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 , Moraxella osloensis , and Staphylococcus haemolyticus . E. faecalis has become a particular problem in hospitals . Species belonging to these genera of bacteria are commonly found on skin, nostrils, or even as part of the normal gut microbiota . 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 . 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 [5-7], 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 . Many of them produce biofilm "slime layers" that serve as environmental protection and make them resistant to both antibiotics  and cleaning regimens . Moreover, this same slime production ability appears to protect them from the mammalian immune system . A number of Pseudomonads, such as P.stutzeri, are known opportunistic pathogens  and have also been implicated in hospital acquired infections [35,36]. P. aeruginosa is also known to be resistant to antibiotics .
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 . 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 . 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 , while Oxalobacter formingenes is found in the human gastrointestinal tract . 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  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 , Stenotrophomonas maltophilia [44,45], Flavobacterium indologenes  and Rothia dentocariosa  (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 . Other pathogenic strains potentially harmful to children that were found included Rothia dentocariosa, known to cause the childhood illness tonsillitis , 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.