Biomedical subjects

Caroline Duchaine

Publications and source records attributed to Caroline Duchaine.

11 recordsLinked to original sources

PubMed2006

Flow cytometry sorting protocol of Bacillus spore using ultraviolet laser and autofluorescence as main sorting criterion.

The ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS), a flow cytometer-like apparatus was developed by the Canadian Department of National Defence for real-time detection of autofluorescence of biological aerosol particles such as bacterial spores. The direct relation between autofluorescence intensity and viability has recently been reported and viable spore are more autofluorescent in UV (Laflamme, Frontiers in Bioscience). The goal of this manuscript is to describe a flow cytometry sorting protocol based on UV-induced autofluorescence. An EPICS ELITE ESP flow cytometer equipped with a UV laser and cell sorter was used to mimic the optical properties of FLAPS and to study the two extremes of a spore population according to its autofluorescence (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA) spores). Bacillus subtilis var niger was used as a surrogate for Bacillus anthracis spores and sorted using autofluorescence intensity as the main criterion. The protocol developed in our laboratory to sort Bacillus spores according to their autofluorescence properties is described. Purity of each sorted population was greater than 95%. Using autofluorescence as the main criterion, we demonstrate that it is possible to separate two distinct spore populations.

Bacillus subtilis↗

PubMed2006

Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579.

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.

Bacillus cereus↗

PubMed2006

Hypersensitivity pneumonitis in a hardwood processing plant related to heavy mold exposure.

Two workers employed in a hardwood floor plant presented symptoms suggestive of hypersensitivity pneumonitis (HP). At that plant, kiln-dried wood often shows moldy growth and is subsequently brought inside for processing. This study evaluated the environment in attempt to identify the causative antigen and verify whether other workers of this and similar plants had or were at risk of developing HP. Dust from dust-removing systems and molds on the surface of wood planks were collected and air samples taken from a sister plant. Blood samples, spirometry, and symptoms' questionnaires were obtained from 11 co-workers. Dense Paecilomyces growth was observed on the surface of the dried processed wood in the index plant. This fungal genus was not detected in the sister plant. An additional worker had symptoms suggestive of HP, and his bronchoalveolar lavage revealed a lymphocytic alveolitis. The 3 confirmed cases of HP and the other 10 workers had positive specific IgG antibodies to Paecilomyces. We report 3 cases of HP out of 13 workers and a 100% sensitization to molds in workers of a hardwood processing plant. This rate is much higher than what is commonly seen in other environments associated with HP. The drying process is suspected of being responsible for the massive Paecilomyces contamination likely responsible for the HP.

Adult↗

PubMed2006

Bioaerosols in peat moss processing plants.

Peat moss is organic matter colonized by large numbers of microorganisms. Storage prior to its processing may result in massive microbial growth. These biological contaminants can become airborne during processing. Our goals were (a) to evaluate concentrations of bioaerosols (inhalable dust, molds, bacteria) in peat moss processing plants that used dust removing systems, and (b) to evaluate the presence of these microorganisms in peat moss. Fourteen plants from Eastern Canada were visited; 3 plants operated all year (all-year mixing plants), and 11 plants functioned only during summer months (seasonal). Air samples were taken throughout the day at different work sites using IOM cassettes for inhalable dust and All-Glass Impinger-30 samplers and Andersen six-stage impactors for microorganisms. Samples of nonprocessed and bagged peat moss (solid material) were also taken and analyzed. A total of 25 work sites for air sampling and 33 solid material samples were analyzed. Air samples contained up to 441.7 mg/m3 of inhalable dust and up to 1.0 x 10(8) CFU/m3 mesophilic molds and 3.3 x 10(5) CFU/m3 bacteria. Seasonal plants were more contaminated with molds and dust than all-year mixing plants. Sieving sites were the most highly contaminated work sites. Airborne dust concentration was significantly correlated with molds and bacteria. Up to 3.8 x 10(7) CFU/g (dry weight) and 4.8 x 10(7) CFU/g (dry weight) molds and bacteria, respectively, were found in the solid material samples. Airborne contaminants did not correlate with solid material content. Despite the use of dust removing systems, peat moss processing plants contain very large amounts of microbially contaminated bioaerosols that do not correlate with the quality of the processed peat. Efficiency of dust removing systems could influence the contamination levels.

Air Pollutants, Occupational↗

PubMed2006

Sensitization to airborne molds and its health effects in peat moss processing plant workers.

The goal of this study was to evaluate the incidence of sensitization to the major molds found in peat dust in workers exposed to stored peat moss and the health impact of this sensitization. Air samples from each plant were obtained to measure the levels of airborne molds, bacteria, and dust. There were 189 workers from 14 peat moss processing plants (3 all-year mixing plants and 11 seasonal plants) recruited for the study. The subjects completed a symptoms questionnaire, underwent spirometric measurements and skin-prick tests, and gave venous blood samples. Blood samples from 43 nonexposed control subjects were also taken. A similar percentage of smokers from both plant types was observed. Twenty-eight percent of the workers tested had a positive serum reaction to at least one of the tested molds. The percentage of positive workers varied from plant to plant, going from none in 4 plants to 14 out of 21 for 1 plant. This variability was not correlated with the airborne levels of molds. FEV tended to be lower in the workers with positive antibodies compared with seronegative workers. IgG positive frequency was higher for those workers employed in the all-year plants, and workers from those plants had lower FEV/FVC than seasonal plant workers. Seasonal plants were more contaminated with molds than all-year mixing plants, suggesting that the duration of exposure may trigger more sensitization than the level of exposure. We conclude that there is a high incidence of mold sensitization in peat moss factory workers and that this sensitization may have a negative respiratory health impact.

Adult↗

PubMed2005

Metalworking fluid with mycobacteria and endotoxin induces hypersensitivity pneumonitis in mice.

BACKGROUND: Human cases of hypersensitivity pneumonitis (HP) have been reported among machinists for over 10 yr. Although mycobacteria have been implicated as causal agents, this has not been established in experimental studies and the mechanisms remain unclear. Other constituents of in-use metalworking fluids (MWFs) may also contribute to the development of lung disease. We investigated the potential for Mycobacterium immunogenum (MI) in MWFs to induce HP. METHODS: Mice were exposed intranasally for 3 wk to MI (isolated from MWFs), Saccharopolyspora rectivirgula (positive control), saline, endotoxin, MWFs spiked with endotoxin and/or MI, used MWFs, and particulate-fortified used MWFs. Responses were assessed 96 h after the last exposure. RESULTS: Mice exposed to MI in MWFs developed lung pathology consistent with HP along with significantly more monocytes and neutrophils in lung lavage, increased CD4+/CD8+ T-lymphocyte ratio, and marked pulmonary lymphocytosis on histologic examination when compared with saline-treated control mice. Mice with Grade 2 or higher pathology (0-4 point scale) exhibited significantly elevated macrophage inflammatory protein-1alpha and IL-10 and a trend toward higher RANTES 96 h after the final dose. Endotoxin coexposure augmented lung pathology. CONCLUSION: MWFs containing mycobacteria induced granulomatous lung lesions, peribronchiolar lymphocytosis, increased cell concentrations in lavage, and up-regulation of several cytokines. These findings are consistent with HP.

Aerosols↗

PubMed2005

Autofluorescence as a viability marker for detection of bacterial spores.

Recent biological terrorism events have indicated that bacterial spores such as Bacillus anthracis are real threat agents. Real time detection of biological agents is possible with the use of an ultraviolet Fluorescent Aerodynamic Particle Sizer (FLAPS) that measures particles' intrinsic fluorescence. It is important to know whether intrinsic fluorescence could be used to estimate agents' viability. Two categories of Bacillus spore populations can be differentiated by the intensity of intrinsic fluorescence emitted by ultraviolet (UV) stimulation : autofluorescent and non-autofluorescent. This study was performed to determine whether intensity of autofluorescence correlates with spore viability. Spores were analyzed using flow cytometer (equipped with a cell sorter) to mimic optical properties of FLAPS. Autofluorescent and non-autofluorescent spores were sorted according to the intensity of autofluorescence emitted following UV stimulation. Culturability, membrane integrity, membrane potential and dipicolinic acid (DPA) content were assessed. Autofluorescent spores were 1.7 times more culturable than the corresponding non-autofluorescent population. Moreover, a small proportion of autofluorescent spores exhibited extracellular membrane damages. Autofluorescent spores also showed higher membrane potential activity and contained higher levels of DPA. In conclusion, this study documents that the overall viability potential of bacterial spores can be assessed by UV flow cytometry used in the FLAPS technology.

Bacillus anthracis↗

PubMed2004

Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye.

Germination of Bacillus anthracis spores is necessary for the transcription of plasmidic genes essential to the infection. Assessing germination potential is crucial to predict the risk associated with pathogenic Bacillus exposure. The aim of this study was to set up a viability assay based on membrane potential in order to predict the earliest germination event of spores. B. cereus and two strains of B. subtilis were used. The spores were isolated with a sodium bromide gradient. Approximately 10(7) spores were incubated at 37 degrees C in tryptic soy broth (TSB). Aliquots were harvested at predetermined times and stained with 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] or with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)]. Fluorescence characteristics were obtained using flow cytometry. The earliest detectable activation of membrane potential occurred after 15 min of incubation in TSB using DiOC(6)(3). Using DiBAC(4)(3), the earliest detectable signal was after 4 h of incubation. Control experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP)-treated spores did not show any change in the fluorescence intensity over time. Since no membrane potential and no germination were detected in CCCP-treated spores, the activation of membrane potential seems to be associated with germination. DiOC(6)(3) can be used as an early membrane potential indicator for spores. DiBAC(4)(3), by contrast, is not a early membrane potential marker.

Bacillus↗

PubMed2004

Six month tracking of microbial growth in a metalworking fluid after system cleaning and recharging.

Large volumes of metalworking fluids (MWFs) are used in manufacturing industries for cooling and lubrication of metal pieces and tools during machining. MWFs accumulate microbial growth through continuous recirculation and reuse. We studied the progression of microbial contamination for 6 months after dumping, cleaning and recharging (DCR) of a large semi-synthetic MWF system managed with several biocides. Fresh, uncontaminated fluid was added to the system after extensive cleaning. The following samples were collected and analyzed: pre-DCR fluid (before system cleaning); neat fluid diluted to 6% with water; in use MWF 12 h and 1, 3 and 6 months post-DCR. Samples were analyzed for total microorganism concentrations by direct counting using fluorescence microscopy and by plate counting on various media (R2A, BHI, Middlebrooks and rose bengal under aerobic conditions). In addition, PCR was performed for the detection of mycobacteria. There was a rapid progression in the total bacterial counts as determined by fluorescence microscopy: 5.7 x 10(7) cells/ml in the pre-DCR used fluid, no measurable bacteria in the neat fluid, 6.9 x 10(6) cells/ml after 12 h and 2.2 x 10(6), 3.6 x 10(8) and 6.1 x 10(8) cells/ml after 1, 3 and 6 months, respectively. On average, only 0.2% of the direct count organisms were quantified on R2A cultures. PCR showed the presence of mycobacteria in the used MWF at 3 and 6 months. Mycobacteria were also identified from cultures on Middlebrooks and R2A. This study demonstrates that standard methods for cleaning MWF systems are inadequate since residual bacteria in the system can rapidly repopulate the newly charged MWF.

DNA, Bacterial↗

PubMed2004

Working Group report 4: exposure assessment for biological agents.

This Working Group was assembled to review and evaluate methods currently used to estimate work site exposures to biological agents and to present recommendations on suitable measurement strategies. The current state of exposure assessment was evaluated for environments with organic dust including agriculture, composting, sewage and waste treatment processing, peat moss harvesting and handling, cotton and textile processing, greenhouse work, and grass seed processing. Methods for measurement of microbial contaminants in indoor environments were also considered. Important methods are emerging that use quantitative PCR for assessment of microbial agents. Difficulties exist with optimization of extraction to yield contaminant-free DNA without significant DNA loss. Advances in assays for microbial agents (e.g., endotoxin and glucans) as well as allergens have increased the utility of exposure assessment for these agents. A crucial area for further development is international harmonization of methodologies to reduce interlaboratory variability and to facilitate establishment of exposure guidelines. Endotoxin exposure assessment using the Limulus amebocyte lysate method is a high priority for harmonization because of its importance as a pulmonary inflammatory agent in many occupational settings.

Air Pollution, Indoor↗

PubMed1999

Saccharopolyspora rectivirgula from Quebec dairy barns: application of simplified criteria for the identification of an agent responsible for farmer's lung disease.

Saccharopolyspora rectivirgula (Micropolyspora faeni) is one of the major agents responsible for farmer's lung disease, a form of hypersensitivity pneumonitis. It is frequently isolated from the air of contaminated barns. The identification of this actinomycete is difficult because most of its phenotypic characteristics are variable and classical tests are not easy to perform on actinomycetes. Fatty acid analysis is very useful for the identification of these strains, but is not available except in some research or reference laboratories. Morphological (microscopic and macroscopic observations), physiological and biochemical tests (growth properties; macromolecules degraded; citrate utilisation and acid production from carbohydrates; resistance to antibiotics, lysozyme and heat), cell wall and fatty acid analyses and IgG analyses with serum from patients with farmer's lung were performed on 12 environmental isolates presumed to be S. rectivirgula and two control strains of S. rectivirgula. From this, a simple and rapid scheme for the identification of this actinomycete is proposed: optimal growth temperature (55 degrees C); colony appearance based on morphology (filamentous) and colour (beige to orange-brown); microscopic morphology (chains of spores on both aerial and substrate mycelium); growth on NaCl 10%; cell-wall analysis (type IV); and the verification of antibody response with serum from a patient with farmer's lung. This last criterion is important to confirm the immunogenicity of the strains identified as S. rectivirgula. This scheme provides an accurate and efficient way of identifying S. rectivirgula strains and evaluating exposure to this bacterium. The study shows the limited value and the lack of reproducibility of some classical biochemical tests.

Actinomycetales Infections↗