Philippe Duquenne

Measurement of Endotoxins in Bioaerosols at Workplace: A Critical Review of Literature and a Standardization Issue

Endotoxins are lipopolysaccharides found in the outer membrane of most Gram-negative bacteria and cyanobacteria. Worker exposure to endotoxins has been shown in a number of work situations and is associated with both respiratory and systemic pathologies. The lack of an occupational exposure limit is mainly due to the absence of a standard protocol at the international level for sampling and analyzing airborne endotoxins. The bibliographic review in this article takes an exhaustive look at the current knowledge on measuring airborne endotoxins. It shows that, despite several reference documents at the international level, the methods used to measure endotoxin exposure differ considerably from one laboratory to another. Standardization is necessary to reduce interlaboratory variability and, ultimately, to improve the use of interstudy data. The bibliographic review presents the current status of standardization for airborne endotoxin measurement methods in the workplace and summarizes areas for further research. This article is both a reference document for all operators wishing to use such methods and a working document to build international consensus around the measurement of airborne endotoxins.

Bioaerosol sampling by a personal rotating cup sampler CIP 10-M

High concentrations of bioaerosols containing bacterial, fungal and biotoxinic matter are encountered in many workplaces, e.g. solid waste treatment plants, waste water treatment plants and sewage networks. A personal bioaerosol sampler, the CIP 10-M (M-microbiologic), has been developed to measure worker exposure to airborne biological agents. This sampler is battery operated; it is light and easy to wear and offers full work shift autonomy. It can sample much higher concentrations than biological impactors and limits the mechanical stress on the microorganisms. Biological particles are collected in 2 ml of liquid medium inside a rotating cup fitted with radial vanes to maintain an air flow rate of 10 l min(-1) at a rotational speed of approximately 7,000 rpm. The rotating cup is made of sterilisable material. The sampled particles follow a helicoidal trajectory as they are pushed to the surface of the liquid by centrifugal force, which creates a thin vertical liquid layer. Sterile water or another collecting liquid can be used. Three particle size selectors allow health-related aerosol fractions to be sampled according to international conventions. The sampled microbiological particles can be easily recovered for counting, incubation or further biochemical analysis, e.g., for airborne endotoxins. Its physical sampling efficiency was laboratory tested and field trials were carried out in industrial waste management conditions. The results indicate satisfactory collection efficiency, whilst experimental application has demonstrated the usefulness of the CIP 10-M personal sampler for individual bioaerosol exposure monitoring.

Assessment of Workers’ Exposure to Bioaerosols in a French Cheese Factory

Hundreds of different cheeses are produced in France, where 23.9kg of cheese were consumed per inhabitant in 2009, when it was ranked the second cheese-consuming nation. To meet this considerable demand, a large number of cheese factories exist where many workers, especially cheese washers, may be exposed to fungal bioaerosols that can lead to adverse toxinic and allergic effects. Airborne bacteria, fragments, or microbial by-products (endotoxins) are also found and contribute to total worker exposure. However, there is almost no published data concerning worker exposure or characteristics of bioaerosols emitted during these activities. Here, we measured the parameters (concentrations, species present, and size distribution) of the culturable fungal bioaerosol emitted in a French natural-rind cheese-maturing cellar. Concentrations of airborne bacteria and endotoxins were also measured. The main tasks were investigated using stationary or personal sampling over three consecutive days. Depending on the work area, high concentrations of culturable mesophilic microorganisms were measured (using closed-face cassettes): from 10(4) to 2×10(8) CFU m(-3) for fungi and from 10(3) to 10(6) CFU m(-3) for bacteria. These concentrations are 10- to 100000-fold higher than those measured at two reference points (indoor and outdoor) that are assumed not to be contaminated by the plants activities. Endotoxin concentrations were between 10 and 300 EU m(-3) in the plant. Exposure was further assessed by identifying the predominant culturable fungi (allergenic Mucor fuscus and Penicillium sp.) and by measuring particle size distributions (cascade impactor). Airborne fungal entities (spores, mycelium strands and fragments, agglomerates, etc.) were found with aerodynamic diameters from 3 to over 20 µm. A metrological approach was used to fully characterize the culturable fungal aerosols generated during cheese maturing in this plant. The results show that workers are exposed to concentrations of airborne culturable fungi, sometimes very high, throughout the manufacturing process. In addition to fungi, culturable bacteria and endotoxins are also present in the work atmosphere. All these microbial organisms thus contribute in a complex manner to total worker exposure. Despite the lack of both occupational exposure limit values and standardized measuring methods, our results suggest that an immunological risk may occur among workers, especially for cheese brushers, cheese washers, and packagers who are the most exposed workers in the factory.

First Metagenomic Survey of the Microbial Diversity in Bioaerosols Emitted in Waste Sorting Plants

Waste sorting activities are source of occupational bioaerosol exposures that are associated with several health disorders. New analytical tools, based on next-generation sequencing (NGS) technologies, provide powerful methods to assess the microbial composition of bioaerosols. The objectives of the study were (i) to assess the feasibility and the repeatability of NGS-based biodiversity measurements and (ii) to study the microbial biodiversity using NGS in bioaerosols emitted in a waste sorting plant (WSP). Three stationary parallel samples were collected in a sorting cabin using closed-face cassettes equipped with polycarbonate membranes. Bacterial and fungal diversity was assessed by sequencing 16S and 18S rDNA genes using either Illumina sequencing or 454 pyrosequencing methods. At sampling point, airborne bacteria were dominated by Proteobacteria, Firmicutes, and Actinobacteria with prevailing genera assigned to unclassified Enterobacteriaceae, Staphylococcus, Acinetobacter, Leuconostoc, Pseudomonas, and Lactobacillus. Airborne fungi were dominated by Ascomycota with prevailing genera assigned to Penicillium, Aspergillus, Rhizopus, Wallemia, and Hemicarpenteles. The NGS biodiversity measurements revealed a higher biodiversity bioaerosols that previously reported for WSP in studies carried out using culture methods followed by identification of microorganisms. These results provide the first survey about taxonomic biodiversity in bioaerosols from WSPs using high-throughput sequencing.

Generation of Actinomycetes Aerosols Containing Spores and Mycelium: Performances of a Liquid Bubbling Aerosolizer

The presence of actinomycetes in many workplaces and their role in the incidence of various respiratory symptoms remains poorly understood and underestimated. A sampling and culture-independent analysis method to measure airborne actinomycetes has yet to be developed and controlled bioaerosols are needed for laboratory investigations. In this article, the performances of a single-pass bubbling aerosolizer were characterized to evaluate the feasibility of generating actinomycetes from a liquid source and to confirm that viability of aerosolized entities was preserved. Six preparation protocols for liquid Thermoactinomycetes vulgaris cultures were compared in terms of culturable flora and total spores concentrations (culture and epifluorescence microscopy) and size distributions (optical counter and cascade impactor) of the bioaerosols generated. Using the best protocol, the generators performances were then validated using three species: Thermoactinomyces vulgaris, Thermobifida fusca, and Streptomyces californicus. Bioaerosols contained a mixture of spores and mycelium and their properties were stable throughout generation (120 min) and were satisfactorily reproducible between runs. Depending on the species generated, the culturable concentrations measured were between 104 and 108 CFU.m−3, with corresponding total spore concentrations between 105 and 109 Spores.m−3. These concentrations cover the ranges measured in the workplace. The generators flexibility should make it possible to produce bioaerosols with other actinomycetes species, and use them in laboratory trials with various objectives and constraints. Copyright 2013 American Association for Aerosol Research

Evolution of microbial aerosol behaviour in heating, ventilating and air-conditioning systems--quantification of Staphylococcus epidermidis and Penicillium oxalicum viability.

The aim of this study was to develop an experimental set-up and a methodology to uniformly contaminate several filter samples with high concentrations of cultivable bacteria and fungi. An experimental set-up allows contaminating simultaneously up to four filters for range of velocities representative of heating, ventilating and air-conditioning systems. The test aerosol was composed of a microbial consortium of one bacterium (Staphylococcus epidermidis) and one fungus (Penicillium oxalicum) and aerosol generation was performed in wet conditions. Firstly, the experimental set-up was validated in regards to homogeneity of the air flows. The bioaerosol was also characterized in terms of number and particle size distribution using two particle counters: optical particle counter Grimm® 1.109 (optical diameters) and TSI APS 3321 (aerodynamic diameters). Moreover, stabilities of the number of particles generated were measured. Finally, concentrations of cultivable microorganisms were measured with BioSamplers (SKC) downstream of the four filters.

On the Identification of Culturable Microorganisms for the Assessment of Biodiversity in Bioaerosols

The Annals of Work Exposures and Health recently published two interesting studies combining the use of culture and molecular methods. The method involves the cultivation of bioaerosol samples on agar media and the pick-up of grown colonies 16S rRNA gene amplification, subsequent cloning, sequencing, and identification of bacterial isolates through the assignment against known gene databases. The aim of the present paper is to discuss the contribution of the proposed method in regards with the already proposed approaches used for identification of cultured bacteria. It details the new proposed method and discusses its contribution to the existing culture-based identification methods. Such methods include macroscopic and microscopic observations, miniature biochemical tests (API® trips, VITEK 2® etc.), chemical methods such as the Fatty Acid Methyl Ester (FAME) and the Matrix-assisted laser desorption/ionization time off light (MALDI-TOF) analysis as well polymerase chain reaction (PCR) followed by sequencing. The proposed method supplements the panel of existing biodiversity ones for cultivated bacteria, especially useful for infectious microorganisms, as well as culture-independent ones. As both culture-based and culture-independent methods could therefore be used for the characterization of the occupational environmental microbiome, further applications in other occupational environments as well as additional comparisons with both culture-based and culture-independent methods would complete its characterization.

Physical performances and kinetics of evaporation of the CIP 10-M personal sampler's rotating cup containing aqueous or viscous collection fluid

ABSTRACT The CIP 10-M personal sampler measures worker exposure to airborne particles by collecting particles in a rotating metal cup containing a few milliliters of a collection fluid. This device is mainly used to sample microorganisms or microbial components to measure bioaerosol concentrations in various occupational environments. Aqueous liquids are generally used, but their rapid evaporation limits the duration of sampling; alternative collection fluids could alleviate this problem. Indeed, the particle-collection efficiency of the rotating cup has not been extensively studied, and the only data available relate to a discontinued model. This study aimed to measure the collection efficiency of the current rotating cup model containing an aqueous (water) or viscous (ViaTrap mineral oil) collection fluid. The kinetics of evaporation confirmed that ViaTrap does not evaporate, making 8-h sampling campaigns in constant volumes feasible. Particles with a wide range of aerodynamic diameters (between around 0.1 and 10 µm) were produced using various test rigs and mono- or polydisperse test aerosols. Both new and older cup models performed similarly, with a collection efficiency of >80% for larger particles (aerodynamic diameters >2.8 µm), progressively decreasing to around 50% for aerodynamic diameters of 2.1 µm; with aerodynamic diameters of <1 µm, the collection efficiency was generally <10%. In physical terms, collection efficiency was unaffected by the type (aqueous or viscous) or volume (between 0 and 3 mL) of collection fluid used. Bias maps indicated that the inhalable fraction may be underestimated in occupational settings, particularly with aerosols mainly composed of particles with aerodynamic diameters of less than around 3 µm. Copyright