Udo Jäckel

Detection of Airborne Bacteria in a German Turkey House by Cultivation-Based and Molecular Methods

Todays large-scale poultry production with densely stocked and enclosed production buildings is often accompanied by very high concentrations of airborne microorganisms leading to a clear health hazard for employees working in such environments. Depending on the expected exposure to microorganisms, work has to be performed under occupational safety conditions. In this study, turkey houses bioaerosols were investigated by cultivation-based and molecular methods in parallel to determine the concentrations and the composition of bacterial community. Results obtained with the molecular approach showed clearly its applicability for qualitative exposure measurements. With both, cultivation-based and molecular methods species of microorganism with a potential health risk for employees (Acinetobacter johnsonii, Aerococcus viridans, Pantoea agglomerans, and Shigella flexneri) were identified. These results underline the necessity of adequate protection measures, including the recommendation to wear breathing masks during work in poultry houses.

Concentration of Bioaerosols in Composting Plants Using Different Quantification Methods

BACKGROUND Bioaerosols (organic dusts) containing viable and non-viable microorganisms and their metabolic products can lead to adverse health effects in exposed workers. Standard quantification methods of airborne microorganisms are mainly based on cultivation, which often underestimates the microbial burden. The aim of the study was to determine the microbial load in German composting plants with different, mainly cultivation-independent, methods. Second purpose was to evaluate which working areas are associated with higher or lower bioaerosol concentrations. METHODS A total of 124 inhalable dust samples were collected at different workplaces in 31 composting plants. Besides the determination of inhalable dust, particles, and total cell numbers, antigen quantification for moulds (Aspergillus fumigatus, Aspergillus versicolor, Penicillium chrysogenum, and Cladosporium spp.) and mites was performed. Concentrations of β-glucans as well as endotoxin and pyrogenic activities were also measured. The number of colony forming units (cfu) was determined by cultivation of moulds and actinomycetes in 36 additional dust samples. RESULTS With the exception of particle numbers, concentrations of all determined parameters showed significant correlations (P < 0.0001; r Spearman: 0.40-0.80), indicating a close association between these exposure markers. Colony numbers of mesophilic moulds and actinomycetes correlated also significantly with data of cultivation-independent methods. Exposure levels showed generally large variations. However, all parameters were measured highest in dusty working areas like next to the shredder and during processing with the exception of Cladosporium antigens that were found in the highest concentrations in the delivery area. The lowest concentrations of dust, particles, antigens, and pyrogenic activity were determined in wheel loader cabins (WLCs), which were equipped with an air filtration system. CONCLUSION It was possible to assess the microbial load of air in composting plants with different quantification methods. Since allergic and toxic reactions may be also caused by nonliving microorganisms, cultivation-independent methods may provide additional information about bioaerosol composition. In general, air filtration reduced the bioaerosol exposure shown in WLCs. Due to the fact that the mechanical processing of compost material, e.g. by shredding or sieving is associated with the generation of high bioaerosol concentrations, there is still a need of improved risk assessment and state-of-the-art protective measures in composting plants.

Eggshells as a source for occupational exposure to airborne bacteria in hatcheries.

ABSTRACT Occupational exposure to high concentrations of airborne bacteria in poultry production is related to an increased risk of respiratory disorders. However, potential sources and formation of hatchery bioaerosols are rarely characterized. In this study, bacterial multiplication on fresh shell fragments from turkey hatching eggs under conditions present in a hatcher incubator was investigated. A 105-fold amplification was observed both by colony count and total cell count gaining 4 × 107 cfu/cells per gram eggshell within 30 hr of incubation. Furthermore, the bacterial community present on eggshells was analyzed by generation of 16S rRNA gene clone libraries and identification of eight isolates. RFLP analysis revealed no shift in community composition during incubation and Enterococcus faecalis and Enterococcus gallinarum were found as the predominant species on turkey eggshells, both have been classified as risk group 2 microorganisms (German TRBA 466). Since Enterococcus spp. were found as predominant species on turkey eggshells, contribution of this genus to bioaerosol formation was demonstrated. During different work activities with poult and eggshell handling concentrations of airborne enterococci up to 1.3 × 104 cfu m−3 were detected. In contrast, no enterococci were identified at a day without poult or eggshell processing. In conclusion, turkey hatching eggs carry a viable specific microflora from breeder flocks to hatcheries. After hatching of turkey poults, hatcher incubators and eggshell fragments provide appropriate conditions for excessive bacterial growth. Thus, high bacterial loads on eggshell fragments are a source of potential harmful bioaersols caused by air flows, poult activity, and handling of equipment.

Detection of Airborne Bacteria in a Duck Production Facility with Two Different Personal Air Sampling Devices for an Exposure Assessment

Prevalent airborne microorganisms are not well characterized in industrial animal production buildings with respect to their quantity or quality. To investigate the work-related microbial exposure, personal bioaerosol sampling during the whole working day is recommended. Therefore, bioaerosol sampling in a duck hatchery and a duck house with two personal air sampling devices, a filter-based PGP and a NIOSH particle size separator, was performed. Subsequent, quantitative and qualitative analyses were carried out with” culture independent methods. Total cell concentrations (TCC) determined via fluorescence microscopy showed no difference between the two devices. In average, 8 × 106 cells/m3 were determined in the air of the duck hatchery and 5 × 107 cells/m3 in the air of the duck house. A Generated Restriction Fragment Length Polymorphism (RFLP) pattern revealed deviant bacterial compositions comparing samples collected with both devices. Clone library analyses based on 16S rRNA gene sequence analysis from the hatcherys air showed 65% similarity between the two sampling devices. Detailed 16S rRNA gene sequence analyses showed the occurrence of bacterial species like Acinetobacter baumannii, Enterococcus faecalis, Escherichia sp., and Shigella sp.; and a group of Staphylococcus delphini, S. intermedius, and S. pseudintermedius that provided the evidence of potential exposure to risk group 2 bacteria at the hatchery workplace. Size fractionated sampling with the developed by the Institute for Occupational Safety and Health of the German Social Accident Insurance (IFA) device revealed that pathogenic bacteria would deposit in the inhalable, the thorax, and possibly alveolar dust fraction according to EN481. TCC analysis showed the deposition of bacterial cells in the third stage (< 1μm) at the NIOSH device which implies that bacteria can reach deep into the lungs and contaminate the alveolus after inhalation. Nevertheless, both personal sampling devices could be recommended for exposure assessment at agricultural workplaces.

Heterogeneity in Cultivation-Based Monitoring of Airborne Bacterial Biodiversity in Animal Farms

Diversity analyses of bioaerosol samples from highly loaded workplaces as found in agricultural production or waste management help to improve the knowledge of exposure levels of workers. However, different used methods resulting in the detection of different bacterial species at the same work places. The present study obviously supports the deviation of received results using cultivation and further isolation approaches. Within the present study, the bacterial community at workplaces was estimated using the powerful tool of 16S rRNA gene sequence analyses after cultivation procedure. To avoid complex isolation procedures, the suitability of cultivation and subsequent cloning procedures was determined in bioaerosols from a duck hatchery. Diversity analysis of one bioaerosol sample, which was prepared independently three times in parallel, resulted in similarity values of 38.5%-57.1%. Further, similarity analysis calculated from three independent bioaerosol samplings on 1 day resulted in 31%-40% similarity. Although similar concentration between 2.22 × 106 and 4.46 × 106 CFU per m3 hatchery air were measured, in a ring-like trail, diversity analyses from six labs differ widely, resulting in 38.9%-78.6% divergence. The present method seems to be very useful for diversity analysis of bioaerosol samples, although heterogeneity in monitoring of airborne bacteria via cultivation was pointed out.

Development of a methodological approach for the characterization of bioaerosols in exhaust air from pig fattening farms with MALDI-TOF mass spectrometry

In this paper, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a cultivation-independent, routinely applicable approach to identify microbial fractions in bioaerosol emission samples. We developed a streamlined protocol in line with the German state-of-the-art impingement sampling guideline. Following isokinetic sampling, a fast and reliable pre-treatment methodology involving a series of cascade filtration steps was implemented, which produced fractions for spectrometric measurement devoid of interfering substances. We sampled the exhaust air from eight pig fattening farms around western Germany, which yielded two sets of samples for both method development and validation. For method development, in total 65 bacterial isolates were produced directly from the exhaust air samples, taxonomically classified by 16S rRNA-Gene sequencing, and subjected to MALDI-TOF analysis. In this way, we could assign fingerprint biomarkers to classified bacterial genera or even species to build up a preliminary reference database. For verification of the novel methodology and application of the reference database, we subjected the second set of exhaust air samples to the developed protocol. Here, 18 out of 21 bacterial species deposited in the database were successfully retrieved, including organisms classified in risk group 2, which might be used to evaluate the pathogenic potential of sampled exhaust air. Overall, this study pursues an entirely new approach to rapidly analyze airborne microbial fractions.