Mallory Vacheyrou

Cultivable microbial communities in raw cow milk and potential transfers from stables of sixteen French farms

The indigenous microflora in raw milk plays an important role in the diversity of cheese flavours and may protect against the growth of pathogens, but the sources of contamination and the factors that might affect the microbial communities in milk are not well known. The objectives of this study were to broaden knowledge of the microbial composition of milk and to assess microbial transfers from the stable to the milk. Air (collected in milking parlour and stable), dust (passively collected using plastic box), cow teat surface, and hay and milk samples were collected in 16 French farms with either stanchion barn or freestall barn configurations and plated on various culture media. Bacterial and fungal colonies were identified using phenotypic and DNA sequencing methods. Results showed that most of the fungal species and environmental bacteria found in the milk were also found in the stable and the milking parlour environments, indicating large microbial transfer from stable to milking parlour then to milk. However, milk from the stanchion barns were more contaminated than milk from freestall barns. Contrasting with other bacterial and fungal species, useful cheese-making bacteria--lactobacilli and PAB--were frequently identified in the milk and on the teat surface but were rarely found in other environments. In conclusion, milk contamination by the stable environment is considerable, even if it is lower in farms with a milking parlour. Besides this environmental contamination, the teat surface remains the main source of useful cheese-making bacteria.

Airborne cultivable microflora and microbial transfer in farm buildings and rural dwellings

Objectives Exposure to environments rich in microorganisms such as farms has been shown to protect against the development of childhood asthma and allergies. However, it remains unclear where, and how, farm and other rural children are exposed to microbes. Furthermore, the composition of the microbial flora is poorly characterised. We tested the hypothesis that farm children are exposed indoors to substantial levels of viable microbes originating from animal sheds and barns. We also expected that environmental microbial flora on farms and in farm homes would be more complex than in the homes of rural control children. Methods Dust samples were collected using passive samplers in the bedrooms of the following groups of children in rural Bavaria, Germany: (i) those living on farms (n=144), (ii) those regularly exposed to farm environments but not living on farms (n=149) and (iii) those never visiting farms (n=150). For farm children, additional samples were collected in animal sheds and barns. All samples were subjected to fungal and bacterial culturing. Results Detectable levels of microorganisms were more often found in samples taken from farm dwellings than from other homes. Farm dwellings also showed higher microbial levels. Microbial counts of farm dwelling samples correlated with the counts in corresponding animal sheds and barns. Conclusions Microorganisms are transported from animal sheds and barns into farm dwellings. Therefore, children living in these environments are exposed when indoors and when visiting animal sheds and barns. Indoor exposure may also contribute to the protective effect of the farm environment.

Assessment of Dust Sampling Methods for the Study of Cultivable-Microorganism Exposure in Stables

ABSTRACT Studies have shown a link between living on a farm, exposure to microbial components (e.g., endotoxins or β-d-glucans), and a lower risk for allergic diseases and asthma. Due to the lack of validated sampling methods, studies of asthma and atopy have not relied on exposure assessment based on culture techniques. Our objective was therefore to compare several dust sampling methods for the detection of cultivable-microorganism exposure in stables. Sixteen French farms were sampled using four different methods: (i) active air sampling using a pump, (ii) passive dust sampling with a plastic box, (iii) dust sampling with an electrostatic dust fall collector (wipe), and (iv) dust sampling using a spatula to collect dust already settled on a windowsill. The results showed that collection of settled dust samples with either plastic boxes or wipes was reproducible (pairwise correlations, 0.72 and 0.73, respectively) and resulted in highly correlated results (pairwise correlation between the two methods, 0.82). We also found that settled dust samples collected with a plastic box correctly reflected the composition of the samples collected in the air of the stable when there was no farmer activity. A loss of microbial diversity was observed when dust was kept for 3 months at room temperature. We therefore conclude that measurement of viable microorganisms within a reasonable time frame gives an accurate representation of the microbial composition of stable air.

Contribution of a Cyclonic-Based Liquid Air Collector for Detecting Aspergillus Fumigatus by QPCR in Air Samples

Fungal infections represent a constant threat in hospitals, especially for high-risk patients hospitalized in hematology and bone marrow transplant units. While routine air sampling to predict the fungal contamination risk is discussed by some authors,(1,2) French hospital guidelines specify that each hospital is responsible for the air it provides to patients and that air control efficiency must be monitored by specific methodologies.(3) Traditional monitoring involves impacted culture media that are incubated for several days so that the fungal species may grow and be identified on the basis of macroscopic and microscopic criteria.(4) A variety of portable air impactors are commercially available and validated,(5,6) but these systems are limited by their link with culture techniques, involving a time-lapse to results of around 7 days and by a maximal sampling rate of 100 L/min. As optimal environmental monitoring requires large sample sizes and fast, accurate detection of microorganisms contained within the samples,(7,8) new techniques concurring with these characteristics are regularly evaluated. Quantitative polymerase chain reaction (QPCR) represents a very attractive alternative to culture techniques as it allows Aspergillus fumigatus, which is the preponderant etiologic agent of invasive aspergillosis, to be specifically detected in under 2 days. Reducing the time to results is key to permit intervention to prevent Aspergillus exposure in immunocompromised patients. In this way, QPCR was previously used to amplify Aspergillus fumigatus DNA on various substrates, such as tap water,(9) carpets,(7) air filters,(10) and impacted lowmelt agar plates.(11) A new device, the Coriolis μ air sampler (Bertin Technologies, Montigny, France), based on a cyclonic system, was recently proposed for collecting large volumes of air on liquid medium quickly. This device was previously assessed for different areas other than fungal aero-contamination monitoring: analysis of pollen grain distribution(12,13) and detection of Pneumocystis jiroveci DNA by QPCR.(14) A preliminary study was carried out to test this cyclonic-based liquid device, combined with QPCR, and to assess its performance for detecting A. fumigatus DNA.

Assessment of pets (cats and dogs) in homes using electrostatic dust collectors and QPCR: new tools to evaluate exposure and risk of allergies

Abstract Contradictory results are found in the literature concerning fungi, bacteria, and pet exposure and the risk of developing asthma. All these allergens have been thoroughly studied separately in cohort studies, and a variety of sampling and analytical methods are used. It is already possible to characterize fungi, mites, and bacteria by QPCR. The aim of our study is to evaluate QPCR systems to quantify the presence of cats and dogs in homes. Twenty-four houses were sampled with an Electrostatic Dust Collector which was analyzed by QPCR. Questionnaires on the presence of pets in homes were completed. The results from QPCR were correlated for real presence of cats and dogs, and highlighted indirect exposure. This study provides a useful screening tool that will be used in future large cohort studies, such as the ELFE cohort study.

Health effects of occupational exposure in a dairy food industry, with a specific assessment of exposure to airborne lactic acid bacteria.

Objective: Lactic acid bacteria (LAB) are used in food industries as probiotic agents. The aim of this study is to assess the potential health effects of airborne exposure to a mix of preblend (LAB and carbohydrate) and milk powder in workers. Methods: A medical questionnaire, lung function tests, and immunologic tests were carried out on 50 workers. Occupational exposure to inhalable dust and airborne LAB was measured. Results: Workers not using respiratory masks reported more symptoms of irritation than workers using protection. Workers from areas with higher levels of airborne LAB reported the most health symptoms and the immune responses of workers to LAB was higher than the immune responses of a control population. Conclusions: Measures to reduce exposure to airborne LAB and milk powder in food industries are recommended.

Positive fungal quantitative PCR and Th17 cytokine detection in bronchoalveolar lavage fluids: Complementary biomarkers of hypersensitivity pneumonitis?

BACKGROUND Interstitial lung disease (ILD) is a large group of diseases, including hypersensitivity pneumonitis (HP), idiopathic pulmonary fibrosis (IPF) and sarcoidosis. OBJECTIVE In this study, we aimed to identify bronchoalveolar lavage fluid (BALF) biomarkers which could be contributive for HP diagnosis. METHODS We analyzed 39 BALF samples from 12 ILD patients with sarcoidosis, 11 with IPF and 16 with HP. We determined the levels of 10 cytokines and carried out quantitative PCR for 10 microorganisms involved in farm-associated or domestic forms of HP. RESULTS IL-8, IL-6, TNFα, IL-17 and IL-23 levels were significantly higher in BALF samples from HP patients (p<0.05, one-way Kruskal-Wallis analysis). QPCR tests for Eurotium amstelodami and Wallemia sebi were positively significantly more frequently for HP patients (p<0.05, one-way Kruskal-Wallis). CONCLUSION The biomarkers identified here can be detected in BALF, which could be routinely obtained as complementary analysis in ILD diagnosis.