Interaction of bacterial communities and indicators of water quality in shoreline sand, sediment, and water of Lake Michigan.

Shoreline sand harbors high concentrations of fecal indicator bacteria (FIB) that may be resuspended into the water column through washing and resuspension. Studies have explored coastal processes that influence this sand-water flux for FIB, but little is known about how microbial markers of contamination or the bacterial community interact in the sand-water interface. In this study, we take a three-tiered approach to explore the relationship between bacteria in sand, sediment, and overlying water at three shoreline sites and two associated rivers along an extended freshwater shoreline. Samples were collected over two years and analyzed for FIB, two microbial source tracking (MST) markers (Catellicoccus marimammalium, Gull2; Bacteroides HF183), and targeted metagenomic 16S rRNA gene analysis. FIB was much higher in sand than in water at all three sites. Gull2 marker was abundant in shoreline sand and water while HF183 marker was mostly present in rivers. Overall bacterial communities were dissimilar between sand/sediment and water, indicating little interaction. Sediment composition was generally unfavorable to bacterial resuspension. Results show that FIB and MST markers were effective estimates of short-term conditions at these locations, and bacterial communities in sand and sediment reflected longer-term conditions. Findings are useful for locating contamination sources and targeting restoration by evaluating scope of shoreline degradation.

Real-Time Water Quality Monitoring at a Great Lakes National Park.

Quantitative polymerase chain reaction (qPCR) was used by the USEPA to establish new recreational water quality criteria in 2012 using the indicator bacteria enterococci. The application of this method has been limited, but resource managers are interested in more timely monitoring results. In this study, we evaluated the efficacy of qPCR as a rapid, alternative method to the time-consuming membrane filtration (MF) method for monitoring water at select beaches and rivers of Sleeping Bear Dunes National Lakeshore in Empire, MI. Water samples were collected from four locations (Esch Road Beach, Otter Creek, Platte Point Bay, and Platte River outlet) in 2014 and analyzed for culture-based (MF) and non-culture-based (i.e., qPCR) endpoints using and enterococci bacteria. The MF and qPCR enterococci results were significantly, positively correlated overall ( = 0.686, < 0.0001, = 98) and at individual locations as well, except at the Platte River outlet location: Esch Road Beach ( = 0.441, = 0.031, = 24), Otter Creek ( = 0.592, = 0.002, = 24), and Platte Point Bay ( = 0.571, = 0.004, = 24). Similarly, MF and qPCR results were significantly, positively correlated ( = 0.469, < 0.0001, = 95), overall but not at individual locations. Water quality standard exceedances based on enterococci levels by qPCR were lower than by MF method: 3 and 16, respectively. Based on our findings, we conclude that qPCR may be a viable alternative to the culture-based method for monitoring water quality on public lands. Rapid, same-day results are achievable by the qPCR method, which greatly improves protection of the public from water-related illnesses.

Prototypic automated continuous recreational water quality monitoring of nine Chicago beaches.

Predictive empirical modeling is used in many locations worldwide as a rapid, alternative recreational water quality management tool to eliminate delayed notifications associated with traditional fecal indicator bacteria (FIB) culturing (referred to as the persistence model, PM) and to prevent errors in releasing swimming advisories. The goal of this study was to develop a fully automated water quality management system for multiple beaches using predictive empirical models (EM) and state-of-the-art technology. Many recent EMs rely on samples or data collected manually, which adds to analysis time and increases the burden to the beach manager. In this study, data from water quality buoys and weather stations were transmitted through cellular telemetry to a web hosting service. An executable program simultaneously retrieved and aggregated data for regression equations and calculated EM results each morning at 9:30 AM; results were transferred through RSS feed to a website, mapped to each beach, and received by the lifeguards to be posted at the beach. Models were initially developed for five beaches, but by the third year, 21 beaches were managed using refined and validated modeling systems. The adjusted R(2) of the regressions relating Escherichia coli to hydrometeorological variables for the EMs were greater than those for the PMs, and ranged from 0.220 to 0.390 (2011) and 0.103 to 0.381 (2012). Validation results in 2013 revealed reduced predictive capabilities; however, three of the originally modeled beaches showed improvement in 2013 compared to 2012. The EMs generally showed higher accuracy and specificity than those of the PMs, and sensitivity was low for both approaches. In 2012 EM accuracy was 70-97%; specificity, 71-100%; and sensitivity, 0-64% and in 2013 accuracy was 68-97%; specificity, 73-100%; and sensitivity 0-36%. Factors that may have affected model capabilities include instrument malfunction, non-point source inputs, and sparse calibration data. The modeling system developed is the most extensive, fully-automated system for recreational water quality developed to date. Key insights for refining and improving large-scale empirical models for beach management have been developed through this multi-year effort.

Relationship and variation of qPCR and culturable Enterococci estimates in ambient surface waters are predictable.

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P < 0.05) at most sites. A linearly decreasing variance of CCE with increasing CFU levels was significant (P < 0.05) or evident for all sites. Both marine and freshwater sites under continuous influence of point-source contamination tended to reveal a relatively constant proportion of CCE to CFU. The consistency in the mean and variance patterns of CCE versus CFU indicates that the relationship of results based on these two methods is more predictable at high CFU levels (e.g., log(10)CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions.

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R=0.650, P<0.0001, N=32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P<0.0001). Culturable enterococci densities at Burns Ditch and the beaches were significantly correlated (R=0.565, P<0.0001, N=32). Culturable E. coli and enterococci densities were significantly correlated (R=0.682, P<0.0001, N=32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R(2)=0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity x wave height (adjusted R(2)=0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results.

Geographic relatedness and predictability of Escherichia coli along a peninsular beach complex of Lake Michigan.

To determine more accurately the real-time concentration of fecal indicator bacteria (FIB) in beach water, predictive modeling has been applied in several locations around the Great Lakes to individual or small groups of similar beaches. Using 24 beaches in Door County, Wisconsin, we attempted to expand predictive models to multiple beaches of complex geography. We examined the importance of geographic location and independent variables and the consequential limitations for potential beach or beach group models. An analysis of Escherichia coli populations over 4 yr revealed a geographic gradient to the beaches, with mean E. coli concentrations decreasing with increasing distance from the city of Sturgeon Bay. Beaches grouped strongly by water type (lake, bay, Sturgeon Bay) and proximity to one another, followed by presence of a storm or creek outfall or amount of shoreline enclosure. Predictive models developed for beach groups commonly included wave height and cumulative 48-h rainfall but generally explained little E. coli variation (adj. R2=0.19-0.36). Generally low concentrations of E. coli at the beaches influenced the effectiveness of model results presumably because of low signal-to-noise ratios and the rarity of elevated concentrations. Our results highlight the importance of the sensitivity of regressors and the need for careful methods evaluation. Despite the attractiveness of predictive models as an alternative beach monitoring approach, it is likely that FIB fluctuations at some beaches defy simple prediction approaches. Regional, multi-beach, and individual beach predictive models should be explored alongside other techniques for improving monitoring reliability at Great Lakes beaches.

Hand-mouth transfer and potential for exposure to E. coli and F+ coliphage in beach sand, Chicago, Illinois.

Beach sand contains fecal indicator bacteria, often in densities greatly exceeding the adjacent swimming waters. We examined the transferability of Escherichia coli and F+ coliphage (MS2) from beach sand to hands in order to estimate the potential subsequent health risk. Sand with high initial E. coli concentrations was collected from a Chicago beach. Individuals manipulated the sand for 60 seconds, and rinse water was analysed for E. coli and coliphage. E. coli densities transferred were correlated with density in sand rather than surface area of an individual's hand, and the amount of coliphage transferred from seeded sand was different among individuals. In sequential rinsing, percentage reduction was 92% for E. coli and 98% for coliphage. Using dose-response estimates developed for swimming water, it was determined that the number of individuals per thousand that would develop gastrointestinal symptoms would be 11 if all E. coli on the fingertip were ingested or 33 if all E. coli on the hand were ingested. These results suggest that beach sand may be an important medium for microbial exposure; bacteria transfer is related to initial concentration in the sand; and rinsing may be effective in limiting oral exposure to sand-borne microbes of human concern.

Seasonal stability of Cladophora-associated Salmonella in Lake Michigan watersheds.

The bacterial pathogens Shigella, Salmonella, Campylobacter, and shiga toxin-producing E. coli (STEC) were recently found to be associated with Cladophora growing in southern Lake Michigan. Preliminary results indicated that the Salmonella strains associated with Cladophora were genetically identical to each other. However, because of the small sample size (n=37 isolates) and a lack of information on spatial-temporal relationships, the nature of the association between Cladophora and Salmonella remained speculative. In this study, we investigated the population structure and genetic relatedness of a large number of Cladophora-borne Salmonella isolates from Lake Michigan (n=133), as well as those isolated from stream and lake water (n=31), aquatic plants (n=8), and beach sands and sediments (n=8) from adjacent watersheds. Salmonella isolates were collected during 2005-2007 between May and August from Lake Michigan beachsheds in Wisconsin, Illinois, and Indiana. The genetic relatedness of Salmonella isolates was examined by using the horizontal, fluorophore-enhanced rep-PCR (HFERP) DNA fingerprinting technique. While the Salmonella isolates associated with Cladophora exhibited a high degree of genetic relatedness (>or=92% similarity), the isolates were not all genetically identical. Spatial and temporal relationships were evident in the populations examined, with tight clustering of the isolates both by year and location. These findings suggest that the relationship between Salmonella and Cladophora is likely casual and is related to input sources (e.g. wastewater, runoff, birds) and the predominant Salmonella genotype surviving in the environment during a given season. Our studies indicate that Cladophora is likely an important reservoir for Salmonella and other enteric bacterial pathogens in Lake Michigan beachsheds, which in turn may influence nearshore water quality.

Environmental occurrence of the enterococcal surface protein (esp) gene is an unreliable indicator of human fecal contamination.

The enterococcal surface protein (esp) gene found in Enterococcus faecalis and E. faecium has recently been explored as a marker of sewage pollution in recreational waters but its occurrence and distribution in environmental enterococci has not been well-documented. If the esp gene is found in environmental samples, there are potential implications for microbial source tracking applications. In the current study, a total of 452 samples (lake water, 100; stream water, 129; nearshore sand, 96; and backshore sand, 71; Cladophora sp. (Chlorophyta), 41; and periphyton (mostly Bacillariophyceae), 15) collected from the coastal watersheds of southern Lake Michigan were selectively cultured for enterococci and then analyzed for the esp gene by PCR, targeting E. faecalis/ E. faecium (esp(fs/fm)) and E. faecium (esp(fm)). Overall relative frequencies for esp(fs/fm) and esp(fm) were 27.4 and 5.1%. Respective percent frequency for the esp(fs/fm) and esp(fm) was 36 and 14% in lake water, 38.8 and 2.3% in stream water, 24 and 6.3% in nearshore sand; 0% in backshore sand; 24.4 and 0% in Cladophora sp.; and 33.3 and 0% in periphyton. The overall occurrence of both esp(fs/fm) and esp(fm) was significantly related (chi2 = 49, P < 0.0001). Post-rain incidence of esp(fs/fm) increased in lake and stream water and nearshore sand. Further, E. coli and enterococci cell densities were significant predictors for esp(fs/fm) occurrence in post-rain lake water, but esp(fm) was not F+ coliphage densities were not significant predictors for esp(fm) or esp(fs/fm) gene incidence. In summary, the differential occurrence of the esp gene in the environment suggests that it is not limited to human fecal sources and thus may weaken its use as a reliable tool in discriminating contaminant sources (i.e., human vs. nonhuman).

Sunlight, season, snowmelt, storm, and source affect E. coli populations in an artificially ponded stream.

Reducing fecal indicator bacteria, such as Escherichia coli (E. coli), in streams is important for many downstream areas. E. coli concentrations within streams may be reduced by intervening ponds or wetlands through a number of physical and biological means. A section of Dunes Creek, a small coastal stream of southern Lake Michigan, was impounded and studied for 30 months from pre-through post-construction of the experimental pond. E. coli reduction became more predictable and effective with pond age. E. coli followed the hydrograph and increased several-fold during both rainfall and snowmelt events. Seasonally, the pond was more effective at reducing E. coli during summer than winter. Late summer, non-solar reduction or inactivation of E. coli in the pond was estimated at 72% and solar inactivation at 26%. E. coli DNA fingerprinting demonstrated that the winter population was genetically more homogeneous than the summer population. Detection of FRNA coliphages suggests that there was fecal contamination during heavy rain events. An understanding of how environmental factors interact with E. coli populations is important for assessing anticipated contaminant loading and the reduction of indicator bacteria in downstream reaches.

Incidence of the enterococcal surface protein (esp) gene in human and animal fecal sources.

The occurrence of the enterococcal surface protein (esp) gene in the opportunistic pathogens Enterococcus faecalis and E. faecium is well-documented in clinical research. Recently, the esp gene has been proposed as a marker of human pollution in environmental waters; however, information on its relative incidence in various human and animal fecal sources is limited. We have determined the occurrence of the esp gene in enterococci from human (n=64) and animal (n=233) fecal samples by polymerase chain reaction using two primer sets: one presumably specific for E. faecium (esp(fm)) and the other for both E. faecalis and E. faecium (esp(fs/fm)). We believe that this research is the first to explore the use of esp(fs/fm) for the detection of human waste in natural environmental settings. The incidence in human sources was 93.1% esp(fm) and 100% esp(fs/fm) in raw sewage influent; 30% for both esp(fm) and esp(fs/fm) in septic waste; and 0% esp(fm) and 80% esp(fs/fm) in active pit toilets. The overall occurrence of the gene in animal feces was 7.7% (esp(fs/fm)) and 4.7% (esp(fm)); animal types with positive results included dogs (9/43, all esp(fm)), gulls (10/34, esp(fs/fm); 2/34, esp(fm)), mice (3/22, all esp(fs/fm)), and songbirds (5/55, all esp(fs/fm)). The esp gene was not detected in cat (0/34), deer (0/4), goose (0/18), or raccoon (0/23) feces. The inconsistent occurrence, especially in septic and pit toilet sewage, suggests a low statistical power of discrimination between animal and human sources, which means a large number of replicates should be collected. Both esp(fm) and esp(fs/fm) were common in raw sewage, but neither one efficiently differentiated between animal and other human sources.

Population structure of Cladophora-borne Escherichia coli in nearshore water of Lake Michigan.

We previously reported that the macrophytic green alga Cladophora harbors high densities (up to 10(6) colony-forming units/g dry weight) of the fecal indicator bacteria, Escherichia coli and enterococci, in shoreline waters of Lake Michigan. However, the population structure and genetic relatedness of Cladophora-borne indicator bacteria remain poorly understood. In this study, 835 E. coli isolates were collected from Cladophora tufts (mats) growing on rocks from a breakwater located within the Indiana Dunes National Lakeshore in northwest Indiana. The horizontal fluorophore enhanced rep-PCR (HFERP) DNA fingerprinting technique was used to determine the genetic relatedness of the isolates to each other and to those in a library of E. coli DNA fingerprints. While the E. coli isolates from Cladophora showed a high degree of genetic relatedness (92% similarity), in most cases, however, the isolates were genetically distinct. The Shannon diversity index for the population was very high (5.39). Both spatial and temporal influences contributed to the genetic diversity. There was a strong association of isolate genotypes by location (79% and 80% for lake- and ditch-side samplings, respectively), and isolates collected from 2002 were distinctly different from those obtained in 2003. Cladophora-borne E. coli isolates represented a unique group, which was distinct from other E. coli isolates in the DNA fingerprint library tested. Taken together, these results indicate that E. coli strains associated with Cladophora may be a recurring source of indicator bacteria to the nearshore beach.

Seasonal persistence and population characteristics of Escherichia coli and enterococci in deep backshore sand of two freshwater beaches.

We studied the shoreward and seasonal distribution of E. coil and enterococci in sand (at the water table) at two southern Lake Michigan beaches-Dunbar and West Beach (in Indiana). Deep, backshore sand (approximately 20 m inland) was regularly sampled for 15 months during 2002-2003. E. coli counts were not significantly different in samples taken at 5-m intervals from 0-40 m inland (P = 0.25). Neither E. coli nor enterococci mean counts showed any correlation or differences between the two beaches studied. In laboratory experiments, E. coli readily grew in sand supplemented with lake plankton, suggesting that in situ E. coil growth may occur when temperature and natural organic sources are adequate. Of the 114 sand enterococci isolates tested, positive species identification was obtained for only 52 (46%), with E. faecium representing the most dominant species (92%). Genetic characterization by ribotyping revealed no distinct genotypic pattern (s) for E. coli, suggesting that the sand population was rather a mixture of numerous strains (genotypes). These findings indicate that E. coli and enterococci can occur and persist for extended periods in backshore sand at the groundwater table. Although this study was limited to two beaches of southern Lake Michigan, similar findings can be expected at other temperate freshwater beaches. The long-term persistence of these bacteria, perhaps independent of pollution events, complicates their use as indicator organisms. Further, backshore sand at the water table may act as a reservoir for these bacteria and potentially for human pathogens.

Modeling the transport and inactivation of E. coli and enterococci in the near-shore region of Lake Michigan.

To investigate the transport and fate of fecal pollution at Great Lakes beaches and the health risks associated with swimming, the near-shore waters of Lake Michigan and two tributaries discharging into it were examined for bacterial indicators of human fecal pollution. The enterococcus human fecal pollution marker, which targets a putative virulence factor--the enterococcal surface protein (esp) in Enterococcus faecium, was detected in 2/28 samples (7%) in the tributaries draining into Lake Michigan and in 6/30 samples (20%) in Lake Michigan beaches. This was indicative of human fecal pollution being transported in the tributaries and occurrence at Lake Michigan beaches. To understand the relative importance of different processes influencing pollution transport and inactivation, a finite-element model of surf-zone hydrodynamics (coupled with models for temperature, E. coli and enterococci) was used. Enterococci appear to survive longer than E. coli, which was described using an overall first-order inactivation coefficient in the range 0.5-2.0 per day. Our analysis suggests that the majority of fecal indicator bacteria variation can be explained based on loadings from the tributaries. Sunlight is a major contributor to inactivation in the surf-zone and the formulation based on sunlight, temperature and sedimentation is preferred over the first-order inactivation formulation.

Cladophora (Chlorophyta) spp. harbor human bacterial pathogens in nearshore water of Lake Michigan.

Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 x 10(3) cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 x 10(2) Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.

Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lakes watershed.

The common occurrence of Escherichia coli in temperate soils has previously been reported, however, there are few studies to date to characterize its source, distribution, persistent capability and genetic diversity. In this study, undisturbed, forest soils within six randomly selected 0.5 m2 exclosure plots (covered by netting of 2.3 mm2 mesh size) were monitored from March to October 2003 for E. coli in order to describe its numerical and population characteristics. Culturable E. coli occurred in 88% of the samples collected, with overall mean counts of 16 MPN g(-1), ranging from < 1 to 1657 (n = 66). Escherichia coli counts did not correlate with substrate moisture content, air, or soil temperatures, suggesting that seasonality were not a strong factor in population density control. Mean E. coli counts in soil samples (n = 60) were significantly higher inside than immediately outside the exclosures; E. coli distribution within the exclosures was patchy. Repetitive extragenic palindromic polymerase chain reaction (Rep-PCR) demonstrated genetic heterogeneity of E. coli within and among exclosure sites, and the soil strains were genetically distinct from animal (E. coli) strains tested (i.e. gulls, terns, deer and most geese). These results suggest that E. coli can occur and persist for extended periods in undisturbed temperate forest soils independent of recent allochthonous input and season, and that the soil E. coli populations formed a cohesive phylogenetic group in comparison to the set of fecal strains with which they were compared. Thus, in assessing E. coli sources within a stream, it is important to differentiate background soil loadings from inputs derived from animal and human fecal contamination.

Occurrence and growth characteristics of Escherichia coli and enterococci within the accumulated fluid of the northern pitcher plant (Sarracenia purpurea L.).

Sarracenia purpurea L., a carnivorous bog plant (also known as the pitcher plant), represents an excellent model of a well-defined, self-contained ecosystem; the individual pitchers of the plant serve as a microhabitat for a variety of micro- and macro-organisms. Previously, fecal indicator bacteria (Escherichia coli and enterococci) were shown as incidental contaminants in pitcher fluid; however, whether their occurrence in pitcher fluid is incidental or common has not been established. The purpose of this study was to investigate the occurrence, distribution, and growth potential of E. coli and enterococci in pitcher plant fluid from a protected bog in northwest Indiana. Escherichia coli and enterococci were recovered in pitcher fluids (n=43 plants), with mean densities (log CFU mL-1) of 1.28+/-0.23 and 1.97+/-0.27, respectively. In vitro experiments showed that E. coli growth in fluid not containing insects or indigenous organisms was directly proportional to the fluid concentration (growth was 10-fold in 24 h in 100% fluid); however, in the presence of other indigenous organisms, E. coli and enterococci were only sustained for 5 days at 26 degrees C. Pulsed-field gel electrophoresis (PFGE) analysis showed that the plant Enterococcus faecalis isolates were genetically distinct from the human isolates; identical PFGE patterns were observed among plant isolates that fell into one of six clonal groups. These findings suggest that (i) E. coli and enterococci occurrence in pitcher plants is rather common in the bog studied, although their originating source is unclear, and (ii) the pitcher fluid contains adequate nutrients, especially carbon and energy sources, to promote the growth of indicator bacteria; however, under natural conditions, the biotic factors (e.g., competition for nutrients) may restrict their growth.

Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan.

Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (+/- standard errors) of 5.3 (+/- 4.8) and 4.8 (+/- 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R(2) = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4 degrees C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.

Growth and survival of Escherichia coli and enterococci populations in the macro-alga Cladophora (Chlorophyta).

The macro-alga Cladophora glomerata is found in streams and lakes worldwide. High concentrations of Escherichia coli and enterococci have been reported in Cladophora along the Lake Michigan shore. The objective of this study was to determine if Cladophora supported growth of these indicator bacteria. Algal leachate readily supported in vitro multiplication of E. coli and enterococci, suggesting that leachates contain necessary growth-promoting substances. Growth was directly related to the concentration of algal leachate. E. coli survived for over 6 months in dried Cladophora stored at 4 degrees C; residual E. coli grew after mat rehydration, reaching a carrying capacity of 8 log CFU g(-1) in 48 h. Results of this study also show that the E. coli strains associated with Cladophora are highly related; in most instances they are genetically different from each other, suggesting that the relationship between E. coli and Cladophora may be casual. These findings indicate that Cladophora provides a suitable environment for indicator bacteria to persist for extended periods and to grow under natural conditions.