Here we report results from a multi-laboratory (n=11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR® Green qPCR method, and two TaqMan® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.
Bibliographical noteFunding Information:
This study was primarily funded by a Clean Beach Initiative grant from the California Water Resources Control Board . It was also funded in part by Oceans and Human Health Center grants from NSF and NIEHS (NSF 0CE0432368/0911373 and NIEHS P50ES12736 , respectively). Additional funding support and research collaboration was also provided by the National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory, Ocean Chemistry Division. The U.S. Environmental Protection Agency, through its Office of Research and Development and the RARE program, partially funded and collaborated in the research described herein. Research collaboration support and additional funding was also provided by the U.S. Geological Survey, Great Lakes Science Center. This work was funded in part by the U.S. Geological Survey (USGS) Ocean Research Priorities Plan (ORPP) . Portions of this work conducted at University College Dublin were funded in part by the Irish EPA under the STRIVE Programme. This work has been subjected to agency administrative review and has been approved for external publication. Any opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the U.S. EPA; therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use. HR was funded via a National Research Council fellowship. This article is Contribution 1737 of the U.S. Geological Survey Great Lakes Science Center.
- Catellicoccus marimammalium
- Fecal indicator bacteria
- Microbial source tracking
- Quantitative PCR
- Water quality monitoring