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To determine if locations within the classroom varied in the proportion of dispersed bacteria from windows (as a potential source of environmental bacteria), we used a second dataset that included the relative contribution of chloroplast DNA to a given sample (as a proxy for pollen deposition). We generated this dataset by including the total sequences minus those assigned to mitochondria, plus those sequences assigned to chloroplasts [17]. This dataset was also rarefied to 200 sequences per sample prior to analysis.
We found a significant difference in bacterial communities between high and low contact surfaces, but not among bacterial communities experiencing different levels of visible light, nor distances from windows (as likely sources of environmental bacteria). Our data suggest the bacterial communities present in a classroom are therefore influenced by actions in the classroom in such a way as to lead different surface types having different bacterial communities. The bacterial communities in inside dust are known to be directly influenced by the number and types of occupants in the building [4]. In keeping with these findings, we found occupancy, or the density of children per classroom, correlated with differences in the bacterial communities of these spaces. The density of students, and thus the higher number of humans as microbial sources may drive the differences we noted in communities across classrooms. A recent study investigating the bacterial community of floors in 499 elementary school classrooms similarly found the number of students per classroom to correlate with differences in bacterial communities [47], a pattern also observed in other bacterial studies of homes [48]. Our findings are aligned with those of other studies of buildings occupied primarily by adults, which found bacterial composition varied among surfaces (fomites), due to how often they were touched or likely to be cleaned [6, 8], or other factors [2, 49].
A limitation of our study is our focus on a relatively small number of samples from classrooms in one city in the southeastern United States. While our findings are consistent with similar studies that investigated university classrooms, elementary schools, and childcare centers in other countries [8, 17, 22], there are limits to what we can assert based on the sample sizes in this study. Therefore, future studies are needed to understand the relationship of preschool age children and their built environments. Such studies would benefit particularly from pairing behavioral data on the frequency of contact with surfaces with these microbial studies, and including best sequencing practices (such as sequencing a mock community to assure results can be compared across studies as accurately as possible). This may ultimately inform our understanding of how different age groups engage with their specific environments while providing new insights into the relationship between the microbes of built environments and occupants.
DEB drafted the manuscript. AAM performed the data analyses and drafted the manuscript. MM coordinated the study and carried out childcare center sampling protocol. JH designed and coordinated the study and carried out childcare center sampling protocol. RRD helped to draft the manuscript. All authors read and approved the final manuscript.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
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