Julie L. Kinzelman

A cross comparison of QPCR to agar-based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs

Molecular methods such as quantitative, real-time polymerase chain reaction (QPCR) are intended to shorten the period between sampling and publicly available results. Cross comparison studies in Racine, WI, USA evaluated QPCR against agar-based (US EPA Method 1600) and defined substrate (IDEXX Colilert-18) methods for the detection and quantification of Escherichia coli and enterococci in a variety of aqueous environments (wastewater, stormwater, and surface water). Regulatory outcomes were also compared based on choice of indicator and method. Positive correlation was seen between QPCR cell equivalents and viable cells through the wastewater treatment process and in all surface water samples (river or freshwater bathing beach) but not in direct stormwater discharge. For surface water samples, correlation improved with the application of a site-specific corrective factor, with regulatory action correctly predicted 98% of the time at bathing beaches. This study suggests the potential utility of QPCR for certain water quality monitoring applications.

Microbes in Beach Sands: Integrating Environment, Ecology and Public Health.

Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.

Interlaboratory Comparison of Real-Time PCR Protocols for Quantification of General Fecal Indicator Bacteria

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Dramatic Improvements in Beach Water Quality Following Gull Removal

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (p < 0.001). Enterococcus spp. and Escherichia coli densities were also significantly reduced during gull control (p < 0.001 and p = 0.012, respectively for culture methods; p = 0.012 and p = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in Enterococcus spp. and E. coli densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (p = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality.

Bacteroidales Diversity in Ring-Billed Gulls (Laurus delawarensis) Residing at Lake Michigan Beaches

ABSTRACT This study investigated the occurrence and diversity of Bacteroidales fecal bacteria in gulls residing in the Great Lakes region. Members of this bacterial order have been widely employed as human and bovine host-specific markers of fecal pollution; however, few studies have focused on gulls, which can be a major source of fecal indicator bacteria and pathogens at beaches. We found a low but consistent occurrence of Bacteroidales in gulls at five beaches in three different counties spanning the Wisconsin shoreline of Lake Michigan. The percentages of gulls positive for Bacteroidales were 4 to 8% at beaches in the southern part of the state and 8 to 50% at beaches in the north. Sequencing of 931 clones from seven gull Bacteroidales 16S rRNA gene libraries revealed a large amount of diversity in both individual and pooled gull fecal samples. Two libraries constructed from pooled gull fecal samples (n = 5 and n = 6) did not have a greater richness of sequences than individual samples, suggesting that even within a single gull diversity is high and an extensive sequencing effort is needed to characterize the populations. Estimates of the numbers of operational taxonomic units (OTUs) for the libraries obtained using different similarity levels revealed a large amount of microdiveristy with a limited number of OTUs at the 95% similarity level. Gull sequences were clustered by the beach from which they were collected, suggesting that there were geographic effects on the distribution of Bacteriodales. More than 53% of the 16S rRNA gene sequences from gulls at the southern beaches were associated with the family Porphyromonadaceae, primarily the genus Parabacteroides, whereas sequences from gulls at the northern beaches were comprised of Bacteroidaceae and Prevotellaceae sequences. Comparison of gull sequences with sequences from goose, canine, raccoon, and sewage sources revealed distinct clusters of closely related gull sequences; however, these sequences were widely dispersed across a dendrogram that included all other sources, including previously characterized gull Bacteroidales from other studies, suggesting that geographic influence or simply sample representation plays a greater role in the observed population structure than strictly the host gut environment.

Success of science-based best management practices in reducing swimming bans—a case study from Racine, Wisconsin, USA

The Great Lakes region possesses over 10,000 miles of shoreline (US EPA and Government of Canada, 1995) which are home to over 1,000 beaches. These beaches represent a recreational outlet for over 30 million people (US EPA and Government of Canada, 1995) and yet many of them remain inaccessible for periods of time each bathing season due to water quality advisories. The reason for these advisories is often elusive to beach managers, hence impeding their ability to craft appropriate mitigation measures. Even when the sources of contamination are known, remediation measures may not be put into practice due to the perception that they are too costly. However, a recent study has demonstrated that investing in environmental improvements which increase the number of days available for swimming in the Great Lakes region by 20% would generate

Evaluation of Beach Grooming Techniques on Escherichia coli Density in Foreshore Sand at North Beach, Racine, WI

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The Green Alga, Cladophora , Promotes Escherichia coli Growth and Contamination of Recreational Waters in Lake Michigan

3 billion dollars in direct economic effects. Therefore, while beach closings and advisories continue to rise overall, some Great Lakes communities have recognized the potential for municipal beaches to generate revenue and increase the quality of life for their citizens and have undertaken comprehensive studies to improve recreational water quality. In Racine, Wisconsin, USA, research conducted to identify pollution sources guided the development of better beach management practices such as ecologically appropriate beach modifications, improved mechanical beach grooming strategies, and the redesign of a major storm water outlet (including installation of a constructed wetland area). Resulting improvements have reduced bathing water quality advisories from 66% of days during the swimming season in 2000 to 5% or less in four consecutive years (2005–2008). These improvements to Racine beaches facilitated Blue Wave certification from the Clean Beaches Council (Washington, DC); thereby restoring public confidence, increasing beach use by the residents and tourists, and expanding the role of the beachfront in the local economy.

The effect of two mechanical beach grooming strategies on Escherichia coli density in beach sand at a southwestern Lake Michigan beach

ABSTRACT Elevated levels of Escherichia coli(E. coli) in bathing waters at North Beach, a popular recreational site in Racine, Wisconsin, have been a persistent problem often resulting in the issuance of poor water quality advisories. Moreover, waterfowl (mostly Larus delawarensis and L. argentatus) in nearshore and offshore areas are common and may serve as non-point sources for bacterial contamination of recreational waters. Current beach management practice involves daily mechanical grooming of the nearshore sand for aesthetics and removal of hazardous debris. However, this practice has not been evaluated in terms of its effects on E. coli loading to beach sand and potential introduction to contiguous swimming water. In this study, we tested E. coli responses to three treatments: mechanical groomer, daily and twice weekly hand raking, and a control (no raking/grooming). A randomized block design consisted of replicated treatments and one control (10 each), for a total of 40 blocks sampled daily for 10 days. Foreshore sand samples were collected by hand coring to an average depth of 10 cm. Median E. coli recovered were 73 (mechanically groomed), 27 (hand-raked daily), 32 (hand-raked twice weekly), and 22 (control) colony-forming units (CFU) per gram dry weight sand. E. coli counts in sand that was groomed were significantly higher than hand rakings and control (p <0.0001), and there was no significant difference between control and raking treatments (p<0.01). This study demonstrates the beach management implications related to grooming efficacy and the importance of understanding non-point sources of bacterial contamination.

Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods.

A linkage between Cladophora mats and exceedances of recreational water quality criteria has been suggested, but not directly studied. This study investigates the spatial and temporal association between Escherichia coli concentrations within and near Cladophora mats at two northwestern Lake Michigan beaches in Door County, Wisconsin. Escherichia coli concentrations in water underlying mats were significantly greater than surrounding water (p < 0.001). Below mat E. coli increased as the stranded mats persisted at the beach swash zone. Water adjacent to Cladophora mats had lower E. coli concentrations, but surpassed EPA swimming criteria the majority of sampling days. A significant positive association was found between E. coli concentrations attached to Cladophora and in underlying water (p < 0.001). The attached E. coli likely acted as a reservoir for populating water underlying the mat. Fecal bacterial pathogens, however, could not be detected by microbiological culture methods either attached to mat biomass or in underlying water. Removal of Cladophora mats from beach areas may improve aesthetic and microbial water quality at affected beaches. These associations and potential natural growth of E. coli in bathing waters call into question the efficacy of using E. coli as a recreational water quality indicator of fecal contaminations.