Enric Robine

Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH)

Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

Detection of fungal development in closed spaces through the determination of specific chemical targets

In addition to the biodegradation problems encountered in buildings, exposure of their occupants to moulds is responsible for numerous diseases: infections (invasive nosocomial aspergillosis), immediate or delayed allergies, food-borne infections and different types of irritation. In this context, the aim of our work has been to determine specific chemical tracers for fungal development on construction materials. More generally, by detecting a specific chemical fingerprint of fungal development, our objective was to propose a microbiological alert system which could control systems and/or procedures for the microbiological treatment of indoor areas. We therefore characterized the chemical emissions from six types of construction material contaminated artificially by moulds. Chemical fingerprints were established for 19 compounds arising specifically from fungal metabolism: 2-ethylhexanoic acid methyl ester, 1-octen-3-ol, 3-heptanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 1,3-octadiene, 2-(5H)-furanone, 2-heptene, alpha-pinene, 2-methylisoborneol, 4-heptanone, 2-methylfuran, 3-methylfuran, dimethyldisulfide, methoxybenzene, a terpenoid and three sesquiterpenes. Determining the origin of these compounds and their specific links with a growth substrate or fungal species made it possible to judge the pertinence of choosing these compounds as tracers. Thus the detecting specific volatile organic compounds emitted as from the second day of fungal growth demonstrated that this approach had the advantage of detecting fungal development both reliably and rapidly before any visible signs of contamination could be detected.

Airborne fungal volatile organic compounds in rural and urban dwellings: Detection of mould contamination in 94 homes determined by visual inspection and airborne fungal volatile organic compounds method

Moulds can both degrade the materials and structures they colonise and contribute to the appearance of symptoms and diseases in the inhabitants of contaminated dwellings. Only few data have compared the levels of contamination in urban and rural environments and the results are not consistent. The aim of this study was to use a fungal contamination index, based on the detection of specific Microbial Volatile Organic Compounds (MVOC), to determine the exposure to moulds of individuals living in urban and rural dwellings. For this purpose, 94 dwellings (47 in an urban setting in Clermont-Ferrand and 47 in rural areas of the Auvergne region, France) were studied. By demonstrating marked disparities between the proportion of visible contamination (19%) and that of active, visible and/or hidden contamination (59%) and the fact that almost all visible contamination was identified by MVOC, we were able to show that use of the index seemed relevant to confirm the actual presence of fungal contamination in a dwelling. Furthermore, it was possible to demonstrate a relationship between moulds and the presence of water on surfaces (condensation, infiltrations, water damage, etc.). A higher proportion of positive fungal contamination index in rural homes was observed compared to the proportion in urban ones (68% versus 49%; p<0.05).

Positive associations between respiratory outcomes and fungal index in rural inhabitants of a representative sample of French dwellings.

Our study aims at estimating exposure to molds at home, based on microbial Volatile Organic Compounds (MVOCs) assessment, and evaluating its effect on respiratory diseases in a representative sample of dwellings. In the framework of a national campaign, indoor pollution was monitored in a sample of the 24 million dwellings of metropolitan France (n=567). 727 subjects answered to a standardized questionnaire on respiratory diseases and had MVOCs sampled in their bedrooms and a fungal index (FI) defined. Among the 431 dwellings with complete data, one out of three was contaminated by molds as assessed by a positive FI: 27.0% in urban, 38.2% in periurban and 34.9% in rural dwellings respectively. Positive associations were observed between fungal index and current asthma (8.6%) and chronic bronchitis-like symptoms (8.4%), especially in rural areas (OR=2.95, 95%CI (1.10; 7.95) and 3.35, 95%CI (1.33; 8.48) respectively). Our study, based on objective assessments of fungal contamination, is in agreement with previous results suggesting mold-related respiratory effects. Moreover associations found among rural population could indicate specific pollution and impact in this environment.

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

ABSTRACT Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.

Stability of airborne microbes in the Louvre Museum over time

The microbial content of air has as yet been little described, despite its public health implications, and there remains a lack of environmental microbial data on airborne microflora in enclosed spaces. In this context, the aim of this study was to characterize the diversity and dynamics of airborne microorganisms in the Louvre Museum using high-throughput molecular tools and to underline the microbial signature of indoor air in this human-occupied environment. This microbial community was monitored for 6 month during occupied time. The quantitative results revealed variations in the concentrations of less than one logarithm, with average values of 10(3) and 10(4) Escherichia coli/Aspergillus fumigatus genome equivalent per m(3) for bacteria and fungi, respectively. Our observations highlight the stability of the indoor airborne bacterial diversity over time, while the corresponding eukaryote community was less stable. Bacterial diversity characterized by pyrosequencing 454 showed high diversity dominated by the Proteobacteria which represented 51.1%, 46.9%, and 38.4% of sequences, for each of the three air samples sequenced. A common bacterial diversity was underlined, corresponding to 58.4% of the sequences. The core species were belonging mostly to the Proteobacteria and Actinobacteria, and to the genus Paracoccus spp., Acinetobacter sp., Pseudomonas sp., Enhydrobacter sp., Sphingomonas sp., Staphylococcus sp., and Streptococcus sp.

Impact of Health on Particle Size of Exhaled Respiratory Aerosols: Case‐control Study

Abstract Individuals with viral infection could possibly emit an infectious aerosol. The distinction between exhaled breaths of infected and healthy individuals should facilitate an understanding of the airborne transmission of infections. In this context, the present study is aimed at distinguishing healthy individuals from symptomatic ones by the study of their exhaled breath. A setup composed of a modified hood connected to an electrical low pressure impactor, which allows for the study of a wide range of particle sizes (from 7 nm to 10 μm), has been developed in order to collect exhaled breaths. This setup has been used with seventy eight volunteers. The results obtained using Principal Component Analysis (PCA) showed that exhaled breaths of individuals without symptoms have statistical similarities and are different from those of individuals with symptoms. This separation was made by the greater proportional emission by individuals with symptoms of particles collected on stages 3 (D 50 = 0.09 μm), 6 (D 50 = 0.38 μm), 8 (D 50 = 0.95 μm), 10 (D 50 = 2.40 μm), and 12 (D 50 = 4.02 μm) of the impactor. There was not a specific size distribution obtained for the individuals with symptoms. As a consequence, further research on the exhaled breath should be undertaken with symptomatic volunteers and would require the analysis of this wide range of particle sizes.

Design optimisation of silicon-based MEMS sensors dedicated to bioaerosols monitoring

Due to recent paradigm changes for the monitoring of air pollution, assessing human exposure to (bio)- aerosols with low-cost sensors is expected to be of growing importance. In this context, our work subsequently investigates the development of Si-based gravimetric instruments. Thus particle-resonator interactions are considered to provide a particle detachment criteria as well as geometrical features ensuring performances both in terms of sensitivity and uniform resonator response. Analytical and numerical methods have been carried out and confronted to experimental results.