Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide.

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock.

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).

Automated Image Analysis for Determination of Antibody Titers Against Occupational Bacterial Antigens Using Indirect Immunofluorescence.

Employees who are exposed to high concentrations of microorganisms in bioaerosols frequently suffer from respiratory disorders. However, etiology and in particular potential roles of microorganisms in pathogenesis still need to be elucidated. Thus, determination of employees' antibody titers against specific occupational microbial antigens may lead to identification of potentially harmful species. Since indirect immunofluorescence (IIF) is easy to implement, we used this technique to analyze immunoreactions in human sera. In order to address disadvantageous inter-observer variations as well as the absence of quantifiable fluorescence data in conventional titer determination by eye, we specifically developed a software tool for automated image analysis. The 'Fluorolyzer' software is able to reliably quantify fluorescence intensities of antibody-bound bacterial cells on digital images. Subsequently, fluorescence values of single cells have been used to calculate non-discrete IgG titers. We tested this approach on multiple bacterial workplace isolates and determined titers in sera from 20 volunteers. Furthermore, we compared image-based results with the conventional manual readout and found significant correlation as well as statistically confirmed reproducibility. In conclusion, we successfully employed 'Fluorolyzer' for determination of titers against various bacterial species and demonstrated its applicability as a useful tool for reliable and efficient analysis of immune response toward occupational exposure to bioaerosols.

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.

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 × 10(6) cells/m(3) were determined in the air of the duck hatchery and 5 × 10(7) cells/m(3) 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 hatchery's 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.

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.

Bioaerosols in swine confinement buildings: A metaproteomic view.

Swine confinement buildings represent workplaces with high biological air pollution. It is suspected that individual components of inhalable air are causatives of chronic respiratory disease that are regularly detected among workers. In order to understand the relationship between exposure and stress, it is necessary to study the components of bioaerosols in more detail. For this purpose, bioaerosols from pig barns were collected on quartz filters and analysed via a combinatorial approach of 16S rRNA amplicon sequencing and metaproteomics. The study reveals the presence of peptides from pigs, their feed and microorganisms. The proportion of fungal peptides detected is considered to be underrepresented compared to bacterial peptides. In addition, the metaproteomic workflow enabled functional predictions about the discovered peptides. Housekeeping proteins were found in particular, but also evidence for the presence of bacterial virulence factors (e.g., serralysin-like metalloprotease) as well as plant (e.g., chitinase) and fungal allergens (e.g., alt a10). Metaproteomic analyses can thus be used to identify factors that may be relevant to the health of pig farmers. Accordingly, such studies could be used in the future to assess the adverse health potential of an occupationally relevant bioaerosol and help consider defined protective strategies for workers.

In Vitro Systems for Toxicity Evaluation of Microbial Volatile Organic Compounds on Humans: Current Status and Trends.

Microbial volatile organic compounds (mVOC) are metabolic products and by-products of bacteria and fungi. They play an important role in the biosphere: They are responsible for inter- and intra-species communication and can positively or negatively affect growth in plants. But they can also cause discomfort and disease symptoms in humans. Although a link between mVOCs and respiratory health symptoms in humans has been demonstrated by numerous studies, standardized test systems for evaluating the toxicity of mVOCs are currently not available. Also, mVOCs are not considered systematically at regulatory level. We therefore performed a literature survey of existing in vitro exposure systems and lung models in order to summarize the state-of-the-art and discuss their suitability for understanding the potential toxic effects of mVOCs on human health. We present a review of submerged cultivation, air-liquid-interface (ALI), spheroids and organoids as well as multi-organ approaches and compare their advantages and disadvantages. Furthermore, we discuss the limitations of mVOC fingerprinting. However, given the most recent developments in the field, we expect that there will soon be adequate models of the human respiratory tract and its response to mVOCs.

Detection of Saccharopolyspora rectivirgula by quantitative real-time PCR.

The thermophilic actinomycete species Saccharopolyspora rectivirgula has been associated with the exogen allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of airborne spores that can be found for example in environments of agricultural production, compost facilities, mushroom cultivation rooms, or rooms with technical air moistening. Because of the medical relevance of S. rectivirgula, a reliable detection system is needed. Therefore, a quantitative real-time polymerase chain reaction (qPCR) primer system was designed, targeting the 16S rRNA gene of the type strain S. rectivirgula DSM 43747(T) and six other S. rectivirgula reference strains. Our investigation showed that S. rectivirgula presumably own four operons of the 16S rRNA gene, which has to be considered for estimation of cell equivalents. Furthermore, the DNA recovery efficiency from these strains was tested in combination with bioaerosol or material sample as well as the influence of non-target DNA to the recovery rate. Results showed a recovery DNA efficiency of 7-55%. The recovery rate of DNA in a mixture with non-target DNA resulted in ∼87%. In summary, a high amplification efficiency using real-time PCR was found, for which estimated concentrations revealed cell numbers of 2.7 × 10(5) cells m(-3) in bioaerosol and 2.8 × 10(6) cells g(-1) fw(-1) in material samples from a duck house. The specificity of the new developed quantification system was shown by generation of two clone libraries from bioarosol samples, from a duck house, and from a composting plant. Totally, the results clearly show the specificity and practicability of the established qPCR assay for detection of S. rectivirgula.

Characterization of bacterial contaminants in the air of a duck hatchery by cultivation based and molecular methods.

Today's large-scale poultry production is often accompanied by high concentrations of airborne microorganisms at working places. However, the microbial communities in those bioaerosols are rarely characterised. In this study, we investigated the bacterial population in bioaerosols from a duck hatchery by both cultivation based and molecular methods and compared the results. Depending on used media, concentrations of airborne culturable bacteria varied between 6 × 10(1) and 7 × 10(6) CFU per m(3) air. The corresponding total cell count of DAPI stained cells was 2 × 10(7) cells per m(3) air. 16S rRNA gene analyses of bacterial isolates and clone libraries revealed a low species richness in hatcheries air, respectively. More than 50% of bacterial isolates were phylogenetically most closely related to bacterial species of the risk group 2 (German TRBA). The sequence composition in clone libraries supported the result of cultivation based approaches, whereby sequences assigned to Staphylococcus, Acinetobacter and Enterococcus are the most common. The high concentration of airborne bacteria which are most closely related to species of potential health risk requires further detailed investigations for these bacterial species.

Application of impedance measurement to investigate in vitro inhalation toxicity of bacteria.

BACKGROUND: Workers of agriculture and intensive life stock farming are exposed to highly contaminated workplaces. Bioaerosol exposures are suspected to trigger respiratory health effects of the workers. So far, risk evaluation of bioaerosols has been assessed through the infectivity of comprising biological agents that is classified in Europe by four risk groups according to the criteria of Directive 2000/54EC of the European Parliament. However, this directive additionally requires the risk assessment of allergenic and toxigenic effects without further elaboration. The aim of our study was to establish an in vitro screening system that is able to measure inhalative toxic effects of bacteria and their metabolites. METHODS: In this study, we analyzed three bacterial toxins and five culture supernatants of selected bacteria with known toxicity as model agents exposed to the lung epithelial cell line NuLi-1. We used electrical cell-substrate impedance sensing (ECIS) method to monitor real-time cell changes and the viability test Prestoblue™. RESULTS: We confirmed concentration dependent cytotoxic effects of the selected toxins in NuLi-1 cells over a period of up to 48 h. Each toxin resulted in a different but specific impedance profile over time according to their mode of action, whereas viability assay showed the metabolic activity of the cells at a chosen time point without revealing any information on their mode of action. Furthermore, dose-response-relationships were monitored. Tested model bacteria (Streptoccous pneumoniae, Acinetobacter radioresistens, Aerococcus viridans, Aeromonas hydrophila) reacted according to their expected toxicity except one bacterium (Enterococcus faecalis). The established assays revealed the concentration dependent onset and intensity of bacterial cytotoxicity and the viability of the cells at 24 h and 48 h exposure. CONCLUSION: Impedance measurement and the viability assay Prestoblue™ in combination are suitable as sensitive screening methods to analyze toxic potential of bacteria and can therefor support the risk assessment of workplaces in terms of the directive 2000/54/EC.

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.

Quantification and identification of culturable airborne bacteria from duck houses.

Employees at agricultural working places are often exposed to complex bioaerosols. Investigations of bioaerosols in duck houses revealed concentrations of cultivable bacteria between 0.4 and 3 x 10(5) colony forming units (CFU) m(-3) on tryptone soy agar, 0.3 and 2 x 10(5) CFU m(-3) on actinomycetes isolation agar, and 0.8 and 5 x 10(3) CFU m(-3) on Middlebrook agar, respectively, when incubated at 25 degrees C. At an incubation temperature of 37 degrees C, 0.6-3 x 10(2) CFU m(-3) were counted on MacConkey agar and 0.3-2 x 10(3) CFU m(-3) on Middlebrook agar, and the concentrations of bacteria on glycerol-arginine agar and oatmeal agar incubated at 50 degrees C varied between 0.1 and 2 x 10(3) and 1 and 7 x 10(3) CFU m(-3), respectively. In addition, high concentrations of cells were observed by fluorescence microscope quantification of cell counts after 4',6-diamidino-2-phenylindol staining with 3-8 x 10(7) cells m(-3). A total of 213 colonies with different morphological appearance were selected and the isolated pure cultures were identified at the genus level using the 16S rRNA gene sequence analyses. In summary, 19 different genera of Actinobacteria, four genera of the Firmicutes, one genus of the Bacteroidetes, and five genera of the Proteobacteria were identified. Several isolates represent new phylogenetic lineages. Based on 16S rRNA gene analyses, some isolates were most closely related to Cellulosimicrobium funkei, Corynebacterium falsenii, Corynebacterium xerosis, Mycobacterium arupense, and Staphylococcus epidermidis, which have been grouped into Risk group 2 of biological agents and may cause negative pulmonary health effects. These bacterial species were present in high concentrations up to 10(4) CFU m(-3). For this reason, we recommend an adequate personal breathing protection at these working places.

Benchmarking the MinION: Evaluating long reads for microbial profiling.

Nanopore based DNA-sequencing delivers long reads, thereby simplifying the decipherment of bacterial communities. Since its commercial appearance, this technology has been assigned several attributes, such as its error proneness, comparatively low cost, ease-of-use, and, most notably, aforementioned long reads. The technology as a whole is under continued development. As such, benchmarks are required to conceive, test and improve analysis protocols, including those related to the understanding of the composition of microbial communities. Here we present a dataset composed of twelve different prokaryotic species split into four samples differing by nucleic acid quantification technique to assess the specificity and sensitivity of the MinION nanopore sequencer in a blind study design. Taxonomic classification was performed by standard taxonomic sequence classification tools, namely Kraken, Kraken2 and Centrifuge directly on reads. This allowed taxonomic assignments of up to 99.27% on genus level and 92.78% on species level, enabling true-positive classification of strains down to 25,000 genomes per sample. Full genomic coverage is achieved for strains abundant as low as 250,000 genomes per sample under our experimental settings. In summary, we present an evaluation of nanopore sequence processing analysis with respect to microbial community composition. It provides an open protocol and the data may serve as basis for the development and benchmarking of future data processing pipelines.

Direct detection of salmonella cells in the air of livestock stables by real-time PCR.

A SYBR Green real-time quantitative polymerase chain reaction (qPCR) assay for specific detection and quantification of airborne Salmonella cells in livestock housings is presented. A set of specific primers was tested and validated for specific detection and quantification of Salmonella-specific invA genes of DNA extracted from bioaerosol samples. Application of the method to poultry house bioaerosol samples showed concentrations ranging from 2.2 x 10(1) to 3 x 10(6) Salmonella targets m(-3) of air. Salmonella were also detected by a cultivation-based approach in some samples, but concentrations were two to three magnitudes lower than the concentrations detected by molecular biological results. Specificity of results was demonstrated by cloning analyses of PCR products, which were exclusively assigned to the genus Salmonella. However, by molecular methods, microorganisms are detected independently of their viability status, leading to an overestimation of concentration. Hence, the survival rate of Salmonella cells was measured on filter surfaces during filtration samplings where 82% of the cells died within 20 min of filtration. The results clearly show the specificity and practicability of the established qPCR assay for analysis and quantification of salmonellae in bioaerosols. The results demonstrate airborne Salmonella workplace concentrations in poultry production of up to 3.3% of 4',6-Diamidino-2-phenylindole-counted total cell numbers.

Occupational Exposure to Antibiotics in Poultry Feeding Farms.

Today, antibiotics are essential for effective treatment of infectious diseases both in human and veterinary medicine. The increasing development and distribution of antibiotic-resistant microorganisms are subject of concern. In some sectors of animal agriculture, such as poultry feeding farms, the application of antibiotics and hence occupational exposure is inevitable. In the past, numerous studies focussed on the occurrence of antibiotic-resistant bacteria in livestock farming, but little attention was paid to the employees. The exposure of workers to antibiotics was the focus of the study detailed in this article. Four biomonitoring campaigns monitoring systemic exposure of workers to antibiotics were run at two farms over four fattening periods. Urine samples of potentially affected employees were sampled and analysed for the antibiotics of interest by liquid chromatography-mass spectrometry. The highest antibiotic concentrations detected in urine samples exceeded the minimum inhibitory concentration of some bacteria strains. In some cases, the amount of antibiotics excreted over a time-period of 24 h indicated the exceedance of the tolerable daily intake.

Analysis of airborne microorganisms, MVOC and odour in the surrounding of composting facilities and implications for future investigations.

Emission and dispersal of microorganisms and odours from composting facilities were studied in a 3-year project at nine different composting facilities in Germany. Measurements were carried out under so-called 'normal-case', i.e. typical local climate conditions and working activities within the facilities, and 'real worst-case' conditions ('drainage flow' conditions) being characterized by the translocation of cold air mostly at night, and containing large amounts of bioaerosols. Highest concentrations of microorganisms were observed during turning of compost with a maximum of 2.4x10(6)cfu m(-3) for thermophilic actinomycetes. Other groups of microorganisms were detected in concentrations of about 10(5)cfu m(-3). During shredding of fresh organic material, the concentrations of all microorganisms reached 10(4)cfu m(-3). Here, odour concentrations turned out to be highest (up to 1,367 odour units (OU)m(-3)). At facilities equipped with a biofilter (odour reduction), a decrease in OU by a factor of 10 was observed. In the surrounding of the facilities, highest concentrations ranged between 10(1)-10(3)cfu m(-3) upwind and from 10(1)-10(4)cfu m(-3) downwind. The specific local meteorological situations must be considered carefully in advance and during sampling. Especially 'drainage flow' situations can lead to high microorganism concentrations (>10(4)-10(5)cfu m(-3) of thermophilic actinomycetes and thermophilic fungi) in the surroundings of composting facilities.