Culture-Independent Characterization of Bacteria and Fungi in a Poultry Bioaerosol Using Pyrosequencing: A New Approach
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Abstract Work in animal production facilities often results in exposure to organic dusts. Previous studies have documented decreases in pulmonary function and lung inflammation among workers exposed to organic dust in the poultry industry. Bacteria and fungi have been reported as components of the organic dust produced in poultry facilities. To date, little is known about the diversity and concentration of bacteria and fungi inside poultry buildings. All previous investigations have utilized culture-based methods for analysis that identify only biota cultured on selected media. The bacterial tag-encoded flexible (FLX) amplicon pyrosequencing (bTEFAP) and fungal tag-encoded flexible (FLX) amplicon pyrosequencing (fTEFAP) are modern and comprehensive approaches for determining biodiversity of microorganisms and have not previously been used to provide characterization of exposure to microorganisms in an occupational environment. This article illustrates the potential application of this novel technique in occupational exposure assessment as well as other settings. An 8-hr area sample was collected using an Institute of Medicine inhalable sampler attached to a mannequin in a poultry confinement building. The sample was analyzed using bTEFAP and fTEFAP. Of the bacteria and fungi detected, 116 and 39 genera were identified, respectively. Among bacteria, Staphylococcus cohnii was present in the highest proportion (23%). The total inhalable bacteria concentration was estimated to be 7503 cells/m 3 . Among the fungi identified, Sagenomella sclerotialis was present in the highest proportion (37%). Aspergillus ochraceus and Penicillium janthinellum were also present in high proportions. The total inhalable fungi concentration was estimated to be 1810 cells/m 3 . These estimates are lower than what has been reported by others using standard epifluorescence microscope methods. However, no study has used non-culture-based techniques, such as bTEFAP and fTEFAP, to evaluate bacteria and fungi in the inhalable fraction of a bioaerosol in a broiler production environment. Furthermore, the impact of this bTEFAP and fTEFAP technology has yet to be realized by the scientific community dedicated to evaluating occupational and environmental bioaerosol exposure. Keywords: Aspergillus ochraceus bioaerosolorganic dustpoultrypyrosequencing ACKNOWLEDGMENTS This study would not have been possible without the assistance of Dr. Michael Pangburn and Ms. Aika Hussain. Grant support was provided for this project from the Southwest Center for Agricultural Health, Injury Prevention and Education (CDC/NIOSH U50 OH07541) at the University of Texas Health Science Center at Tyler.Keywords:
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Studying the distribution characteristics of bioaerosols and their interaction with the environment is crucial for dairy farms. The distribution of aerosols differs in dairy farming from farming of other livestock, and their sensitivity to environmental factors varies across sites. Field experiments were conducted in an intensive commercial dairy farm in Northern China to investigate the horizontal and vertical distribution of culturable bacterial bioaerosols. Concentration levels and particle size ranges were analyzed, and the impact of multiple environmental factors on culturable bacterial bioaerosols was assessed. Significant variations in culturable bacterial bioaerosol concentrations were observed across eight functional zones, ranging from 1.14 × 103 to 7.35 × 103 CFU/m3. Culturable bacterial bioaerosols exhibited consistent carrier distribution patterns across six different size ranges. Vertical analysis revealed significantly higher culturable bacterial bioaerosol concentrations at a 1 m height compared to 4 m (p < 0.05), while similar size distributions were observed at different heights of the same sampling location. The top three environmental factors influencing culturable bacterial bioaerosol concentrations were PM100 concentration, wind direction, and air temperature. This study provides insights into the distribution characteristics of culturable bacterial bioaerosols on dairy farms and their response to environmental factors. The findings serve as a reference for evaluating bioaerosol emissions and establishing daily disinfection management measures on dairy farms.
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Biofilters have long been considered as an effective technology for the abatement of odour and more recently, bioaerosols at enclosed waste management facilities. However, there is a lack of quantitative data on the influence of biofilter operating parameters on bioaerosol removal effectiveness. It is imperative to develop a better understanding of biofilter design and effective performance monitoring techniques especially if they are to continue to control all emissions and achieve their full potential. This study investigated the performance of pilot-scale biofilters for removal of bioaerosols from waste airstreams in a materials recovery facility based in Leeds, UK, with the objectives of assessing impact of gas phase residence time on bioaerosol control; evaluating net bioaerosol emitting potentials of biofilters and assessing the effect of inlet concentration on bioaerosol control; as well as assessing size distribution of bioaerosol particles in air exhausted from biofilters in order to relate these to the tidal volume inhaled by humans. A six-stage Andersen sampler was used to measure the concentrations of four groups of bioaerosols (Aspergillus fumigatus, total fungi, total mesophilic bacteria and Gram negative bacteria) in the airstream before and after passing through the biofilters over a period of 11 months. The biofilters achieved a removal efficiency up to 97% for A. fumigatus, 94% for total fungi, 86% for total mesophilic bacteria and 85% for Gram negative bacteria, provided that the inlet concentration was high (103 – 105 cfu m-3), which is the case for most waste treatment facilities. The performance was highly variable at low inlet concentration with some cases showing an increase in outlet concentrations, suggesting that biofilters had the potential to be net emitters of bioaerosols. The gas phase residence time did not appear to have any statistically significant impact on bioaerosol removal efficiency. Particle size distribution varied between the inlet and outlet air, with the outlet having a greater proportion of smaller sized particles that represent a greater human health risk as they can penetrate deep into the respiratory system where gaseous exchange occurs. However, the outlet concentrations were low and would further be diluted by wind in full scale applications. In conclusion, this study shows that biofilters designed and operated for odour degradation can also achieve significant bioaerosol control in waste gas.
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Despite of an important concern, human bioaerosol emission into subway is not well and directly characterized. Here, we used bioaerosol detector and next generation sequencing methods to investigate time-dependent bioaerosol size distributions in Beijing subway system between March and April, 2015. In contrast to weekends, weekday microbial aerosol results exhibited strong time dependence with higher bacterial and fungal aerosol levels up to 2083 CFU m–3 and 483 CFU m–3 observed, respectively, for the peak hours. During the peak hour (17:30–18:30), bioaerosol emissions at 0.8–3 µm was detected, while about three times higher concentration levels were observed compared to those during the off-peak hour (22:00–23:00). Similar bioaerosol size distributions were observed between ventilation outlets and subway platform air. During off-peak hours, subway bioaerosols had similar size distributions with the outside air. Sequence results revealed a vast array of airborne microbial species which varied from one station to another, but with Aspergillus spp. as dominant fungal species, and Staphylococcus, Pseudomonas as primary bacterial genera including human opportunistic ones. Our results provide direct online observations of human contributions to subway size-resolved bioaerosols, and enhancing ventilation system might help for controlling the exposure especially during the peak-hours.
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Indoor bioaerosols have recently received considerable interest because of their impact on health. In this study, concentrations of bioaerosols in relation to airborne particulate matter in various indoor environments were investigated. The comparative performance of two common biosamplers, including the single-stage Andersen impactor and the all-glass impinger (AGI) for bioaerosol sampling, was also evaluated. The average levels of airborne bacteria and fungi sampled by Andersen were 516 and 176 colony forming units (CFU) m–3 and by AGI were 163 and 151 CFU m–3, respectively. The highest bacterial levels were measured in residence apartments. The most predominant bacteria were belonged to Staphylococcus sp. and Arthrobacter sp. The Andersen impactor appeared to yield fungal concentrations that were comparable to the results obtained using the AGI biosampler. Meanwhile, Andersen impactor counts for bacteria were significantly higher than those obtained by AGI. Particle count data generated by the optical particle counter indicated that 95% of airborne particles were < 1 µm in diameter. Statistical analysis revealed a significant correlation between particle counts of PM1 and concentrations of culturable airborne bacteria measured with the both bioaerosol samplers. Based on these results, the Andersen impactor performed much better than the AGI for sampling airborne bioaerosols in low-contaminated indoor environments. Accurate measurement of microbial concentrations in indoor environments should be performed by bioaerosol monitoring; however, combining particle counting with bioaerosol sampling could provide prompt information about rapid variations of air quality.
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Accurate exposure assessments are needed to evaluate health hazards caused by airborne microorganisms and require air samplers that efficiently capture representative samples. This highlights the need for samplers with well-defined performance characteristics. While generic aerosol performance measurements are fundamental to evaluate/compare samplers, the added complexity caused by the diversity of microorganisms, especially in combination with cultivation-based analysis methods, may render such measurements inadequate to assess suitability for bioaerosols. Specific performance measurements that take into account the end-to-end sampling process, targeted bioaerosol and analysis method could help guide selection of air samplers. Nine different samplers (impactors/impingers/cyclones/ electrostatic precipitators/filtration samplers) were subjected to comparative performance testing in this work. Their end-to-end cultivation-based biological sampling efficiencies (BSEs) and PCR-/microscopy-based physical sampling efficiencies (PSEs) relative to a reference sampler (BioSampler) were determined for gram-negative and gram-positive vegetative bacteria, bacterial spores, and viruses. Significant differences were revealed among the samplers and shown to depend on the bioaerosol's stress–sensitivity and particle size. Samplers employing dry collection had lower BSEs for stress-sensitive bioaerosols than wet collection methods, while nonfilter-based samplers showed reduced PSEs for 1 μm compared to 4 μm bioaerosols. Several samplers were shown to underestimate bioaerosol concentration levels relative to the BioSampler due to having lower sampling efficiencies, although they generally obtained samples that were more concentrated due to having higher concentration factors. Our work may help increase user awareness about important performance criteria for bioaerosol sampling, which could contribute to methodological harmonization/standardization and result in more reliable exposure assessments for airborne pathogens and other bioaerosols of interest. Copyright 2014 American Association for Aerosol Research
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Transport of infectious particles through the air has the potential to contaminate the indoor environment creating reservoirs of infectious material on surfaces. There is evidence that typical nursing activities can release large quantities of bacteria including MRSA into the hospital air, which may lead to surface contamination thereby increasing opportunities for further spread. Air sampling studies were conducted over a period of 5 days on a four-bed bay in a respiratory ward. Results showed that sampled bioaerosols are more likely to be carried on large particles >5 µm in diameter, and that the relationship between bioaerosols and particle size varies when respiratory interventions are in use. Increased activity in the hospital bay was shown to correlate to increased concentrations of bioaerosols whereas sedentary visitors did not. In particular, the occurrence of patient washing that occurred behind closed curtains correlated to large values of bioaerosol release. Floor cleaning generated large number of particles, but with no significant increase in sampled bioaerosols. This provides valuable information for understanding when and where bioaerosols are released on a hospital ward which may inform future research into physical segregation of patients and the definition of bioaerosol sources in computer simulations.
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Bioaerosols consisting of biologically originated airborne particles such as microbes, metabolites, toxins, and fragments of microorganisms are present ubiquitously in our living environment. The international interests in bioaerosols have rapidly increased because of their many potential health effects. Thus, accurate and fast detection of total bioaerosols in different environments has become an important task for safeguarding against biological threats and broadening the pool of bioaerosol knowledge. To quickly evaluate the total bioaerosol concentration, we developed a localized surface plasmon resonance biosensor based on succinimidyl-ester-functionalized gold nanoislands (SEF–AuNIs) for quantitative bioaerosol detection. The detection limit of our proposed SEF–AuNI sensors for model bacteria Escherichia coli and Bacillus subtilis can go to 0.5119 and 1.69 cells/mL, respectively. To demonstrate the capability of this bioaerosol sensing technique, we tested aerosol samples collected from Bern (urban station), Basel (suburban station), and Rigi mountain (rural and high altitude station) in Switzerland and further investigated the correlation with endotoxin and PM10. The results substantiated that our SEF–AuNI sensors could be a reliable candidate for total bioaerosol detection and air quality assessment.
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Bioaerosols (or biological aerosols) consist of aerosol particles that originate biologically either as fully active component or as whole or part of inactive fragments. They are ubiquitously present in the atmospheric environment. They are the least investigated pollutants due to their complex structure and composition. The effects of bioaerosols, originating due to the processes, such as wastewater management, handling of sludge, composting, municipal solid waste, and animal facilities, on human health are well recognized. Proper identification, quantification, impacts and exposure threshold levels are essential to understand the nature and impact of bioaerosols on human health and climate. In this communication, we determine the inhalable (PM2.5) particulate matter concentration and embedded bioaerosol (bacteria and fungi) levels over a Municipal Solid Waste (MSW) landfill site in relation to surrounding upwind and downwind locations in Nagpur, India. Measurements were made using an Airmetrics MiniVol air sampler and bioaerosols were analyzed by adopting the culture-based method. A total of 23 fungal and 17 bacterial morphotypes were found in this study. The results showed dominance of bacterial bioaerosol over fungal bioaerosol at the landfill site. The bioaerosol levels were higher at the landfill than the upwind and downwind sites. The bioaerosols did not show any correlation to the PM2.5. In summary, the results indicate abundance of PM2.5, containing both bacterial and fungal bioaerosols, which can pose human health hazards over the study region. In our knowledge, it is the first study of bioaerosols at the landfill site in Nagpur, India.
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