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Development and evaluation of 16S rRNA gene targeting Enterococcus genus- and species-specific assays
RYU, H., M. Henson, M. Elk, S. GLASSMEYER, AND J. W. SANTO-DOMINGO. Development and evaluation of 16S rRNA gene targeting Enterococcus genus- and species-specific assays . Presented at 112th American Society for Microbiology General Meeting, San Francisco, CA, June 16 - 19, 2012.
To inform the public.
Enterococci have been widely used as indicators of fecal pollution in recreational waters. Most studies enumerate enterococci using culture-based techniques that are time consuming and do not provide information on the identity of enterococci species within a given sample. Although there are a few enterococci species-specific assays available, only a handful of them have been used in environmental studies. Moreover, these assays have been tested against a limited number of environmental enterococci strains. To address these issues we performed 16S rRNA gene sequence analysis and used several currently available (n=2) and newly developed genus- and species-specific assays (n=13) targeting enterococci species 16S rRNA gene. The specificity of the assays was evaluated against eight of the most commonly isolated Enterococcus species and 15 non-enterococci species. Additionally, the assays were tested against 439 presumptive Enterococcus environmental strains isolated from 15 different geographic locations. Phylogenetic analysis showed that most of the environmental isolates (91%) were indeed enterococci species, whereas others were classified as non-enterococci bacteria (7%) or potentially novel enterococci species (2%). E. casseliflavus (34%) and E. faecalis (25%) were the most dominant species, while E. mundtii, E. faecium, and E. hirae (i.e., >99% identity) were also identified but to a lesser extent. The genus-specific assays successfully amplified >98% of the Enterococcus strains, but it cross-amplified with some of 20 non-enterococcus bacteria tested (15-35%). The species-specific assays showed relatively high amplification rates with targeted-species (>95%), although some assays cross-amplified with non-enterococci species (<10%) and with non-targeted enterococci species (11-20%). The prevalence of genus marker was in agreement with the combined number of signals observed for the E. casseliflavus, E. faecalis, and E. faecium markers in environmental waters (699) and most fecal (1400) samples tested. However, in shore bird fecal samples (n=587), 55% and 3% were positive for genus- and species-specific assays. Overall, the results suggest that genus and species-specific assays could be used in environmental monitoring studies, with the exception of shorebird impacted waters.