Anne Mériaux

Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods

ABSTRACT Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using theLimulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.

Assessment of Particulates and Bioaerosols in Eastern Canadian Sawmills

The purpose of this study was to quantify and identify the airborne contamination in eastern Canadian sawmills. Seventeen sawmills were chosen to cover a wide range of size, geographic distribution, and wood species processed. Within each sawmill different work sites (debarking, sawing, sorting, or planing) were studied separately. Area sampling was performed for exposure assessment. Microbial contaminants were assessed with all-glass impingers 30 and six-stage Andersen microbial samplers; appropriate selective media and culture conditions for bacteria, thermophilic actinomycetes, molds, and yeasts were used. Inhalable dust, endotoxins, temperature, and humidity also were measured. Penicillium species were the most predominant molds with up to 40 different Penicillium species identified. Debarking was the working site most highly contaminated by molds, bacteria, and endotoxins (p=0.0001). At this working site mold levels reached a maximum of 1.5 x 10(6) CFU/m3, whereas the median values for culturable bacteria and endotoxin were 21,620 CFU/m3 and 1,081 endotoxin units/m3, respectively. Planing sites were the most highly dust contaminated (median: 3.0 mg/m3) (p <0.05). Sawmills of eastern Canada contain airborne biological contaminants that vary between working sites, and their microflora is different from that previously described in European sawmills.

Human pathogens and tetracycline-resistant bacteria in bioaerosols of swine confinement buildings and in nasal flora of hog producers

Swine confinement buildings in eastern Canada are enclosed and equipped with modern production systems to manage waste. Bioaerosols of these swine confinement buildings could be contaminated by human pathogens and antimicrobial resistant bacteria which could colonize exposed workers. We therefore wanted to analyze bioaerosols of swine confinement buildings and nasal flora of Canadian hog producers to evaluate possible colonization with human pathogens and tetracycline-resistant bacteria. Culturable and non-culturable human pathogens and tet genes were investigated in the bioaerosols of 18 barns. The nasal passages of 35 hog producers were sampled and total DNA was extracted from the calcium-alginate swabs to detect, by PCR, Campylobacter, C. perfringens, Enterococcus, E. coli, Y. enterocolitica, tetA/tetC, tetG and ribosomal protection protein genes. Airborne culturable C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were present in the bioaerosols of 16, 17, 11 and 6 of the 18 facilities. Aerosolized total (culturable/non culturable) Campylobacter, C. perfringens, Enterococcus, E. coli and Y. enterocolitica were detected in 10, 6, 15, 18 and 2 barns, respectively. Tet genes were found in isolates of culturable human pathogens. TetA/tetC, tetG and ribosomal protection protein genes were detected in the bioaerosols of all 18 studied buildings. Campylobacter, C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were found respectively in 4, 9, 17, 14 and one nasal flora of workers. One and 10 workers were positive for tetA/tetC and tetG genes, respectively. In swine confinement buildings, hog producers are exposed to aerosolized human pathogens and tetracycline-resistant bacteria that can contaminate the nasal flora.

Airborne Microflora in Quebec Dairy Farms: Lack of Effect of Bacterial Hay Preservatives

Pediococcus pentosaceus is a lactic-acid producing bacterium inoculated in hay to prevent hay deterioration. This study sought to verify the effect of this treatment on the barn microenvironment. Air samples were obtained from 19 barns using bacterial hay treatment and from 18 control barns with six-stage Andersen samplers and all-glass impingers. Appropriate culture media were used for the recovery and identification of microorganisms. Endotoxins were measured with chromogenic Limulus amoebocyte lysate assay. Median values (respectively for treated and untreated hay barns) were: 5.28 x 10(5) and 3.84 x 10(5) colony-forming units (CFU)/m3 for total bacteria; 3.18 x 10(6) and 4.5 x 10(6) CFU/m3 for molds; 1.36 x 10(3) and 1.74 x 10(3) endotoxin units/m3 for endotoxin levels; and 1.03 x 10(3) and 3.00 x 10(3) CFU/m3 for Saccharopolyspora rectivirgula. No viable P. pentosaceus were recovered. The presence of S. rectivirgula, the causative agent for farmers lung, was not influenced by the hay treatment. Since no significant difference was observed in any of the airborne contaminants, this type of hay treatment probably does not protect farmers from the respiratory effect of ambient microbial contaminants.

Random Amplified Ribosomal DNA Restriction Analysis for Rapid Identification of Thermophilic Actinomycete-like Bacteria Involved in Hypersensitivity Pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.

The importance of combining air sampling and surface analysis when studying problematic houses for mold biodiversity determination

Houses that underwent water damages are often responsible for heath problems of the occupants. Since there is no universally used protocol for the analysis, we wanted to verify the usefulness of surface sampling versus air sampling for the evaluation of mold diversity in problematic houses and the value of the number of visible mold growth zones to predict air quality. Seventeen houses were sampled for culturable molds in the air and on the surfaces showing contamination. We compared the mold taxa found in the air and on the surfaces and verified the correlation between the number of moldy surfaces and airbone mold concentration. This study demonstrated that, surprisingly, some of the so called wet spore molds (e.g. Stachybotrys) were found more often from air than surface samples whereas, some dry spore molds (e.g. Asp. fumigatus) was more easily isolated from surface samples. There was a good correlation between the number of visible mold growth zones and the concentration of airborne molds. We conclude that air and surface sampling are necessary to evaluate mold diversity in problematic houses and that the number of mold growth zones is a good predictor of airborne mold concentration.

Metalworking fluid-related aerosols in machining plants.

Respiratory problems are observed in machinists using soluble metalworking fluid (MWF). Evidences suggest that these problems could be related to the aerosolized microorganisms and their byproducts from MWF. To establish MWF aerosol exposure thresholds and to better understand their effect on human health, these aerosols must be fully characterized. This article evaluates airborne microorganisms and aerosols from soluble MWF in the working environment. Air quality parameters (endotoxin levels, culturable airborne microorganisms, fluid mist, inhalable dust and air exchange rates) were evaluated at 44 sites, in 25 shops in Quebec, Canada. Microorganism concentrations were also measured in MWF. Culturable airborne bacteria concentrations were low, ranging from 1.2 × 101 to 1.5 × 103 CFU (colony forming units) m− 3, even for metalworking fluid highly contaminated by bacteria (up to 2.4 × 109 CFU mL− 1). Inhalable dust varied between < 0.1 to 2.6 mg m− 3, while air exchange rates were mostly below the standard (4 h− 1) for this type of workplace, between 0.6 to 14.2 h− 1. Only nine of 44 sites respected the suggested minimum value for air exchange rates. Fluid mist ranged from 0.02 to 0.89 mg m− 3, which is below the threshold limit value (TLV) (ACGIH) of 5 mg m− 3. Airborne endotoxin concentrations ranged from undetectable to 183 EU m− 3 (endotoxin units), showing no correlation with airborne microorganisms or inhalable dust. Most workstations respected the suggested minimum values for fluid mist and showed low concentrations of airborne endotoxin, culturable microorganisms and inhalable dust despite fluid contamination, even when air exchange rates were below the recommendations. Airborne Pseudomonas pseudoalcaligenes was recovered from many sites at significant concentrations. Health-associated risks following exposure to this microorganism should be further investigated.

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 farmers 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 farmers 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 farmers 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.

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

Aims:  To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity.

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 × 108 CFU/m3 mesophilic molds and 3.3 × 105 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 × 107 CFU/g (dry weight) and 4.8 × 107 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.