Jeotgalicoccus schoeneichii sp. nov. isolated from exhaust air of a pig barn.

A Gram-staining-positive, non-motile, non-spore-forming, coccus (strain 140805-STR-02T) was isolated from exhaust air of a pig barn on Columbia Blood Agar Base (Oxoid) supplemented with 5 % defibrinated horse blood, Streptococcus selective supplement and 0.5 mg erythromycin l-1. The strains shared high 16S rRNA gene sequence similarity to Jeotgalicoccus pinnipedialis (98.6 %) but only a maximum of 94 % sequence similarity to all other species of the genus Jeotgalicoccus. DNA-DNA hybridisation values between strain 140805-STR-02T and J. pinnipedialis CIP 107946T were 60.3 % (reciprocal, 51.2 %). The quinone system of 140805-STR-02T contained predominantly menaquinone MK-7 and minor amounts of MK-6. The polar lipid profile of strain 140805-STR-02T contained the major compounds diphosphatidylglycerol and phosphatidylglycerol and four unidentified lipids present in minor to moderate amounts. In the polyamine pattern spermidine and spermine were predominant. The fatty acid profile comprising iso-C15 : 0 and anteiso-C15 : 0 as major fatty acids, and was in congruence with those reported for other species of the genus Jeotgalicoccus and thus supported the affiliation of strain 140805-STR-02T to this genus. The results of physiological and biochemical tests allowed a clear phenotypic differentiation of strain 140805-STR-02T from the most closely related species. Strain 140805-STR-02T represents a novel species, for which the names Jeotgalicoccus schoeneichii sp. nov. is proposed, with the type strain 140805-STR-02T (=LMG 29445T=CCM 8667T).

Rothia aerolata sp. nov., isolated from exhaust air of a pig barn.

A Gram-stain-positive, coccoid, oxidase-negative, non-motile isolate from exhaust air of a pig barn, collected on 17 September 2014 and designated strain 140917-MRSA-09T, was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to Rothia amarae, Rothia terrae and Rothia endophytica (all <97.8 %). The G+C content of the genomic DNA was 58.9 mol %. The quinone system consisted of the major menaquinones MK-8 and MK-7. The polar lipid profile of strain 140917-MRSA-09T contained the major lipids diphosphatidylglycerol and phosphatidylglycerol and moderate amounts of dimannosylglyceride and trimannosyldiacylglycerol. The polyamine pattern was composed of the major amines putrescine and spermidine. In the fatty acid profile, iso- and anteiso-branched acids predominated (anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0). The strain showed a chemoheterotrophic metabolism and was able to grow aerobically well on nutrient-rich media at temperatures from 15-36 °C (weak at 42 °C), pH 5.5-9.5 and NaCl concentrations ranging from 0 to 7 % (w/v). Growth under anaerobic conditions was weak. Physiological traits as well as unique traits in the quinone pattern and the fatty acid pattern distinguished strain 140917-MRSA-09T from the most closely related species. All these data showed that strain 140917-MRSA-09T is a representative of a novel species of the genus Rothia, for which we propose the name Rothia aerolata sp. nov. The type strain is 140917-MRSA-09T (=LMG 29446T=CCM 8669T).

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

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.

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 ß-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.

Quantification of Saccharopolyspora rectivirgula in composting plants: assessment of the relevance of S. rectivirgula.

Exposure to bioaerosols in composting plants can lead to negative health effects on compost workers. Health complaints vary between cough, irritation of the eyes and the skin, sinusitis, or dyspnea among others. It is fact that compost materials harbor high concentrations of microorganisms, which were aerosolized during handling compost. Within the present study, total cell numbers between 3.4 × 10(4) and 1.6 × 10(8) cell counts per m(3) air were determined after 4',6-Diamidin-2-phenylindol DAPI staining in 124 samples from German composting plants. Special attention should be paid to some specific microorganisms, which are able to cause health complaints. Saccharopolyspora rectivirgula, known to be one of the major causes of extrinsic allergic alveolitis (EAA, also called hypersensitivity pneumonitis, HP), was often found in environments of agricultural production, where the classical form of EAA ('farmer's lung disease') is common, but also in composting plants. In Germany, cases are known where workers had to terminate their work due to this disease. However, up to now, the relevance of S. rectivirgula at composting plants is unexplained. This study showed that high concentrations of airborne S. rectivirgula were found in composting plants similar to that found in agricultural production. Altogether, in 86.7% of the 124 analyzed samples, S. rectivirgula was detected using quantitative real-time polymerase chain reaction (PCR). Estimated concentrations ranged between 1.24 × 10(2) cell counts of S. rectivirgula per cubic meter air next to the rotted residues and 1.5 × 10(7) cell counts next to a converter. Furthermore, our methodical proceedings were verified. To analyze DNA extraction limits through the amount of cells within one sample, the DNA concentration was compared with total cell counts (TCCs). Altogether, when TCC was <1.4 × 10(5) cells per DNA extraction assay, no DNA was measurable; when TCC reached 3.5 × 10(6) cells, DNA was always detectable by fluorometric method. To overcome limitation of DNA measurement using fluorometric method, samples without measurable DNA were inserted in a PCR assay with universal primers. Results showed that a gain of 37% was possible, when samples were additionally analyzed by universal PCR. Hence, cell counts >2.0 × 10(6) cells were necessary to measure DNA concentrations in 90% of the analyzed samples, whereas cell counts <3.0 × 10(5) are sufficient to detect PCR products. Therefore, sampling of bioaerosols should be done in consideration of the expected cell count per cubic meter air. Note, to get measurable DNA using a fluorometer, >3.5 × 10(6) cells must be sampled for one DNA extraction assay. With this study, the real-time PCR approach for the detection of S. rectivirgula at workplaces in compost plants was revised, and the results revealed that this method is suitable for occupational exposure measurements.

Multiplexed Workplace Measurements in Biogas Plants Reveal Compositional Changes in Aerosol Properties.

Anaerobic digestion is an emerging technology producing energy from renewable resources or food waste. Exposure screenings, comprising hazardous substances and biological agents, at different workplaces are necessary for a comprehensive overview of potential hazards in order to assess the risk of employees in biogas plants. In order to analyse these parameters, workplace measurements were conducted in seven full-scale anaerobic digesters. Personal and stationary sampling was performed for inhalable and respirable particles, volatile organic compounds, ammonia, hydrogen sulphide, carbon monoxide, and carbon dioxide. Furthermore, concentrations of the total cell count, endotoxins, and fungi-down to species level-were determined in comparison to windward air. Sequencing of the 16S rRNA genes was utilized for the determination of the bacterial composition inside the biogas plants. Measurements of hazardous substances show hardly values reaching the specific occupational exposure limit value, except ammonia. An approximate 5-fold increase in the median of the total cell count, 15-fold in endotoxins, and 4-fold in fungi was monitored in the biogas plants compared with windward air. Specifying the comparison to selected workplaces showed the highest concentrations of these parameters for workplaces related to delivery and cleaning. Strikingly, the fungal composition drastically changed between windward air and burdened workplaces with an increase of Aspergillus species up to 250-fold and Penicillium species up to 400-fold. Sequence analyses of 16S rRNA genes revealed that many workplaces are dominated by the order of Bacillales or Lactobacillales, but many sequences were not assignable to known bacteria. Although significant changes inside the biogas plant compared with windward air were identified, that increase does not suggest stricter occupational safety measures at least when applying German policies. However, exposure to biological agents revealed wide ranges and specific workplace measurements should be conducted for risk assessment.

Detection of airborne bacteria in a German turkey house by cultivation-based and molecular methods.

Today's 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.

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.