that mannitol promoted adherence of two representative B. multivorans strains. However, whilst this enhanced adherence was largely EPS-dependent in an environmental isolate, it was EPS-independent within a CF outbreak strain, suggesting strain-to-strain variation in adhesins. Genome sequencing of the outbreak strain enabled the identification of two distinct loci encoding putative fimbrial and afimbrial adhesins. The putative fimbriae-encoding locus was found to be widely distributed amongst clinical and environmental B. multivorans. In contrast, the locus encoding the putative afimbrial adhesin (of the filamentous haemagglutinin family, FHA) was restricted to clinical isolates. Both loci contributed to biofilm formation and mucin adherence. Furthermore, we report that mannitol promoted expression of both loci, and that the locus encoding the putative FHA-family adhesin is a key determinant of the enhanced adherence observed following growth in mannitol. Our studies provide the first characterization, to our knowledge, of B. multivorans adhesins, and in so doing highlight the strain-dependent role of EPS in the Bcc and the difficulties in assigning phenotypic traits to Bcc EPS due to the wider response to mannitol. Our observations also highlight the need to monitor the microbiological effects of inhaled mannitol therapy in Bcc-infected CF patients. © 2013 SGM.
Abstract.
2010
Clarke DJ, Ortega XP, Mackay CL, Valvano MA, Govan JRW, Campopiano DJ, Langridge-Smith P, Brown AR (2010). Subdivision of the bacterioferritin comigratory protein family of bacterial peroxiredoxins based on catalytic activity.
Biochemistry,
49(6), 1319-1330.
Abstract:
Subdivision of the bacterioferritin comigratory protein family of bacterial peroxiredoxins based on catalytic activity
Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across allmembers of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia.We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity froma 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity. © 2010 American Chemical Society.
Abstract.
2009
Clarke DJ, Mackay CL, Campopiano DJ, Langridge-Smith P, Brown AR (2009). Interrogating the molecular details of the peroxiredoxin activity of the Escherichia coli bacterioferritin comigratory protein using high-resolution mass spectrometry.
Biochemistry,
48(18), 3904-3914.
Abstract:
Interrogating the molecular details of the peroxiredoxin activity of the Escherichia coli bacterioferritin comigratory protein using high-resolution mass spectrometry.
Bacterioferritin comigratory protein (BCP) is a bacterial thioredoxin-dependent thiol peroxidase that reduces a variety of peroxide substrates. Using high-resolution Fourier transform ion cyclotron resonance mass spectrometry coupled with top-down fragmentation techniques, we have analyzed the mechanistic details of hydrogen peroxide reduction by E. coli BCP. We show here that catalysis occurs via an atypical two-cysteine peroxiredoxin pathway. A transient sulfenic acid is initially formed on Cys-45, before resolution by the formation of an intramolecular disulfide bond between Cys-45 and Cys-50. This oxidized BCP intermediate is shown to be a substrate for reduction by thioredoxin, completing the catalytic cycle. Although we invoke Cys-50 in the catalytic cycle of Escherichia coli bacterioferritin comigratory protein (BCP), a previous study had shown that this residue was not absolutely required for peroxiredoxin activity. In order to explain these apparently conflicting phenomena, we analyzed the reaction of a C50S BCP mutant with peroxide. We show that this mutant BCP enzyme adopts a different and novel mechanistic pathway. The C50S BCP mutant reacts with peroxide to form a sulfenic acid on Cys-45, in the same manner as wild-type BCP. However, the nascent intermediate is then resolved by reaction with Cys-45 from a second BCP molecule, resulting in a dimeric intermediate containing an intermolecular disulfide bond. We further show that this novel resolving complex is a substrate for reduction by thioredoxin. The importance of our results in furthering the understanding of catalysis within BCP family is discussed.
Abstract.
Author URL.
2008
Bartholdson SJ, Brown AR, Mewburn BR, Clarke DJ, Fry SC, Campopiano DJ, Govan JR (2008). Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.
Microbiology,
154(Pt 8), 2513-2521.
Abstract:
Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.
The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.
Abstract.
2007
Ortega XP, Cardona ST, Brown AR, Loutet SA, Flannagan RS, Campopiano DJ, Govan JR, Valvano MA (2007). A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability.
J Bacteriol.,
189(9), 3639-3644.
Abstract:
A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability
Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N
Abstract.
Brown AR, Govan JRW (2007). Assessment of fluorescent in situ hybridization and PCR-based methods for rapid identification of Burkholderia cepacia complex organisms directly from sputum samples.
J Clin Microbiol,
45(6), 1920-1926.
Abstract:
Assessment of fluorescent in situ hybridization and PCR-based methods for rapid identification of Burkholderia cepacia complex organisms directly from sputum samples.
Several species within the Burkholderia cepacia complex (BCC) have emerged as significant opportunistic pathogens of patients with cystic fibrosis (CF). BCC infection is typically associated with a poor clinical prognosis and decreased survival. These factors, combined with the existence of highly transmissible epidemic strains, have resulted in strict segregation of BCC- and non-BCC-infected patients to minimize cross infection. Accurate and rapid diagnosis of infections is essential to enable appropriate patient management. However, the rapidly evolving taxonomy of BCC poses a considerable challenge to diagnostics. In the present study, we assessed a commercially available fluorescent in situ hybridization (FISH) assay (seaFAST Cystic Fibrosis I kit) and a novel rRNA gene-based PCR assay for the rapid identification of BCC-positive sputa, irrespective of the BCC species. We report that, while the FISH assay fails to identify all BCC species, it does identify the majority of species, including the two most clinically relevant species, B. multivorans and B. cenocepacia. The sensitivity of the assay applied to sputum was limited by nonspecific background fluorescence. While sputum processing was optimized to minimize background, the resulting sensitivity for BCC detection was 8 x 10(5) CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 10(4) CFU/ml when applied to sputum. This novel rRNA gene-based assay is currently the most sensitive BCC-specific PCR assay for the detection of BCC direct from clinical samples and as such is a valuable addition to the field of BCC diagnostics.
Abstract.
Author URL.
Brown AR, Blanco ARA, Miele G, Hawkins SA, Hopkins J, Fazakerley JK, Manson J, Clinton M (2007). Differential expression of erythroid genes in prion disease.
Biochem Biophys Res Commun,
364(2), 366-371.
Abstract:
Differential expression of erythroid genes in prion disease.
We previously reported reduced expression of erythroid-associated factor (ERAF) within haematopoietic tissues of rodent scrapie models, suggesting an unrecognized role for the erythroid lineage in prion disease. In the present study, we compared the expression of a panel of erythroid genes within four murine scrapie models and five virus infection models with parallels to prion disease pathogenesis. We report that differential expression of erythroid genes is not limited to ERAF, and is a common feature of murine scrapie, dependent on host expression of cellular prion protein. In contrast, erythroid gene expression was not altered following virus infection. Whilst these results further implicate cells of the erythroid lineage in the peripheral pathogenesis of prion disease, analysis of blood from BSE-infected cattle and scrapie-infected sheep reveals that the extent of differential expression of erythroid genes within peripheral blood is not sufficient to provide a discriminatory diagnostic test.
Abstract.
Author URL.
Govan JRW, Brown AR, Jones AM (2007). Evolving epidemiology of Pseudomonas aeruginosa and the Burkholderia cepacia complex in cystic fibrosis lung infection.
Future Microbiol,
2(2), 153-164.
Abstract:
Evolving epidemiology of Pseudomonas aeruginosa and the Burkholderia cepacia complex in cystic fibrosis lung infection.
The morbidity and mortality of patients with cystic fibrosis (CF) is primarily determined by chronic and debilitating lung infections caused by a surprisingly narrow spectrum of bacterial pathogens. Pseudomonas aeruginosa is by far the most prevalent life-threatening CF pathogen. In the absence of aggressive early therapy, it infects the majority of adult patients and determines long-term survival. The epidemiology of CF pulmonary infections continues to evolve. Amongst the most recent CF pathogens to have emerged are a group of closely related bacteria, known as the Burkholderia cepacia complex. These organisms are a particular challenge due to inherent antibiotic resistance, the potential for patient-to-patient spread, and the risk of 'cepacia syndrome', a rapid fulminating pneumonia sometimes accompanied by bacteremia. Strict cross-infection control was prompted by early epidemiological experience of the B. cepacia complex and is essential in the management of all CF pathogens.
Abstract.
Author URL.
2005
Brown AR, Rebus S, McKimmie CS, Robertson K, Williams A, Fazakerley JK (2005). Gene expression profiling of the preclinical scrapie-infected hippocampus.
Biochem Biophys Res Commun,
334(1), 86-95.
Abstract:
Gene expression profiling of the preclinical scrapie-infected hippocampus.
The molecular events that underlie prion disease neuropathology remain poorly defined. Within the hippocampus of the ME7/CV mouse scrapie model, profound CA1 neuronal loss occurs between 160 and 180 days post-infection (dpi). To elucidate the molecular events that may contribute to this neuronal loss, we have applied Affymetrix high-density oligonucleotide probe arrays to the study of ME7-infected hippocampal gene expression at 170 dpi. The study has identified 78 genes that are differentially expressed greater than 1.5-fold within the preclinical ME7-infected hippocampus prior to the profound late stage glial cell activation. The results indicate oxidative and endoplasmic reticulum (ER) stress, activated ER and mitochondrial apoptosis pathways, and activated cholesterol biosynthesis within the scrapie-infected hippocampus, and offer insight into the molecular events which underlie the neuropathology.
Abstract.
Author URL.
2004
Brown AR, Webb J, Rebus S, Williams A, Fazakerley JK (2004). Identification of up-regulated genes by array analysis in scrapie-infected mouse brains.
Neuropathol Appl Neurobiol,
30(5), 555-567.
Abstract:
Identification of up-regulated genes by array analysis in scrapie-infected mouse brains.
The major neuropathological features of the transmissible spongiform encephalopathies (TSEs) are well documented, however, the underlying molecular events are poorly defined. We have applied cDNA expression arrays and quantitative RT-PCR to the study of gene expression in the brain, and more specifically in the hippocampus, of the well-characterized ME7/CV mouse model of scrapie. The number of genes showing consistent, scrapie-associated changes in expression was limited, and was primarily restricted to glial-associated genes. Increased expression of genes encoding glial fibrillary acidic protein, vimentin, complement component 1q (alpha and beta polypeptides), cathepsin D, clusterin and cystatin C was evident in the hippocampus from 170 days after inoculation (dpi), with expression increasing thereafter to terminal disease (225-235 dpi). Elevation of gene expression preceded clinical disease by approximately 30 days, and coincided with a 20-day period in the ME7/CV model during which 50% of the CA1 hippocampal neurones are lost. Increased expression of cystatin C, an inhibitor of lysosomal cysteine proteases, is a novel finding in the context of TSE neuropathology and was confirmed by Western analysis and immunocytochemistry.
Abstract.
Author URL.
2003
Brown AR, Webb J, Rebus S, Walker R, Williams A, Fazakerley JK (2003). Inducible cytokine gene expression in the brain in the ME7/CV mouse model of scrapie is highly restricted, is at a strikingly low level relative to the degree of gliosis and occurs only late in disease.
J Gen Virol,
84(Pt 9), 2605-2611.
Abstract:
Inducible cytokine gene expression in the brain in the ME7/CV mouse model of scrapie is highly restricted, is at a strikingly low level relative to the degree of gliosis and occurs only late in disease.
The temporal course of cerebral cytokine gene expression was investigated in the ME7/CV murine scrapie model to determine any association with neuropathological events. Analysis by RNase protection assay (RPA) demonstrated no transcripts for ILs 2, 3, 4, 5, 6, 7, 10, 12p40 and 13, granulocyte macrophage colony-stimulating factor, IFN-gamma or lymphotoxin-alpha at any time during the course of this disease. Transcripts for transforming growth factor-beta 1 were constitutively expressed in both control and scrapie-infected brain and were elevated at terminal disease. RPA and quantitative real-time RT-PCR detected low levels of transcripts for IL-1 alpha, IL-1 beta and TNF alpha in scrapie-infected brain but only IL-1 beta was elevated consistently in all mice studied. Although glial cell activation within the hippocampus was evident from 100 days post-infection (p.i.), elevated IL-1 beta transcripts (and immunoreactivity) were evident from 180 days p.i. around the time of hippocampal pyramidal neuron loss, and increased steadily thereafter to reach a 3.5-fold increase at terminal disease. Even at their maximum, levels of these transcripts were disproportionately low relative to the degree of glial cell activation. It is concluded that cytokine gene expression in the ME7 scrapie-infected mouse brain, relative to the degree of reactive gliosis, is highly restricted, temporally late and disproportionately low.
Abstract.
Author URL.
2001
Brown AR, Townsley AC, Amyes SG (2001). Diversity of Tn1546 elements in clinical isolates of glycopeptide-resistant enterococci from Scottish hospitals.
Antimicrob Agents Chemother,
45(4), 1309-1311.
Abstract:
Diversity of Tn1546 elements in clinical isolates of glycopeptide-resistant enterococci from Scottish hospitals.
The Tn1546-related elements of 48 Van glycopetide-resistant enterococci were compared. Ten distinct Tn1546 types were identified with variation primarily due to IS1542 and IS1216V-like insertions. Clonal isolates frequently differed in their Tn1546 type, indicating instability of Tn1546-related elements. A putative hybrid promoter was identified, generated upstream of vanR by the insertion of IS1542. The presence of this hybrid promoter was associated with constitutive expression of the van genes and elevated teicoplanin resistance.
Abstract.
Author URL.
2000
Nelson RR, McGregor KF, Brown AR, Amyes SG, Young H (2000). Isolation and characterization of glycopeptide-resistant enterococci from hospitalized patients over a 30-month period.
J Clin.Microbiol.,
38(6), 2112-2116.
Abstract:
Isolation and characterization of glycopeptide-resistant enterococci from hospitalized patients over a 30-month period
In February 1996, a Hospital Infection Control Practices Advisory Committee-style screening program was commenced to isolate and subsequently characterize glycopeptide-resistant enterococci (GRE) from patients at a hospital trust in Glasgow, Scotland. Over the next 30 months, GRE were isolated from 154 patients. GRE were isolated from patients in traditionally high-risk areas such as the renal unit and intensive care unit and also in areas considered to be lower risk, including medical wards and associated long-stay geriatric hospitals. The majority (90%) of isolates were Enterococcus faecium vanB. The remaining isolates consisted of seven E. faecalis (vanA), three E. gallinarum (vanC), and a further six E. faecium (five vanA, one both vanA and vanB) isolates. Analysis of SmaI-digested DNA by pulsed-field gel electrophoresis revealed that 34 of 40 (85%) VanB E. faecium isolates were identical or closely related, while 11 of 13 (85%) VanA GRE were distinct. High-level aminoglycoside resistance was seen in less than 8% of isolates. VanB E. faecium isolates were almost uniformly resistant to ampicillin and tetracycline. In this study, GRE have been isolated over a prolonged period from a broad range of patients. Glycopeptide resistance within the study hospital trust appeared to be mainly due to the clonal dissemination of a single strain of E. faecium VanB
Abstract.
1998
Brown AR, Amyes SG, Paton R, Plant WD, Stevenson GM, Winney RJ, Miles RS (1998). Epidemiology and control of vancomycin-resistant enterococci (VRE) in a renal unit.
J Hosp.Infect.,
40(2), 115-124.
Abstract:
Epidemiology and control of vancomycin-resistant enterococci (VRE) in a renal unit
This study reports an outbreak of infection and colonization caused by vancomycin-resistant enterococci (VRE) in the renal service of a large teaching hospital. The polymerase chain reaction and pulsed-field gel electrophoresis were used to study the epidemiology of 26/34 strains of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium from the outbreak in comparison with five strains from other hospitals in Edinburgh and the Borders, and three from other wards in the Royal Infirmary. The study revealed a heterogeneous population of vancomycin-resistant E. faecalis. Over 60% of E. faecium isolates had matching pulsed-field gel electrophoresis patterns and all of these were of VanA phenotype. These results suggest that clonal spread of VanA phenotype E. faecium within and possibly between hospitals is the major vancomycin-resistant enterococcal problem in Edinburgh. Screening of patients and isolation of colonized and infected patients appear to have been successful in controlling the spread of VRE
Abstract.