Airborne fungal and bacterial microbiome in classrooms of elementary schools during the COVID-19 pandemic period: Effects of school disinfection and other environmental factors.

The aim of the study was to examine the effects of environmental factors including disinfection on airborne microbiome during the coronavirus disease 2019 pandemic, we evaluated indoor and outdoor air collected from 19 classrooms regularly disinfected. Extracted bacterial and fungal DNA samples were sequenced using the Illumina MiSeq™ platform. Using bacterial DNA copy number concentrations from qPCR analysis, multiple linear regressions including environmental factors as predictors were performed. Microbial diversity and community composition were evaluated. Classrooms disinfected with spray ≤1 week before sampling had lower bacterial DNA concentration (3116 DNA copies/m3 ) than those >1 week (5003 copies/m3 ) (p-values = 0.06). The bacterial DNA copy number concentration increased with temperature and was higher in classrooms in coastal than inland cities (p-values <0.01). Bacterial diversity in outdoor air was higher in coastal than inland cities while outdoor fungal diversity was higher in inland than coastal cities. These outdoor microbiomes affected classroom microbial diversity but bacterial community composition at the genus level in occupied classrooms were similar between coastal and inland cities. Our findings emphasize that environmental conditions including disinfection, climate, and school location are important factors in shaping classroom microbiota. Yet, further research is needed to understand the effects of modified microbiome by disinfection on occupants' health.

Investigation of bacterial and fungal communities in indoor and outdoor air of elementary school classrooms by 16S rRNA gene and ITS region sequencing.

The advent of high-throughput sequencing methods allowed researchers to fully characterize microbial community in environmental samples, which is crucial to better understand their health effects upon exposures. In our study, we investigated bacterial and fungal community in indoor and outdoor air of nine classrooms in three elementary schools in Seoul, Korea. The extracted bacterial 16S rRNA gene and fungal ITS regions were sequenced, and their taxa were identified. Quantitative polymerase chain reaction for total bacteria DNA was also performed. The bacterial community was richer in outdoor air than classroom air, whereas fungal diversity was similar indoors and outdoors. Bacteria such as Enhydrobacter, Micrococcus, and Staphylococcus that are generally found in human skin, mucous membrane, and intestine were found in great abundance. For fungi, Cladosporium, Clitocybe, and Daedaleopsis were the most abundant genera in classroom air and mostly related to outdoor plants. Bacterial community composition in classroom air was similar among all classrooms but differed from that in outdoor air. However, indoor and outdoor fungal community compositions were similar for the same school but different among schools. Our study indicated the main source of airborne bacteria in classrooms was likely human occupants; however, classroom airborne fungi most likely originated from outdoors.

Development of analytical methods for the determination of 3-hydroxy fatty acids and muramic acid as bacterial markers in airborne particles and settled dust.

Inhalation of airborne bacteria in indoor environments is known to be associated with respiratory diseases. Analytical methods for the determination of 3-hydroxy fatty acids (3-OHFAs) and muramic acid (MA) as chemical markers of gram-negative and gram-positive bacteria, respectively, were developed for airborne particle and dust samples in this study. 3-OHFAs as markers of endotoxin were released and esterified during the hydrolysis process under methanolic acid conditions, and their hydrolysates, i.e., 3-OHFA methyl esters, were cleaned up by solid-phase extraction using silica sorbent that provided more effective separation from interferents than polymeric sorbent through elution pattern. The SPE eluent was analyzed by GC-MS/MS measurement after the trimethylsilylation reaction. The recovery of the method ranged from 82.1 % to 103.2 %, with a limit of detection ranging from 0.5 to 1.1 ng/filter and good linearity (R2 > 0.991). For the analysis of MA, muramic acid methyl ester (MAME), a product formed during methanolic hydrolysis, was selected as a specific marker of peptidoglycan. It was the first proposed compound identified and confirmed with MS and MS/MS spectra using high-resolution measurement. In particular, the measurement of MAME providing 12.5 times greater sensitivity than MA with the application of the LC-MS/MS method is one of the notable findings of this study. The recovery by simple liquid extraction was 99.4 % following the removal of the hydrophobic matrix and neutralization with solvent reconstruction. The method displayed a LOD of 0.7 ng/filter and linearity (R2) of 0.997 through a simple pretreatment process. Both developed methods were applied and evaluated by determining 3-OHFAs and MA in airborne particles collected from multipurpose facilities and settled dust in the laboratory and office.