Culture-independent analysis of bacterial diversity in a child-care facility - PubMed (original) (raw)

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

Lesley Lee et al. BMC Microbiol. 2007.

Abstract

Background: Child-care facilities appear to provide daily opportunities for exposure and transmission of bacteria and viruses. However, almost nothing is known about the diversity of microbial contamination in daycare facilities or its public health implications. Recent culture-independent molecular studies of bacterial diversity in indoor environments have revealed an astonishing diversity of microorganisms, including opportunistic pathogens and many uncultured bacteria. In this study, we used culture and culture-independent methods to determine the viability and diversity of bacteria in a child-care center over a six-month period.

Results: We sampled surface contamination on toys and furniture using sterile cotton swabs in four daycare classrooms. Bacteria were isolated on nutrient and blood agar plates, and 16S rRNA gene sequences were obtained from unique (one of a kind) colony morphologies for species identification. We also extracted DNA directly from nine representative swab samples taken over the course of the study from both toy and furniture surfaces, and used "universal" 16S rRNA gene bacterial primers to create PCR-based clone libraries. The rRNA gene clones were sequenced, and the sequences were compared with related sequences in GenBank and subjected to phylogenetic analyses to determine their evolutionary relationships. Culturing methods identified viable bacteria on all toys and furniture surfaces sampled in the study. Bacillus spp. were the most commonly cultured bacteria, followed by Staphylococcus spp., and Microbacterium spp. Culture-independent methods based on 16S rRNA gene sequencing, on the other hand, revealed an entirely new dimension of microbial diversity, including an estimated 190 bacterial species from 15 bacterial divisions. Sequence comparisons and phylogenetic analyses determined that the clone libraries were dominated by a diverse set of sequences related to Pseudomonas spp., as well as uncultured bacteria originally identified on human vaginal epithelium. Other sequences were related to uncultured bacteria from wastewater sludge, and many human-associated bacteria including a number of pathogens and opportunistic pathogens. Our results suggest that the child-care facility provided an excellent habitat for slime-producing Pseudomonads, and that diaper changing contributed significantly to the bacterial contamination.

Conclusion: The combination of culture and culture-independent methods provided powerful means for determining both viability and diversity of bacteria in child-care facilities. Our results provided insight into the source of contamination and suggested ways in which sanitation might be improved. Although our study identified a remarkable array of microbial diversity present in a single daycare, it also revealed just how little we comprehend the true extent of microbial diversity in daycare centers or other indoor environments.

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Figures

Figure 1

Results of Bayesian phylogenetic analysis of _Pseudomonas_-related 16S rRNA gene sequences representing the most common clones found in PCR-amplified libraries from swabs samples of toys and surfaces. Cloned sequences are indicated by "CCTR" (Children's Center Toddler Room) or "CCIR" (Children's Center Infant Room) prefixes followed by the date of sampling and whether the sequence was obtained from a toy (T) or a surface (S). The phylogeny includes sequences of closely related cultured and uncultured organisms. GenBank accession numbers are presented next to the names. The values above the branches indicate the Bayesian posterior probabilities (above 0.5) under the specified model of evolution for each node (see Methods for details). Maximum Parsimony and Maximum Likelihood analyses produced highly similar tree topologies. MP bootstrap values were similarly high at nodes well-supported by Bayesian analysis.

Figure 2

Results of Bayesian phylogenetic analysis of 16s rRNA gene sequences from other phylogenetic groups of bacteria found in PCR-amplified clone libraries from swabs samples of toys and surfaces. Cloned sequences are indicated by "CCTR" (Children's Center Toddler Room) or "CCIR" (Children's Center Infant Room) prefixes followed by the date of sampling and whether the sequence was obtained from a toy (T) or a surface (S). GenBank accession numbers are presented next to the names. The tree also includes sequences from some of the cultured isolates in Table 1. The values above the branches indicate the Bayesian posterior probabilities (above 0.5) under the specified model of evolution for each node. Maximum Parsimony and Maximum Likelihood analyses produced highly similar tree topologies. MP bootstrap values were similarly high at nodes well-supported by Bayesian analysis.

Figure 3

Graphical representation of common bacterial-type abundance found in the six clone libraries made from furniture surface swabs based on 16S rRNA gene sequence identification. Pseudomonads and uncultured Oxalobacteraceae were consistently found on all surfaces at high levels. Bacillus species were also common, though at lower abundance. One date in particular, Jan 25th,(Toddler Room 2) had a particularly high concentration of _Streptococcus_-related sequences.

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Culture-independent analysis of bacterial diversity in a child-care facility - PubMed (original) (raw)