Contribution of Manure-Spreading Operations to Bioaerosols and Antibiotic Resistance Genes' Emission.

Manure spreading from farm animals can release antibiotic-resistant bacteria (ARB) carrying antimicrobial resistance genes (ARGs) into the air, posing a potential threat to human and animal health due to the intensive use of antibiotics in the livestock industry. This study analyzed the effect of different manure types and spreading methods on airborne bacterial emissions and antibiotic resistance genes in a controlled setting. Cow, poultry manure, and pig slurry were spread in a confined environment using two types of spreaders (splash plate and dribble bar), and the resulting emissions were collected before, during, and after spreading using high-volume air samplers coupled to a particle counter. Total bacteria, fecal indicators, and a total of 38 different subtypes of ARGs were further quantified by qPCR. Spreading poultry manure resulted in the highest emission rates of total bacteria (1011 16S gene copies/kg manure spread), Archaea (106 16S gene copies/kg manure), Enterococcus (105 16S gene copies/kg manure), and E. coli (104 16S gene copies/kg manure), followed by cow manure and pig slurry with splash plates and the dribble bar. Manure spreading was associated with the highest rates of airborne aminoglycoside genes for cow and poultry (106 gene copies/kg manure), followed by pig slurry (104 gene copies/kg manure). This study shows that the type of manure and spreading equipment can affect the emission rates of airborne bacteria, and ARGs.

Antimicrobial Resistance in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes.

Antimicrobial resistance (AMR) is continuing to grow across the world. Though often thought of as a mostly public health issue, AMR is also a major agricultural and environmental problem. As such, many researchers refer to it as the preeminent One Health issue. Aerial transport of antimicrobial-resistant bacteria via bioaerosols is still poorly understood. Recent work has highlighted the presence of antibiotic resistance genes in bioaerosols. Emissions of AMR bacteria and genes have been detected from various sources, including wastewater treatment plants, hospitals, and agricultural practices; however, their impacts on the broader environment are poorly understood. Contextualizing the roles of bioaerosols in the dissemination of AMR necessitates a multidisciplinary approach. Environmental factors, industrial and medical practices, as well as ecological principles influence the aerial dissemination of resistant bacteria. This article introduces an ongoing project assessing the presence and fate of AMR in bioaerosols across Canada. Its various sub-studies include the assessment of the emissions of antibiotic resistance genes from many agricultural practices, their long-distance transport, new integrative methods of assessment, and the creation of dissemination models over short and long distances. Results from sub-studies are beginning to be published. Consequently, this paper explains the background behind the development of the various sub-studies and highlight their shared aspects.

Production of composted recycled manure solids from a Canadian dairy farm: Impact on microbial air quality in experimental conditions.

Recycled manure solids (RMS) produced in dairy farms from fresh manure need to be sanitized before using them as bedding material. However, the impact on air quality of composting RMS remains unknown. Four RMS composting methods were tested during a 10-day aging of piles in experimental chambers: static windrow (SW), turned windrow (TW), SW following drum composting for 24 h (DC24) or SW following drum composting for 72 h (DC72). Air samples were collected using a SASS®3100 Dry Air Sampler on days 0 (pilling of the RMS), 5, and 10. Bacteria (16S rRNA genes), Penicillium/Aspergillus, A fumigatus, and 11 human pathogenic bacteria (e.g. Klebsiella pneumonia) were quantified by qPCR while endotoxins and dust particles were, respectively, measured by LAL assays and with a DustTrakTM DRX Aerosol Monitor. On day 0, RMS produced by SW and TW yielded the lowest concentrations of airborne bacteria, while DC24 resulted in the lowest levels of Penicillium/Aspergillus and dust particles. SW method led on day 5 to the lowest concentration of bacteria and Penicillium/Aspergillus, and DC24 and DC72 to the lowest concentration of airborne dust. On day 10, SW and TW piles were associated with the lowest levels of Penicillium/Aspergillus and dust particles. A significant difference was observed between concentration of airborne bacteria, Penicillium/Aspergillus and endotoxins before and during the turnover of TW piles. None of the studied human pathogens was detected in the air samples. Results of the present study suggest that SW and TW are the most promising methods for the production of composted RMS with respect to microbial air quality. However, the experimental chambers do not accurately represent commercial dairy barns and further research on these composting methods is necessary. Finally, the study highlights that bedding material and its management may be determinant factors for air quality in dairy barns.Implications: The research evaluated the impact on microbial air quality of composting recycled manure solids (RMS) produced from fresh cow manure. RMS need to be composted or sanitized before using them as bedding material for animals. The impact on animal health of RMS still needs to be confirmed, while the effect on air quality and the health of dairy farmers is unknown. In the present study, microbial air quality associated with four RMS composting methods was investigated. Data revealed that two methods resulted in lower aerosolization of dust particles, endotoxins, molds, and bacteria.

Exposure to indoor air contaminants in school buildings with and without reported indoor air quality problems.

Reported indoor air quality (IAQ) complaints are common even in relatively new or renovated school buildings in Finland. However, detecting the causes for complaints with commonly used indoor air measurements is difficult. This study presents data on perceived and measured IAQ in six comprehensive school buildings in Finland. The aim of this study was to discover the possible differences of perceived and measured IAQ between schools with reported IAQ complaints and schools without reported IAQ complaints. The initial categorisation of schools with ('problematic schools') and without ('comparison schools') complaints was ensured via a validated indoor climate survey and a recently developed online questionnaire, which were completed by 186 teachers and 1268 students from the six schools. IAQ measurements of physical parameters, gaseous pollutants, particulate matter and bioaerosols were conducted in four problematic school buildings (26 classrooms) and two comparison school buildings (12 classrooms). Using air sampling as well as exhaust air filters and classroom settled dust to detect the presence of elevated concentrations of airborne cultivable microbes and pathogenic, toxigenic and mycoparasitic Trichoderma strains were the most indicative methods in distinguishing problematic schools from comparison schools. Other IAQ-related measurements did not detect clear differences between problematic and comparison schools, as the concentration levels were very low. The results indicate that the complaints reported by occupants could have been related to excess moisture or mould problems that had not been found or repaired. Ventilation pressure condition investigations and simultaneous exhaust and supply air filter dust culture should be addressed precisely in future studies.

Bioaerosols Play a Major Role in the Nasopharyngeal Microbiota Content in Agricultural Environment.

Background: Bioaerosols are a major concern for public health and sampling for exposure assessment purposes is challenging. The nasopharyngeal region could be a potent carrier of long-term bioaerosol exposure agents. This study aimed to evaluate the correlation between nasopharyngeal bacterial flora of swine workers and the swine barns bioaerosol biodiversity. Methods: Air samples from eight swine barns as well as nasopharyngeal swabs from pig workers (n = 25) and from a non-exposed control group (n = 29) were sequenced using 16S rRNA gene high-throughput sequencing. Wastewater treatment plants were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. Results: A multivariate analysis showed air samples and nasopharyngeal flora of pig workers cluster together, compared to the non-exposed control group. The significance was confirmed with the PERMANOVA statistical test (p-value of 0.0001). Unlike the farm environment, nasopharynx samples from wastewater workers did not cluster with air samples from wastewater treatment plants. The difference in the microbial community of nasopharynx of swine workers and a control group suggest that swine workers are carriers of germs found in bioaerosols. Conclusion: Nasopharynx sampling and microbiota could be used as a proxy of air sampling for exposure assessment studies or for the determination of exposure markers in highly contaminated agricultural environments.

Organic components of airborne dust influence the magnitude and kinetics of dendritic cell activation.

Bioaerosol exposure in highly contaminated occupational settings is associated with an increased risk of disease. Yet, few determinants allow for accurate prediction of the immunopathogenic potential of complex bioaerosols. Since dendritic cells are instrumental to the initiation of immunopathological reactions, we studied how dendritic cell activation was modified in response to individual agents, combined microbial agents, or air sample eluates from highly contaminated environmental settings. We found that combinations of agents accelerated and enhanced the activation of in vitro-generated murine bone marrow-derived dendritic cell cultures, when compared to individual agents. We also determined that endotoxins are not sufficient to predict the potential of air samples to induce bone marrow-derived dendritic cell activation, especially when endotoxin levels are low. Importantly, bone marrow-derived dendritic cell activation stratified samples from three environmental settings (swine barns, dairy barns, and wastewater treatment plants) according to their air quality status. As a whole, these results support the notion that the interplay between bioaerosol components impacts on their ability to activate dendritic cells and that bone marrow-derived dendritic cell cultures are promising tools to study the immunomodulatory impact of air samples and their components.

Detection of Streptococcus suis in bioaerosols of swine confinement buildings.

Streptococcus suis is an important swine pathogen that can cause septicemia, meningitis, and pneumonia. Also recognized as an emerging zoonotic agent, it is responsible for outbreaks of human infections in Asian countries. Serotype 2 is the predominant isolate from diseased animals and humans. The aerosolization of S. suis in the air of swine confinement buildings (SCB) was studied. The presence of S. suis in bioaerosols was monitored in SCB where cases of infection had been reported and in healthy SCB without reported infections. Using a quantitative-PCR (qPCR) method, we determined the total number of bacteria (1 × 10(8) to 2 × 10(8) airborne/m(3)), total number of S. suis bacteria (4 × 10(5) to 10 × 10(5) airborne/m(3)), and number of S. suis serotype 2 and 1/2 bacteria (1 × 10(3) to 30 × 10(3) airborne/m(3)) present in the air. S. suis serotypes 2 and 1/2 were detected in the air of all growing/finishing SCB that had documented cases of S. suis infection and in 50% of healthy SCB. The total number of bacteria and total numbers of S. suis and S. suis serotype 2 and 1/2 bacteria were monitored in one positive SCB during a 5-week period, and it was shown that the aerosolized S. suis serotypes 2 and 1/2 remain airborne for a prolonged period. When the effect of aerosolization on S. suis was observed, the percentage of intact S. suis bacteria (showing cell membrane integrity) in the air might have been up to 13%. Finally S. suis was found in nasal swabs from 14 out of 21 healthy finishing-SCB workers, suggesting significant exposure to the pathogen. This report provides a better understanding of the aerosolization, prevalence, and persistence of S. suis in SCB.

Endotoxins in indoor air and settled dust in primary schools in a subtropical climate.

Endotoxins can significantly affect the air quality in school environments. However, there is currently no reliable method for the measurement of endotoxins, and there is a lack of reference values for endotoxin concentrations to aid in the interpretation of measurement results in school settings. We benchmarked the "baseline" range of endotoxin concentration in indoor air, together with endotoxin load in floor dust, and evaluated the correlation between endotoxin levels in indoor air and settled dust, as well as the effects of temperature and humidity on these levels in subtropical school settings. Bayesian hierarchical modeling indicated that the concentration in indoor air and the load in floor dust were generally (<95th percentile) <13 EU/m(3) and <24,570 EU/m(2), respectively. Exceeding these levels would indicate abnormal sources of endotoxins in the school environment and the need for further investigation. Metaregression indicated no relationship between endotoxin concentration and load, which points to the necessity for measuring endotoxin levels in both the air and settled dust. Temperature increases were associated with lower concentrations in indoor air and higher loads in floor dust. Higher levels of humidity may be associated with lower airborne endotoxin concentrations.

Potentially pathogenic bacteria and antimicrobial resistance in bioaerosols from cage-housed and floor-housed poultry operations.

BACKGROUND: Antibiotics are used in animal confinement buildings, such as cage-housed (CH) and floor-housed (FH) poultry operations, to lower the likeliness of disease transmission. In FH facilities, antibiotics may also be used at sub-therapeutic levels for growth promotion. Low levels of antibiotic create a selective pressure toward antimicrobial resistance (AMR) in chicken fecal bacteria. OBJECTIVE: The objective of this study was to compare bacteria and AMR genes in bioaerosols from CH and FH poultry facilities. METHODS: Bioaerosols were collected from 15 CH and 15 FH poultry operations, using stationary area samplers as well as personal sampling devices. Bacteria concentrations were determined by genus- or species-specific quantitative polymerase chain reaction (PCR) and AMR genes were detected using endpoint PCR. RESULTS: Enterococcus spp., Escherichia coli, and Staphylococcus spp. were significantly higher in bioaerosols of FH poultry operations than CH bioaerosols (P < 0.001) while Clostridium perfringens was significantly higher in area bioaerosols of CH operations than FH area bioaerosols (P < 0.05). Campylobacter spp. were detected only in bioaerosols of FH facilities. Zinc bacitracin resistance gene, bcrR, erythromycin resistance gene, ermA, and tetracycline resistance gene, tetA/C, were more prevalent in bioaerosols of FH facilities than CH bioaerosols (P < 0.01, P < 0.01, and P < 0.05, respectively). CONCLUSIONS: Most bacteria are more concentrated and most AMR genes are more prevalent in bioaerosols of FH poultry operations, where growth-promoting antibiotics may be used.

Bacterial diversity characterization of bioaerosols from cage-housed and floor-housed poultry operations.

BACKGROUND: Although bioaerosols from both cage-housed (CH) and floor-housed (FH) poultry operations are highly concentrated, workers from CH operations have reported a greater prevalence of respiratory symptoms. OBJECTIVE: The objective of this study was to directly compare bacteria, both quantitatively and qualitatively, in bioaerosols from CH and FH poultry facilities. METHODS: Bioaerosols were collected from fifteen CH and fifteen FH poultry operations, using stationary area samplers as well as personal sampling devices. Dust, endotoxin and bacteria were quantified and bacterial diversity was investigated using PCR followed by denaturing gradient gel electrophoresis (DGGE). RESULTS: Dust (p<0.001), endotoxin (p<0.05) and bacteria (p<0.05) were significantly higher in personal bioaerosols of FH poultry operations than CH bioaerosols. Although dust and endotoxin did not differ significantly between area and personal samples within each barn type, clustering analysis of DGGE profiles of bacteria revealed that area and personal samples shared less than 10% similarity. These data suggest that area samples are not representative of personal bacteria exposures, which may be affected by worker movement, bacteria carried on the worker and worker location. Personal DGGE profiles from CH and FH operations shared less than 20% similarity and composite analysis showed that bacteria were more prevalent in personal samples from CH bioaerosols than FH bioaerosols. CONCLUSIONS: Bacteria concentration and diversity are significantly different between bioaerosols from CH and FH poultry operations.

Human pathogens and tetracycline-resistant bacteria in bioaerosols of swine confinement buildings and in nasal flora of hog producers.

Swine confinement buildings in eastern Canada are enclosed and equipped with modern production systems to manage waste. Bioaerosols of these swine confinement buildings could be contaminated by human pathogens and antimicrobial resistant bacteria which could colonize exposed workers. We therefore wanted to analyze bioaerosols of swine confinement buildings and nasal flora of Canadian hog producers to evaluate possible colonization with human pathogens and tetracycline-resistant bacteria. Culturable and non-culturable human pathogens and tet genes were investigated in the bioaerosols of 18 barns. The nasal passages of 35 hog producers were sampled and total DNA was extracted from the calcium-alginate swabs to detect, by PCR, Campylobacter, C. perfringens, Enterococcus, E. coli, Y. enterocolitica, tetA/tetC, tetG and ribosomal protection protein genes. Airborne culturable C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were present in the bioaerosols of 16, 17, 11 and 6 of the 18 facilities. Aerosolized total (culturable/non culturable) Campylobacter, C. perfringens, Enterococcus, E. coli and Y. enterocolitica were detected in 10, 6, 15, 18 and 2 barns, respectively. Tet genes were found in isolates of culturable human pathogens. TetA/tetC, tetG and ribosomal protection protein genes were detected in the bioaerosols of all 18 studied buildings. Campylobacter, C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were found respectively in 4, 9, 17, 14 and one nasal flora of workers. One and 10 workers were positive for tetA/tetC and tetG genes, respectively. In swine confinement buildings, hog producers are exposed to aerosolized human pathogens and tetracycline-resistant bacteria that can contaminate the nasal flora.

Biological activities of respirable dust from Eastern Canadian peat moss factories.

Bacteria, moulds, endotoxin and quartz from respirable dust of agricultural and industrial buildings are typically incriminated for the respiratory health decline of exposed workers despite that dust being an undefined mixture and quantification methods of aerosolized bacteria, moulds or endotoxin not being standardized yet. We developed an in vitro alveolar epithelial cell system in which biological activities of peat moss factories' dust might be correlated to bacteria, mould, endotoxin and quartz concentrations of the analyzed samples. Following exposure, interleukin-8 protein secretion, necrosis and apoptosis of the exposed A549 cells were monitored respectively with ELISA on cell supernatants, trypan blue exclusion and DNA fragmentation detection by flow cytometry. Respirable dust was collected with liquid impingers and respirable quartz with 10mm Dorr-Oliver cyclones. We quantified mesophilic bacteria, mesophilic moulds and endotoxins from liquid impinger samples. No correlation was observed between biological activities of dust and bacteria, mould, endotoxin or quartz concentrations under our experimental conditions. Our speculation is that simple measurements, such as dust concentrations, may not be adequate indicators of the human respiratory health hazard for a given environment.

Presence of zoonotic pathogens in physico-chemically characterized manures from hog finishing houses using different production systems.

Hog production has been intensified in Eastern Canada, by 50% over the last 20years. Wastes are now managed with conventional production systems (slatted floor), litter systems or source separation systems. We studied the presence of total and fecal coliforms, Campylobacter, Clostridium perfringens, Enterococcus, Escherichia coli, Salmonella, Yersinia enterocolitica, Giardia and Cryptosporidium in the manure of all of these production systems. The concentrations of the studied zoonotic pathogens did not differ between the conventional and the litter systems investigated. The source separation system yielded separated solid and liquid fractions. Total and fecal coliforms, C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were found in the solids and the liquid fractions of a source separation system.Campylobacter species were not detected in the liquid fractions. Zoonotic pathogens are therefore, found in the manure of Eastern Canadian hog finishing houses equipped either with conventional, sawdust litter or source separation systems. Pathogens inactivation will require changes in manure management practices or the use of manure disinfection methods.

Airborne porcine circovirus in Canadian swine confinement buildings.

Porcine circovirus type 2 has been linked to many diseases, such as postweaning multisystemic wasting syndrome and can be found in most commercial swine confinement buildings around the world. Although the exact role of the virus in the appearance of disease in animals is not fully understood, the mechanisms responsible for the transmission of the virus are currently believed to happen mostly by contact. Nevertheless, the possibility of airborne transmission cannot be rejected. This study investigated the presence of the virus, total bacteria and total dusts in aerosols. Air samples were taken with gelatin filters in swine confinement buildings and were analyzed by quantitative polymerase chain reaction. Interestingly, concentrations of airborne PCV2 of up to 10(7) genomes per cubic meter of air were detected. Airborne dust concentrations were correlated to airborne concentrations of PCV2 and total bacteria. Although the infectivity potential of the airborne viral loads were not evaluated, it is clear that the virus can become airborne in detectable concentrations in commercial swine confinement building environments. The significance of this finding in an epidemiological point of view will need further investigation.

Impact of production systems on swine confinement buildings bioaerosols.

Hog production has been substantially intensified in Eastern Canada. Hogs are now fattened in swine confinement buildings with controlled ventilation systems and high animal densities. Newly designed buildings are equipped with conventional manure handling and management systems, shallow or deep litter systems, or source separation systems to manage the large volumes of waste. However, the impacts of those alternative production systems on bioaerosol concentrations within the barns have never been evaluated. Bioaerosols were characterized in 18 modern swine confinement buildings, and the differences in bioaerosol composition in the three different production systems were evaluated. Total dust, endotoxins, culturable actinomycetes, fungi, and bacteria were collected with various apparatuses. The total DNA of the air samples was extracted, and quantitative polymerase chain reaction (PCR) was used to assess the total number of bacterial genomes, as a total (culturable and nonculturable) bacterial assessment. The measured total dust and endotoxin concentrations were not statistically different in the three studied production systems. In buildings with sawdust beds, actinomycetes and molds were found in higher concentrations than in the conventional barns. Aspergillus, Cladosporium, Penicillium, and Scopulariopsis species were identified in all the studied swine confinement buildings. A. flavus, A. terreus, and A. versicolor were abundantly present in the facilities with sawdust beds. Thermotolerant A. fumigatus and Mucor were usually found in all the buildings. The culturable bacteria concentrations were higher in the barns with litters than in the conventional buildings, while real-time PCR revealed nonstatistically different concentrations of total bacteria in all the studied swine confinement buildings. In terms of workers' respiratory health, barns equipped with a solid/liquid separation system may offer better air quality than conventional buildings or barns with sawdust beds. The impact of ventilation rates, air distribution, or building design still has to be explored.

Culture-independent characterization of archaeal biodiversity in swine confinement building bioaerosols.

It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 10(8) 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.

Culture-independent approach of the bacterial bioaerosol diversity in the standard swine confinement buildings, and assessment of the seasonal effect.

The bacterial bioaerosol community of eight swine confinement buildings (SCB) was monitored during two visits in the winter, and one during the summer. To our knowledge, culture-independent approaches and molecular biology tools such as biomass quantification and biodiversity analyses have never been applied to swine building bioaerosol analyses. Total DNA of each sample was extracted and analysed by quantitative real-time polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis using primers targeting the bacterial 16S rRNA gene. Even though the total bacterial concentration was higher in winter than in summer, the total bacterial concentration for both seasons was 100 to1000 times higher than the total cultural bacteria. The concentration of bioaerosol was influenced by the temperature indoors, which was regulated with an electronic fan system driving warm air and particles outside of the SCB. Comparison of the DGGE profiles showed the same biodiversity in each SCB during both seasons. The phylogenetic analysis revealed a large number of sequences (93.8%) related to Gram-positive anaerobic bacteria, such as Clostridia, and dominated by the Clostridia cluster I (C. disporicum) and the Clostridia cluster XI (C. glycolycum). The bioaerosol diversity also contained also a low proportion of Bacteroidetes and Lactobacillales-Streptococcales sequences. Analyses of the global community and phylotype diversity showed that the main source of bioaerosols could come from the pig manure slurry.