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In this study, we used culture and culture-independent methods to determine the …

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- Culture-independent analysis of bacterial diversity in a child-care facility

Child-care facilities appear to provide a setting with many opportunities for exposure and transmission of bacteria and viruses [1-4]. Preschool aged children are often sick with illnesses of unknown origins, and young children have not yet mastered the sanitary cleaning habits present among most adults in our society. Moreover, children have had less exposure to microorganisms, making them more likely to catch and transmit pathogens or opportunistic pathogens, and perhaps more likely to suffer ill effects from contact in densely populated facilities. Recent studies of microbial diversity in indoor environments using molecular methods have revealed considerable bacterial contamination and underscored how little we know about such contamination [5-7]. Understanding the potential public health risks in daycare centers requires a better understanding of microbial diversity in these settings. This is particularly important given the increasing reliance of working parents on daycare facilities for childcare [4].

Culture-based studies of human indoor environments have shown that significant levels of bacteria are present in seemingly innocuous areas such as office buildings, residential homes, and children's schools and daycare centers [8-10]. According to these surveys, low DNA G+C content, Gram-positive bacteria, such as Bacillus cereus, Bacillus licheniformis, Brevibacillus brevis and Staphyloccus spp. along with a few Gram negative species including Chryseomonas spp. and Pantoea spp. tend to predominate [8,11,12]. Indoor culturing studies have also identified the presence of bacteria from the order Actinomycetes, including Rhodococcus fasclans, Arthrobacter pascens, and Corynebacterium spp. [8,11].

Recently, culture-independent molecular studies have greatly expanded our understanding of the bacterial diversity that can be present in indoor environments. The molecular methods we performed in this study included PCR amplification of 16S rRNA genes conducted on DNA extracted directly from our environmental samples. Culture-independent methods have been able to offer a much more complete view of the bacteria present in ordinary everyday surroundings such as indoor pools, shower curtains, and airplane bathrooms; these same methods should prove equally effective for use in daycare settings [5-7]. In some cases, culture-independent methods have identified the source of illness when the microbes were unknown or not currently culturable [5,13].

In an environment so potentially rich in microbial diversity, culturing methods readily identify bacteria with known growth requirements and these methods are necessary to prove the viability of microorganisms in the environment. However, previous work has shown that [14,15]. Indeed, the development of culture-independent methods based on the 16S rRNA gene used in conjunction with phylogenetic analysis has revealed an abundant array of previously unknown and uncultured microbes, including entirely new bacterial divisions [13-18]. The 16S rRNA gene is particularly useful for molecular analysis and identification of organisms due to its high level of information content, conserved nature, and it's presence in all cellular microorganisms [15]. Researchers have also begun to use culture-independent methods to study human biology [19-22], complex diseases [13,23], and human environments [5-7]. Collectively these studies have exposed a remarkable array of microorganisms, many of them with no cultured representatives. In the case of human environments, many potentially opportunistic pathogens have been identified [5-7].

In this study, we surveyed the bacterial diversity present in a daycare facility using both culture and culture-independent methods to analyze samples taken from various toys and surfaces (e.g., counter-tops). This allowed us to gauge the overall complexity of bacterial diversity, determine viability of bacteria, and see how the diversity and abundance changed over time. A total of four rooms were sampled over a six-month period. Sampling was alternated between two toddler rooms and two infant rooms every one to two weeks. Of these samples, DNA was successfully extracted directly from nine swabs, and the samples were subjected to both culture and culture-independent analysis. The facility tested in this case had specific disinfection protocols in place for daily cleaning of the rooms and washing of the toys that the children have played with or come into contact with during the course of the day. Cleaning protocols (e.g., cleaning surfaces with 10% bleach) were followed diligently by the staff in this daycare facility, which placed a high premium on cleanliness.

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