Alan R. Brown

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.

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.

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.

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.

Assessment of Fluorescent In Situ Hybridization and PCR-Based Methods for Rapid Identification of Burkholderia cepacia Complex Organisms Directly from Sputum Samples

ABSTRACT 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 × 105 CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 104 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.

Photoinitiated electron-transfer reactions across the interface between two immiscible electrolyte solutions

A system is described in which photoinitiated electron transfer across the interface between two immiscible electrolyte solutions has been demonstrated. Ion-transfer reactions following electron transfer can be neglected, as the standard potentials of transfer of all reactants and products for the system are known.

Diversity of Tn1546 Elements in Clinical Isolates of Glycopeptide-Resistant Enterococci from Scottish Hospitals

ABSTRACT 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 ofvanR by the insertion of IS1542. The presence of this hybrid promoter was associated with constitutive expression of the van genes and elevated teicoplanin resistance.

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 all members 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 from a 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.

Infrared studies of CH and CD stretching anharmonicity

Progress is reported in the analysis of CH and CD stretching overtones and their accompanying Fermi resonances in C2H4, C2H3D, CH2CD2 and C2HD3. CH stretching overtone data have been obtained for a number of partially deuterated alkanes, halogenated alkanes, MeCN, MeC2H, MeNH2, Me2NH, MeOH and Me2O. Values of anharmonicity constants xii obtained from these are compared with the results of local-mode studies. A provisional Fermi resonance analysis of CH(CD3)3 is reported. The possiblity of a relationship between xii, ωi and CH dissociation energy is explored.

Photochemical ion transfer across the interface between two immiscible electrolyte solutions

Irradiation of the interface between two immiscible electrolyte solutions (ITIES) by visible or UV light can give rise to a photocurrent [l-3] or photopotential [4]. This effect has been ascribed either to the photoexcited electron transfer between a sensitizer in one solvent phase and an electron acceptor in the other solvent phase [1,3], or to the transfer of an ionic product of the homogeneous photoassociated electron transfer reaction [2]. Eventually, the non-radiative dissipation of the absorbed light can result in a microscopic thermal gradient at the ITIES bringing about a change in the interfacial potential difference - a sort of ~e~~lect~c effect [2]. The purpose of this communication is to show that the base electrolyte ions themselves can yield a photocurrent which is closely related to their excited states. This applies in particular to aromatic ions like tetraarylborate anions or tetraarylarsonium cations. The interface between water and 1,2-~chloroeth~e (geometric area of 2.5 cm2) was formed in an all-glass four-electrode cell [5], with the organic solvent phase placed in the bottom part of the cell. The top of the cell was left open, so that the light from a 150 W xenon arc source (Applied Photophysics, Model 4060) reflected by a mirror at 45’ passed only through the layer of the non-absorbing aqueous phase prior to reaching the interface. Mon~hromatic light was obtained from a monochromator (Applied Photophysics) placed between the xenon lamp and the reflection mirror. The incident intensity of the monochromatic light was measured by means of a calibrated photodiode detector (Macam Photometrics, Model SD