Review of biological risks associated with the collection of municipal wastes.

In many countries, the management of household waste has recently changed with an increased focus upon waste sorting resulting in lower collection frequency for some waste fractions. A consequence of this is the potential for increased growth of microorganisms in the waste before collection, which can lead to an increased exposure via inhalation for waste collection workers. Through a review of the literature, we aimed to evaluate risks caused by waste collecting workers' exposure to bioaerosols and to illuminate potential measures to reduce the exposure. Across countries and waste types, median exposure to fungi, bacteria, and endotoxin were typically around 104 colony forming units (cfu)/m3, 104 cfu/m3, and 10 EU/m3, respectively. However, some studies found 10-20+ times higher or lower median exposure levels. It was not clear how different types of waste influence the occupational exposure levels. Factors such as high loading, ventilation in and cleaning of drivers' cabs, increased collection frequency, waste in sealed sacks, and use of hand sanitizer reduce exposure. Incidences of gastrointestinal problems, irritation of the eye and skin and symptoms of organic dust toxic syndrome have been reported in workers engaged in waste collection. Several studies reported a correlation between bioaerosol exposure level and reduced lung function as either a short or a long term effect; exposure to fungi and endotoxin is often associated with an inflammatory response in exposed workers. However, a better understanding of the effect of specific microbial species on health outcomes is needed to proceed to more reliable risk assessments. Due to the increasing recycling effort and to the effects of global warming, exposure to biological agents in this working sector is expected to increase. Therefore, it is important to look ahead and plan future measures as well as improve methods to prevent long and short-term health effects.

One-year follow-up of microbial diversity in bioaerosols emitted in a waste sorting plant in France.

Bioaerosols emitted in waste sorting plants (WSP) can induce some adverse health effects on the workers such as rhinitis, asthma and hypersensitivity pneumonitis. The composition of these bioaerosols is scarcely known and most of the time assessed using culture-dependent methods. Due to the well-known limitations of cultural methods, these biodiversity measurements underestimate the actual microbial taxon richness. The aim of the study was to assess the airborne microbial biodiversity by using a sequencing method in a French waste sorting plant (WSP) for one year and to investigate the main factors of variability of this biodiversity. Static sampling was performed in five areas in the plant and compared to an indoor reference (IR), using closed-face cassettes (10 L.min-1) with polycarbonate membranes, every month for one year. Environmental data was measured (temperature, relative humidity). After DNA extraction, microbial biodiversity was assessed by means of sequencing. Bacterial genera Staphylococcus, Streptococcus, Prevotella, Lactococcus, Lactobacillus, Pseudomonas and fungal genera Wallemia, Cladosporium, Debaryomyces, Penicillium, Alternaria were the most predominant airborne microorganisms. Microbial biodiversity was different in the plant compared to the IR and seemed to be influenced by the season.

Protein stability during nebulization: Mind the collection step!

Inhaled protein therapeutics meet a growing interest for the treatment of respiratory diseases. In liquid aerosols, proteins face stresses that may generate instabilities, such as physicochemical denaturations, aggregation and loss of activity. Monitoring protein stability is thus crucial but implies collection of aerosol droplets before analysis. Many aerosol collection methods may be used, still their interference on protein stability is unknown. In this study, we compared the impact of six aerosol samplers on the stability of a model monoclonal antibody (Ig1), aerosolized with a mesh nebulizer. Ig1 stability was assessed for aggregation and biological activity. The six aerosol samplers generated distinct aggregation profiles for Ig1 at all size scales; counts of micron-sized particles varied by a factor of 100. The heterogeneity did not impact Ig1 activity, which was not significantly changed after nebulization. To extrapolate these results, we evaluated the impact of two samplers on three other proteins. Depending on the protein, samplers gave discordant aggregation and/or activity profiles, sometimes in the reverse trend as compared to Ig1. In conclusion, aerosol samplers interfere with protein stability; this impact depends both on the samplers and the protein, highlighting the importance of using the same collection device throughout the aerosol development process.

Effects of plant features on symptoms and airway inflammation in compost workers followed over 18 months.

This study investigated the plant features associated with increased irritation symptoms and levels of inflammation markers among compost workers (CWs). Ninety CWs were followed over 18 months, using questionnaires on respiratory symptoms, fractional exhaled nitric oxide measurements, spirometry, a methacholine bronchial challenge test, and quantification of specific immunoglobulins E (IgE) and G. CWs in plants processing the highest quantities of waste exhibited more airway irritation symptoms. So did the CWs in partially and fully indoor plants as compared to those in plants entirely outdoors. Working in sewage sludge versus green waste plants and having a high level of exposure were associated with higher levels of different IgE. The duration of employment decreased the FEV1 by 16 ml per year. Working in an indoor plant is linked to symptoms and inflammation markers in CWs.

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.

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.

Endotoxin deposits on the inner surfaces of closed-face cassettes during bioaerosol sampling: a field investigation at composting facilities.

A set of 270 bioaerosol samples was taken from 15 composting facilities using polystyrene closed-face filter cassettes (CFCs). The objective was to measure the quantity of endotoxin deposits on the inner surfaces of the cassettes (sometimes referred to as 'wall deposits'). The results show that endotoxins are deposited on the inner surfaces of the CFCs through sampling and/or handling of samples. The quantity of endotoxins measured on inner surfaces range between 0.05 (the limit of detection of the method) and 3100 endotoxin units per cassette. The deposits can represent a large and variable percentage of the endotoxins sampled. More than a third of the samples presented a percentage of inner surface deposits >40% of the total quantity of endotoxins collected (filter + inner surfaces). Omitting these inner surface deposits in the analytical process lead to measurement errors relative to sampling all particles entering the CFC sampler, corresponding to a developing consensus on matching the inhalable particulate sampling convention. The result would be underestimated exposures and could affect the decision as to whether or not a result is acceptable in comparison to airborne concentration limits defined in terms of the inhalability convention. The results of this study suggest including the endotoxins deposited on the inner surfaces of CFCs during analysis. Further researches are necessary to investigate endotoxin deposits on the inner cassette surfaces in other working sectors.

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 plant's 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.

Exposure to airborne endotoxins among sewer workers: an exploratory study.

Exploratory bioaerosol sampling was performed in order to assess exposure to airborne endotoxins during sewer work. Personal samples were collected in underground sewer pipes using 37-mm closed-face cassettes containing fibreglass filters (CFC-FG method) or polycarbonate filters (CFC-PC method). Endotoxins were quantified using the limulus amoebocyte lysate assay. Concentrations of airborne endotoxins at sewer workplaces (16-420 EU m(-3)) were higher than those measured outside the sewer network (0.6-122 EU m(-3)). Sewer worker exposure to airborne endotoxins depended on the workplace and on the tasks. Exposure levels were the highest for tasks involving agitation of water and matter, especially for 'chamber cleanup' and 'pipes cleanup' with a high-pressure water jet. Airborne endotoxin levels at the workplace tended to be higher when CFC-FG was used as the sampling method rather than CFC-PC. The adjusted mean of the measured concentrations for CFC-PC represents 57% of the mean observed with CFC-FG. The number of samples collected in the descriptive study was too low for drawing definitive conclusions and further exposure investigations are needed. Therefore, our exploratory study provides new exposure data for the insufficiently documented sewer working environment and it would be useful for designing larger exposures studies.

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.

Development and evaluation of a method for the quantification of airborne Thermoactinomyces vulgaris by real-time PCR.

Actinomycetes are ubiquitous and some can be potentially pathogenic for humans when present in the air of some working areas. It's notably the case for Thermoactinomyces vulgaris in composting facilities where aerial concentrations can reach high values of more than 10(7) CFU·m(-3). Workers exposure to these inhalable bioaerosols can be the source of various diseases. The literature reveals a lack of knowledge about risk assessment: there is neither dose-effects relationship for most agents, or threshold limit value. The objectives of this study were to develop and standardize a method to quantify workers exposure to bioaerosols. We have developed and evaluated a method to quantify airborne T. vulgaris based on DNA extraction of aerial microbial communities and qPCR. Four DNA extraction protocols were compared, and primers and a hydrolysis probe were designed for specific amplification of the target species (gyrB gene). This method was compared to traditional methods based on viable or cultivable counting by epifluorescence microscopy or plating on selective media. The method was applied on environmental bioaerosols sampled under real exposure conditions in composting plants. We demonstrate that the method to quantify T. vulgaris in bioaerosols is specific, sensitive and repeatable. We demonstrate the occurrence and quantified T. vulgaris in the atmosphere of composting facilities with concentrations ranging from 3×10(2) to 3×10(6)×m(-3).

Documentation of bioaerosol concentrations in an indoor composting facility in France.

Bioaerosol concentrations were investigated in a totally indoor composting facility processing fermentable household and green wastes to assess their variability. Stationary samples were collected by filtration close to specific composting operations and then were analysed for cultivable mesophilic bacteria, thermophilic bacteria, mesophilic fungi, thermophilic fungi, endotoxins and total airborne bacteria (DAPI-staining). Indoor concentrations exceeded the background levels, between 500 and 5400 EU m(-3) for endotoxins, 10(4) and 10(6) CFU m(-3) for cultivable bacteria and generally below 10(5) CFU m(-3) for airborne cultivable fungi. No significant (p > 0.05) differences were observed between the indoor composting operations. Successive 30 minute bioaerosol samples were collected to investigate the variation of cultivable mesophilic microorganisms over the work shift. Concentrations of mesophilic bacteria and fungi varied up to 1 log unit depending on the time at which they were collected in the day. Total airborne particles, counted using an optical particle counter, were present at up to 10(8) particles m(-3) and several concentration peaks were noted. Values for total airborne bacteria were roughly 70-fold higher than cultivable bacteria. These results raise the question of the sampling strategy (duration of sampling; number of samples to be collected) used in similar studies. They provide new bioaerosol concentration data in a composting facility and suggest that the filtration sampling method might be a useful tool for exposure measurements in that occupational environment.

Bacterial community composition of biological degreasing systems and health risk assessment for workers.

Biological degreasing system is a new technology based on the degradation capabilities of microorganisms to remove oil, grease, or lubricants from metal parts. No data is available about the potential biological health hazards in such system. Thus, a health risk assessment linked to the bacterial populations present in this new degreasing technology is, therefore, necessary for workers. We performed both cultural and molecular approaches in several biological degreasing systems for various industrial contexts to investigate the composition and dynamics of bacterial populations. These biological degreasing systems did not work with the original bacterial populations. Indeed, they were colonized by a defined and restricted group of bacteria. This group replaced the indigenous bacterial populations known for degrading complex substrates. Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, and Pantoea agglomerans were important members of the microflora found in most of the biological degreasing systems. These bacteria might represent a potential health hazard for workers.

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.