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Response to Comment on “Environmental Occurrence of the Enterococcal Surface Protein (esp) Gene is an Unreliable Indicator of Human Fecal Contamination” We appreciate the opportunity to respond to comments by Scott et al. (1) regarding our papers on the enterococcal surface protein (esp) gene frequency in human and animal fecal samples (Whitman et al. 2007) and in forested and aquatic environments (Byappanahalli et al., 2008) (2, 3). Their research represents the type of work that is sorely needed in the field of public health protection through detection of human waste. It represents creative research that begs for confirmation and extension, and it was within that spirit that we undertook follow-up investigations of their work. Their primary concern is with our conclusion that the esp gene associated with Enterococcus faecium, espfm, is an inconsistent marker of human fecal contamination and, thus, an undependable molecular marker of human waste. We are confident of our findings, which have also been validated by Alexandria Boehm’s laboratory at Stanford University (4). Their abstract states that “... E. faecium variant of the esp gene is not human-specific”, and “... the use of the esp gene for microbial source tracking applications may not be appropriate at all recreational beaches.” It should be emphasized that our studies were quite robust. In all, we analyzed 233 animal fecal samples (representing 29 different species), 64 human fecal samples (2) (Whitman et al. 2007), and 452 environmental samples (3) (Byappanahalli et al. 2008). In contrast, Scott et al. 2005 (5) analyzed 102 animal samples (8 different species) and 65 human fecal samples. Whitman et al. 2007 (2) concur that espfm occurs reliably in raw sewage samples (93.1%) but has much lower frequency in other human waste samples. We agree that 100% accuracy is not practical in microbial source tracking applications, yet the espfm occurrence in septage haulers (septic trucks) and pit toilets was quite low in our studies: 30% and 0%, respectively. It should be noted that septic trucks generally carry fecal wastes from family tanks, collecting domestic waste over a 3-8 year period, and also mobile home parks and restaurants among others, so sampling was not limited. On occasions, sewage from small municipalities was also negative for the espfm gene in our study. In addition, espfm failed to detect a heavy sewage bypass by multiple municipalities into a receiving river and beaches. Scott et al. (1) claim that the absence of espfm alone indicates no human pollution in one of our streams (Dunes Creek). We found more than 10000 enterococci per 100 mL in almost all 28 water samples collected throughout Dunes Creek, 60% of which had espfs/fm and 38% F+ coliphage. We previously reported high occurrence of F+ coliphage in this creek after comparable rainfall (6). Thus, human contamination does seem likely and our conclusion is a sequitur. In their comments, Scott et al. (1) suggest that our enterococci counts may have been insufficient to detect the much rarer esp gene. We performed esp gene assays using membrane filters with the highest number of colonies, often from human or animal feces with enterococci counts of usually 103 or above per filter. Further, water samples, where we expected the presence of espfm, were well in excess of the U.S. Environmetal Protection Agency criteria for safe swimming water. The espfm absence in diluted sewage within lake 6436 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 16, 2009
and river water after bypass, despite concentrations of fecal indicator bacteria (FIB) up to 105, undermines the reliability of this marker. We assert that a reliable sewage marker should be positive when enterococci densities are within ranges observed in the environment and especially after a sewage spill. Our conclusion that “differential occurrence of esp gene variants in the environment is due to lack of human specificity,” is supported by our data and other similar findings (relevant citations are indicated): 1. Variants of the E. faecium esp gene have been found in animal fecal sources: dogs (7), dogs and gulls (2), and dogs, gulls, horses, sea lions, and seals (4), and espfs/fm as well in pigs (8, 9), gulls, songbirds, mice (2), and birds (9). 2. Recent investigations by Boehm’s laboratory (4) support our research that espfm can be found in enterococci derived from the environment (37% occurrence in samples, including sand, surf zone, groundwater, storm drain, etc.) as well as animal fecal sources. 3. The espfm gene does not consistently occur in all human fecal sources such as pit toilets, 0% (2), septic systems, 30-67% (2, 10), and some small treatment plants (2). 4. The sequences of the espfm gene from human and animal fecal sources were virtually the same, g97% (3) and >99% (4). Research by Scott et al. (5) and others is commendable and is leading us toward a useful human marker. While espfm may not adequately distinguish human contamination in natural environments, it does not diminish our collective efforts to refine the discovery of a reliable, sensitive, and specific marker for microbial source-tracking and detection of human pollution. We agree that the most useful detection system will ultimately be a suite of markers and surrogates, and we look forward to working concurrently toward that end.
Literature Cited (1) Scott, T. M.; Harwood, V. J.; Ahmed, W.; Masago, Y.; Rose, J. B. Comment on “Environmental occurrence of the enterococcal surface protein (esp) gene is an unreliable indicator of human fecal contamination”. Environ. Sci. Technol. 2009, DOI: 1021/es901282x. (2) Whitman, R. L.; Przybyla-Kelly, K.; Shively, D. A.; Byappanahalli, M. N. Incidence of the enterococcal surface protein (esp) gene in human and animal fecal sources. Environ. Sci. Technol. 2007, 41, 6090–6095. (3) Byappanahalli, M. N.; Przybyla-Kelly, K.; Shively, D. A.; Whitman, R. L. Environmental occurrence of the enterococcal surface protein (esp) gene is an unreliable indicator of human fecal contamination. Environ. Sci. Technol. 2008, 42, 8014– 8020. (4) Layton, B. A.; Walters, S. P.; Boehm, A. B. Distribution and diversity of the enterococcal surface protein (esp) gene in animal hosts and the Pacific coast environment. J. Appl. Microbiol. 2009, 106, 1521–1531. (5) Scott, T. M.; Jenkins, T. M.; Lukasik, J.; Rose, J. B. Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution. Environ. Sci. Technol. 2005, 39, 283–287. (6) Whitman, R. L.; Przybyla-Kelly, K.; Shively, D. A.; Nevers, M. B.; Byappanahalli, M. N. Sunlight, season, snowmelt, storm, and source affect E. coli populations in an artificially ponded stream. Sci. Total Environ. 2008, 390, 448–455. (7) Harada, T.; Tsuji, N.; Otsuki, K.; Murase, T. Detection of the esp gene in high-level gentamicin resistant Enterococcus faecalis strains from pet animals in Japan. Vet. Microbiol. 2005, 106, 139–143. 10.1021/es901795g
Not subject to U.S. Copyright. Publ. 2009 Am. Chem. Soc.
Published on Web 07/17/2009
(8) Hammerum, A. M.; Jensen, L. B. Prevalence of esp, encoding the enterococcal surface protein, in Enterococcus faecalis and Enterococcus faecium isolates from hospital patients, poultry, and pigs in Denmark. J. Clin. Microbiol. 2002, 40, 4396. (9) Poeta, P.; Costa, D.; Saenz, Y.; Klibi, N.; Ruiz-Larrea, F.; Rodrigues, J.; Torres, C. Characterization of antibiotic resistance genes and virulence factors in faecal enterococci of wild animals in Portugal. J. Vet. Med. 2005, 52, 396–402. (10) Ahmed, W.; Stewart, J.; Powell, D.; Gardner, T. Evaluation of the host-specificity and prevalence of enterococci surface protein (esp) marker in sewage and its application for
sourcing human fecal pollution. J. Environ. Qual. 2008, 37, 1583–1588.
Muruleedhara N. Byappanahalli* and Richard L. Whitman Great Lakes Science Center, Lake Michigan Ecological Research Station, U.S. Geological Survey, 1100 N. Mineral Springs Road, Porter, Indiana 46304 ES901795G
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