Characterisation of Bioaerosols from Six Pig Farms Utilising Culture and Culture-Independent Methodologies

Farmers working with livestock are exposed to a mixture of different bacterial and fungal species in the form of bioaerosols. The microbial communities that compose these bioaerosols are known to be detrimental to workers’ health and in some cases cause asthma and other airway maladies. Furthermore, pig farm workers often carry antibiotic resistant bacteria, such as Methicillin-Resistant Staphyloccous aureus (MRSA). MRSA is able to spread through the air in stables as a component of bioaerosols, and therefore it is crucial for workers’ health to understand the level of exposure to potentially pathogenic microbes to help prevent disease. During this study, amplicon sequencing of the bacterial 16S rRNA gene V4 region and the fungal ITS region using Illumina’s MiSeq platform, as well as matrix-assisted laser desorption/ionisation time of flight mass spectroscopy (MALDI-TOF MS) were used for the identification of culturable, and non-culturable bacteria and fungi. Samples from from settling airborne dust were collected from pig farm stables of different types (care, farrowing, weaning, and slaughter) from different regions (Zealand, Jutland, and Funen) of Denmark. Electrostatic dust fall collectors (EDCs) were utilised for the collection of settling airborne dust from six swine farm stables. The settled dust was extracted from the cloths in an extraction buffer before being suspended in glycerol and frozen at -80°C until further use. Dilution series were made from 10-1 to 10-5 before being plated onto either nutrient agar or dichloran glycerol plates and incubated at 25°C & 10°C. Culturing and identification by MALDI-TOF MS revealed a large diversity in bacterial and fungal species with Aerococcus viridans, Staphylococcus equorum, Leuconostoc mesenteroides, and Staphylococcus saprophyticus being the most dominant bacterial species. Interestingly, although S. aureus and MRSA had been previously detected when using selective media, they were not observed when samples were cultured on nutrient agar plates. This result could be due to lower concentrations of antibiotic resistant and non-resistant strains of S. aureus in comparison to other species, thereby masking the presence of antibiotic resistant and non-resistant S. aureus. Sequencing revealed a greater diversity of bacterial and fungal species when compared to MALDI-TOF MS analyses, with the most common bacterial species being members of the genera Staphylococcus, Clostridium, Lactobacillus, and Turicibacter. The most common fungal genera observed were Debaryomyces, Alternaria, and Aspergillus, whose members include known plant pathogens, possibly originating from the feed.Inter- and intrafarm samples clustered differently in Principle Component Analysis (PCA) plots indicating that several small, but important, variables have an influence on the microflora in pig farms. The vast majority of the species identified are not considered human pathogens, but may contribute to the overall inflammatory potential of the dust.This study highlights the need for multiple testing parameters to assess the full diversity of bacteria and fungi in environmental samples due to intrinsic limitations of different methods.