Construction of aminoglycoside-sensitive Burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay

Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

Interactions of Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria with epithelial and phagocytic cells

Burkholderia cenocepacia is a member of the B. cepacia complex (Bcc), a group of opportunistic bacteria that infect the airways of patients with cystic fibrosis (CF) and are extraordinarily resistant to almost all clinically useful antibiotics. Infections in CF patients with Bcc bacteria generally lead to a more rapid decline in lung function, and in some cases to the 'cepacia syndrome', a virtually deadly exacerbation of the lung infection with systemic manifestations. These characteristics of Bcc bacteria contribute to higher morbidity and mortality in infected CF patients. In the last 10 years considerable progress has been made in understanding the interactions between Bcc bacteria and mammalian host cells. Bcc isolates can survive either intracellularly within eukaryotic cells or extracellularly in host tissues. They survive within phagocytes and respiratory epithelial cells, and they have the ability to breach the respiratory epithelium layer. Survival and persistence of Bcc bacteria within host cells and tissues are believed to play a key role in pulmonary infection and to contribute to the persistent inflammation observed in patients with CF. This review summarizes recent findings concerning the interaction between Bcc bacteria and epithelial and phagocytic cells.

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.

Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase

The lpcA locus has been identified in Escherichia coli K12 novobiocin-supersensitive mutants that produce a short lipopolysaccharide (LPS) core which lacks glyceromannoheptose and terminal hexoses. We have characterized lpcA as a single gene mapping around 5.3 min (246 kilobases) on the E. coli K12 chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant plasmids containing only lpcA restored a complete core LPS in the E. coli strain chi711. We show that this strain has an IS5-mediated chromosomal deletion of 35 kilobases that eliminates lpcA. The LpcA protein showed discrete similarities with a family of aldose/ketose isomerases and other proteins of unknown function. The isomerization of sedoheptulose 7-phosphate, into a phosphosugar presumed to be D-glycero-D-mannoheptose 7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA+ and of lpcA mutants containing the recombinant lpcA gene. We concluded that LpcA is the phosphoheptose isomerase used in the first step of glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain glyceromannoheptose in the inner core LPS, indicating that LpcA is an essential component in a conserved biosynthetic pathway of inner core LPS.

Immunological variants of the aerobactin-cloacin DF13 outer membrane protein receptor IutA among enteric bacteria

Mouse monoclonal antibodies (MAbs) were generated against a 76-kDa IutA receptor of pathogenic avian Escherichia coli 15972. Six of the eight IutA-specific MAbs isolated (AB1 to AB6) were shown to be directed toward membrane-exposed conformational epitopes, although they did not interfere with the uptake of ferric aerobactin and cloacin DF13 as assessed by competition experiments with purified ligands. The two remaining IutA MAbs (AB9 and AB10) recognized linear epitopes buried in the IutA molecule. The panel of IutA MAbs was used to characterize IutA variants occurring in strains of E. coli, Klebsiella pneumoniae, Enterobacter spp., and Shigella spp., resulting in the identification of four immunological groups of IutAs. MAb AB9 defined an epitope conserved in all IutA variants. In addition, the panel of IutA MAbs served to identify the presence of IutA in wild-type bacteria grown in the presence of diphenylamine to reduce the expression of O-specific polysaccharide.

A PLATE ASSAY FOR THE DETECTION OF ORGANOPHOSPHONATE MINERALIZATION BY ENVIRONMENTAL BACTERIA, AND ITS MODIFICATION AS AN ACTIVITY STAIN FOR IDENTIFICATION OF THE CARBON-PHOSPHORUS BOND-CLEAVAGE ENZYME PHOSPHONOACETATE HYDROLASE

A replica plate screening technique, based on the acid molybdate assay for detection of phosphate has been developed to permit the detection of microorganisms capable of mineralizing organophosphonates. The method was further adapted as the basis of an activity stain for the detection of the carbon - phosphorus bond cleavage enzyme phosphonoacetate hydrolase in PAGE gels.

The impact of antibiotic use on the incidence and resistance pattern of extended-spectrum beta-lactamase-producing bacteria in primary and secondary healthcare settings

Aims: The objective of the present study was to study the relationship between hospital antibiotic use, community antibiotic use and the incidence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in hospitals, while assessing the impact of a fluoroquinolone restriction policy on ESBL-producing bacteria incidence rates. METHODS: The study was retrospective and ecological in design. A multivariate autoregressive integrated moving average (ARIMA) model was built to relate antibiotic use to ESB-producing bacteria incidence rates and resistance patterns over a 5 year period (January 2005-December 2009). Results: Analysis showed that the hospital incidence of ESBLs had a positive relationship with the use of fluoroquinolones in the hospital (coefficient = 0.174, P= 0.02), amoxicillin-clavulanic acid in the community (coefficient = 1.03, P= 0.03) and mean co-morbidity scores for hospitalized patients (coefficient = 2.15, P= 0.03) with various time lags. The fluoroquinolone restriction policy was implemented successfully with the mean use of fluoroquinolones (mainly ciprofloxacin) being reduced from 133 to 17 defined daily doses (DDDs)/1000 bed days (P <0.001) and from 0.65 to 0.54 DDDs/1000 inhabitants/day (P= 0.0007), in both the hospital and its surrounding community, respectively. This was associated with an improved ciprofloxacin susceptibility in both settings [ciprofloxacin susceptibility being improved from 16% to 28% in the community (P <0.001)] and with a statistically significant reduction in ESBL-producing bacteria incidence rates. Discussion: This study supports the value of restricting the use of certain antimicrobial classes to control ESBL, and demonstrates the feasibility of reversing resistance patterns post successful antibiotic restriction. The study also highlights the potential value of the time-series analysis in designing efficient antibiotic stewardship. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.

Visuomotor transformation for interception:catching while fixating

Catching a ball involves a dynamic transformation of visual information about ball motion into motor commands for moving the hand to the right place at the right time. We previously formulated a neural model for this transformation to account for the consistent leftward movement biases observed in our catching experiments. According to the model, these biases arise within the representation of target motion as well as within the transformation from a gaze-centered to a body-centered movement command. Here, we examine the validity of the latter aspect of our model in a catching task involving gaze fixation. Gaze fixation should systematically influence biases in catching movements, because in the model movement commands are only generated in the direction perpendicular to the gaze direction. Twelve participants caught balls while gazing at a fixation point positioned either straight ahead or 14 degrees to the right. Four participants were excluded because they could not adequately maintain fixation. We again observed a consistent leftward movement bias, but the catching movements were unaffected by fixation direction. This result refutes our proposal that the leftward bias partly arises within the visuomotor transformation, and suggests instead that the bias predominantly arises within the early representation of target motion, specifically through an imbalance in the represented radial and azimuthal target motion.

Uptake, translocation and transformation of arsenate and arsenite in sunflower (Helianthus annuus):formation of arsenic-phytochelatin complexes during exposure to high arsenic concentrations

The aim of the study was to determine the time-dependent formation of arsenic-phytochelatin (As-PC) complexes in the roots, stems and leaves of an arsenic-nontolerant plant (Helianthus annuus) during exposure to 66 mol l(-1) arsenite (As(III)) or arsenate (As(V)). We used our previously developed method of simultaneous element-specific (inductively coupled plasma mass spectrometry, ICP-MS) and molecular-specific (electrospray-ionization mass spectrometry, ES-MS) detection systems interfaced with a suitable chromatographic column and eluent conditions, which enabled us to identify and quantify As-PC complexes directly. Roots of As-exposed H. annuus contained up to 14 different arsenic species, including the complex of arsenite with two (gamma-Glu-Cys)(2)-Gly molecules [As((III))-(PC(2))(2)], the newly identified monomethylarsonic phytochelatin-2 or (gamma-Glu-Cys)(2)-Gly CH(3)As (MA((III))-PC(2)) and at least eight not yet identified species. The complex of arsenite with (gamma-Glu-Cys)(3)-Gly (As((III))-PC(3)) and the complex of arsenite with glutathione (GSH) and (gamma-Glu-Cys)(2)-Gly (GS-As((III))-PC(2)) were present in all samples (roots, stems and leaves) taken from plants exposed to As. The GS-As((III))-PC(2) complex was the dominant complex after 1 h of exposure. As((III))-PC(3) became the predominant As-PC complex after 3 h, binding up to 40% of the As present in the exposed plants. No As-PC complexes were found in sap (mainly xylem sap from the root system), in contrast to roots, stems and leaves, which is unequivocal evidence that As-PC complexes are not involved in the translocation of As from root to leaves of H. annuus.