Child-care facilities appear to provide a setting with many opportunities for exposure and transmission of bacteria and viruses 1 2 3 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 6 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 9 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 6 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 <1% of bacterial species in a given environment are culturable suggesting that a vast majority of the true diversity may be missed if studies rely solely on culturing 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 14 15 16 17 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 20 21 22 , complex diseases 13 23 , and human environments 5 6 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 6 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.

Table 1 details the total number of cultured isolates obtained over the course of the study based on the16S rRNA gene sequence analysis and visible colony morphology. The lysozyme extraction protocol effectively isolated bacterial DNA from all the colonies picked from plates. Typical DNA yields for bacterial colonies were in the range of 0.6 to 82.4 ng μl^-1. Table 2 shows the results of culturing on the environmental swab samples taken between September 2005 – April 2006. For all days sampled except October 6, 2005, colonies grew on either blood or nutrient agar plates. This means that there were large numbers of viable bacteria consistently present on the surfaces and toys sampled at the daycare center. We isolated as many as 29 putative bacterial species. We considered it a potentially different species of bacteria if it had a unique 16S rRNA gene sequence or a clearly distinct morphology or both (Table 2). Bacillus species were the most commonly culture-isolated bacteria, followed by Staphylococcus spp. Culture methods identified at least 29 viable bacterial species on toy and furniture surfaces over the 6 months of the study (Table 1). Species of Bacillus were isolated every day of sampling (Table 2), and we identified as many as 15 different distinct morphologies over the course of the study (Table 1).

The lysozyme DNA extraction protocol proved effective for direct swab extractions and yielded DNA in the range of 5.1 to 14.7 ng μl^-1. Bead-beating methods are typically preferred for isolating environmental DNA because the mechanical shearing of cells by the beads helps extract DNA from particularly "tough" bacterial cells such as bacterial vegetative cells (e.g., Pseudomonas putida), bacterial endospores (e.g., Bacillus spp.), and fungal conidia (e.g., Fusarium moniliforme) 24 . However, our attempts with bead-beating methods failed to isolate sufficient DNA from the swabs (data not shown), whereas the lysozyme method yielded sufficient amounts of DNA for PCR.

Figure 1 and 2 show the results of phylogenetic analysis of selected sequences from the 453 clones obtained from the nine libraries relative to sequences from cultured and uncultured bacteria from other studies. Based on our survey of nine 16S rRNA gene PCR-clone libraries we identified as many as 190 bacterial species (1% divergence; 141 at the more conservative 3% divergence level), most of them with no cultured representatives. The clone library sequence coverage ranged from 48% to 65% (average 54%) for the nine clone libraries. Most sequences found in both groups appeared to be uncultured bacterial species. Members of the Pseudomonadaceae and the Oxalobacteraceae predominated in the clone libraries (Fig. 3). Pseudomonads were particularly abundant and were on every surface sampled on every sampling occasion (Fig. 3).

Since the main purpose of this study was to identify the types of organisms in the daycare center, one-directional sequencing of the first part of the small-subunit rRNA gene, which includes the most variable part of the gene, was enough for our purposes. Sequencing bi-directionally would have been ideal in terms of reducing error, but would have doubled the sequencing costs and added little to our understanding of the diversity. Also, we trimmed the sequences to around ~500 bp in length, and edited out the most problematic part of the reads. Any errors that remained would have had little impact on the phylogenetic analysis. However, we have made glycerol stocks of all the clone libraries we created, which are available from the authors upon request.

The alignment of the edited and trimmed sequences proved straightforward, and the alignment was checked by confirming complementary base-pairing in known stem regions of the alignment. A total of 78 sequences were deposited in GenBank, including sequences from both culture isolates (Table 1) and representative clone library sequences used in the phylogenetic analysis (Fig. 1, 2). Approximately 500 nucleotide positions, corresponding to E. coli positions 25 to 534, were used for all the phylogenetic analyses. There was strong support for the majority of the phylogenetic relationships as judged by both Bayesian posterior probabilities (Fig. 1, 2) and MP bootstrap support (not shown). Bayesian, MP and ML methods produced highly similar tree topologies. The differences in tree topologies produced by the various methods were in regions of the trees not supported by either posterior probabilities (< 0.5) or MP bootstrap values (<50%). The phylogenetic analysis allowed us to easily identify the position of our cloned environmental sequences within known bacterial divisions (Fig. 1, 2). Using this information, and the Fastgroup II dereplication information, we were able to assess the relative abundance of various sequences in clone libraries and these are presented in Figure 3.

Our combination of culturing and culture-independent techniques revealed a remarkable diversity of bacteria contaminating every surface sampled in the daycare facility. Bacillus spp. were particularly common (Table 1 and 2). Bacillus endospores disperse rapidly through the air and are ubiquitous in soils and other environments 25 26 so their abundant presence here was not alarming. Staphylocccus species were also repeatedly isolated, as were species of Pseudomonas and Microbacterium (Table 1 and 2). A number of the isolates were potential pathogens and opportunistic pathogens, including Enterococcus faecalis 27 , Moraxella osloensis 28 , and Staphylococcus haemolyticus 29 . E. faecalis has become a particular problem in hospitals 27 . Species belonging to these genera of bacteria are commonly found on skin, nostrils, or even as part of the normal gut microbiota 30 . Normal shedding of these surfaces, along with the attached bacteria, may explain their abundance in indoor human environments 9 30 .

Our culturing results appeared highly similar to culture-based studies of other indoor environments 8 9 . Specifically we found ~90% of the same bacterial genera as another culture-based study of a daycare setting 8 . Most of these studies sampled airborne bacteria in environments, such as daycare centers, schools, and office buildings 8 9 11 12 . Our results suggest diversity found in air-sampling studies is very similar to that of surface sampling methods and may be a reasonable substitute for costly air-sampling methods at least in terms of determining microbial diversity.

Although the culture-based methods discovered a number of bacterial species and confirmed the viability of bacteria on surfaces in the daycare facility, these methods identified only a small fraction of the true bacterial diversity (~3%). The culture-independent portion of our analysis revealed a whole new dimension of largely unexplored microbial diversity present in a daycare center environment. Similar to other culture-independent studies in human environments 5 6 7 , we uncovered an extraordinary diversity of bacteria from 16 bacterial divisions or sub-divisions that included many bacterial species without cultured representatives (Fig. 1, 2, 3).

The largest proportion of sequences found in the clone libraries came from two groups: the Pseudomonadaceae and the Oxalobacteraceae (Fig. 3). Pseudomonads comprise an extremely diverse array of bacteria that grow on numerous carbon sources and are often associated with spoilage 31 . Many of them produce biofilm "slime layers" that serve as environmental protection and make them resistant to both antibiotics 32 and cleaning regimens 33 . Moreover, this same slime production ability appears to protect them from the mammalian immune system 32 . A number of Pseudomonads, such as P.stutzeri, are known opportunistic pathogens 34 and have also been implicated in hospital acquired infections 35 36 . P. aeruginosa is also known to be resistant to antibiotics 37 .

The predominance of a diverse array of Pseudomonads in the daycare appears to be quite consistent with the nature of the environment. The constant spillage of food and liquids, spread over every surface reachable by children, would make a perfect growth medium for Pseudomonads 31 . This particular daycare center had very rigorous cleaning policies. However, the natural resistance of Pseudomonads to cleaning may actually have served to increase their abundance relative to other bacteria. Given the abundance of Pseudomonads in our clone libraries, it is somewhat surprising that we did not find more species growing on nutrient agar plates (Table 2). Another published culture-based study of a daycare facility showed a lack of diversity on nutrient agar plates 8 . However, growth on media other than blood or nutrient agar was not tried. It is possible that had a media specifically designed to culture Pseudomonads been selected, our results may have been different. In addition, our inability to grow these bacteria on agar plates may be a result of the fact that so many of the Pseudomonas spp.-related sequences came from uncultured bacteria (1–3% divergence from cultured species; Fig. 2).

The other most consistently abundant group, based on clone library sequence analysis, included a large collection of uncultured species in the Oxalobacteraceae family. According to the research literature, the Oxalobacteraceae include numerous bacterial species with diverse habitats. For example, many Collimonas and Herbaspirillum spp. are soil dwelling bacteria 38 , while Oxalobacter formingenes is found in the human gastrointestinal tract 39 . Unfortunately, our uncultured species appeared to belong to a novel group of Oxalobacteraceae leaving us with little information concerning their source or habitats, apart from a rather basic understanding of their phylogenetic relationships. However, a recent culture-independent study of human-associated microbial communities 40 allowed us to identify the human vaginal epithelium as a likely source of a large number of these sequences (Fig. 1, 3). An intensive sequencing effort by Hyman et al. (2005) revealed a tremendous diversity of uncultured bacteria associated with the vaginal epithelium, and many of the 16S rRNA gene sequences obtained from surface samples, especially within the Oxalobacteraceae, were closely related to these published sequences (Fig. 2). Sequences in our clone libraries from nine other bacterial divisions were also nearly identical to bacterial sequences isolated from the vaginal epithelium (Fig. 2). We also note that we found many sequences of apparently uncultured bacteria related to uncultured bacteria identified from molecular studies of wastewater sludge (Fig. 2; 41 42 ).

The predominance of sequences in our libraries related to bacteria found in the vaginal epithelium and in wastewater sludge suggests that a significant proportion of the bacterial contamination in daycares results from frequent diaper changes. This conclusion is supported by the discovery of cultured bacteria known to reside in the human intestine (e.g., E. coli, Enterococcus faecalis; Fig. 2). Since we know so little about the uncultured bacteria, we cannot say whether they pose a particular health threat. However, the significant diversity of so many human-associated bacteria contaminating various toys and surfaces suggests that enteropathogenic bacteria could be easily spread in daycare settings. Given the fact that we did not achieve full sampling coverage of the sequence diversity for any of the samples (average clone library sequence coverage ~54%), we expect that many more bacterial species may be found in daycare settings. These results not only demonstrate how little is known about microbial diversity of indoor environments, but also emphasizes the need for a much more complete understanding of microbes associated with humans, which appeared to be the source of most of the contamination.

In addition to the sequences from uncultured bacteria, we also identified a number of sequences in the culture-independent molecular analysis with close relatives known to be pathogens or opportunistic pathogens that were not found through culturing. These include sequences related to Streptococcus mutans, S. mitis, Chryseobacterium indologenes 43 , Stenotrophomonas maltophilia 44 45 , Flavobacterium indologenes 46 and Rothia dentocariosa 47 (Fig. 1, 2). The January 25 sampling date in particular had a particularly high abundance of Streptococcus-related species (Fig. 3). This sampling was right in the middle of the cold and flu season and was around the time a large number of the kids were kept home due to illness (Daycare staff, pers. comm.). The fact that we found so few of these species through standard culturing approaches supports that notion that culture-independent molecular analyses provide a powerful additional means for studying indoor environments and possibly identifying infectious agents.

The results of this study should also be examined from a public health viewpoint, in order to assess which organisms have the greatest potential to cause illness in children. Streptococcus pneumoniae and Staphylococcus epidermidis were identified in several samples, these represent known pathogens that can cause pneumonia and meningitis in children 48 49 . Stenotrophomonas maltophilia was also found, this bacteria has been known to cause infections and bacteremia in pediatric patients who have contracted it as a nonsocomial infection during a hospital stay 50 51 . The presence of Stenotrophomonas maltophilia was also somewhat distressing due to the recent emergence of an antibiotic resistant strain 52 . Other pathogenic strains potentially harmful to children that were found included Rothia dentocariosa, known to cause the childhood illness tonsillitis 53 , Enterococcus faecalis, a bacteria associated with urinary tract infections 54 55 , and Shigella flexnerii, which causes the common childhood ailment of acute diarrhea 56 57 .

Although culture-independent methods have proven highly useful for uncovering a vast array of new microbes in many environments, including the daycare center, a number of authors have pointed out that methods based on amplifying 16S rRNA gene sequences using "universal" primers may not accurately reflect the true underlying diversity of a given environment 58 59 . Problems such as PCR-bias, ribosomal DNA copy number and the efficiency of DNA extraction procedures all have the potential to significantly skew abundance estimates and there may not be a direct relationship between the number of sequences of a particular type in a clone library and the number of organisms in the environment.

Nevertheless, as demonstrated in this paper, the culture-independent methods do allow for a much more comprehensive assessment of microbial diversity than culturing alone and provide an approximation of the relative diversity in the samples. Given the proper set of growing conditions, many of the uncultured bacteria could be isolated using culture-based methods. For example, by culturing for a longer period of time (five or more days), by culturing at a broader range of temperatures (e.g., 30°C), or by using alternative media (e.g., low-nutrient medium R2A) we might have isolated more types of bacteria. Longer incubation times would have been particularly helpful for growing organisms that tend to live in biofilms. Indeed, the culture-independent methods provide an excellent complement to the culturing approaches. Before the study, we did not expect to find such a diverse array of Pseudomonadaceae and Oxalobacteraceae, but with the knowledge gained from the culture-independent methods we can now adjust our culturing methods to grow these organisms.

The diversity of bacteria in the daycare environment appears to be a rich combination of bacterial species associated with both humans and the outside environment (e.g., Bacillus in soils). Given the extremely high bacterial diversity, and the relatively low sequence coverage we achieved in this preliminary study (~54%), the overall diversity is almost certainly higher than we report. Our results suggest that the microbial diversity associated with human environments remains extremely poorly characterized. In terms of public health, we believe greater attention needs to be paid to the microbial contamination of environments (e.g., daycare centers, nursing homes and hospitals) that take care of the most vulnerable members of society. In terms of the child-care facilities per se, our results suggest that diaper changing stations should be moved further away from the play areas, and that more efforts should be focused on removing tough biofilms. Faster and more comprehensive culture-independent methods, such as environmental microarrays and metagenomic approaches, could help better understand the public health risks in these environments.

LL collected half of the samples; performed most of the culturing; and identified isolates based on 16S rRNA gene sequencing. LL also created 3 of the 9 clone libraries; cleaned, edited and aligned all the sequence data; performed much of the Bioinformatics analyses and wrote early drafts of the manuscript. ST collected the other half of the samples, created 6 of the 9 clone libraries and helped with the sequence analyses. STK designed the study, wrote and edited the manuscript, completed the phylogenetic analyses and created the figures. All authors read and approved the final manuscript.