Corrigendum: Gut Microbiota Associated With Different Sea Lamprey (Petromyzon marinus) Life Stages.

[This corrects the article DOI: 10.3389/fmicb.2021.706683.].

Gut Microbiota Associated With Different Sea Lamprey (Petromyzon marinus) Life Stages.

Sea lamprey (SL; Petromyzon marinus), one of the oldest living vertebrates, have a complex metamorphic life cycle. Following hatching, SL transition into a microphagous, sediment burrowing larval stage, and after 2-10+ years, the larvae undergo a dramatic metamorphosis, transforming into parasitic juveniles that feed on blood and bodily fluids of fishes; adult lamprey cease feeding, spawn, and die. Since gut microbiota are critical for the overall health of all animals, we examined the microbiota associated with SLs in each life history stage. We show that there were significant differences in the gut bacterial communities associated with the larval, parasitic juvenile, and adult life stages. The transition from larval to the parasitic juvenile stage was marked with a significant shift in bacterial community structure and reduction in alpha diversity. The most abundant SL-associated phyla were Proteobacteria, Fusobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Firmicutes, with their relative abundances varying among the stages. Moreover, while larval SL were enriched with unclassified Fusobacteriaceae, unclassified Verrucomicrobiales and Cetobacterium, members of the genera with fastidious nutritional requirements, such as Streptococcus, Haemophilus, Cutibacterium, Veillonella, and Massilia, were three to four orders of magnitude greater in juveniles than in larvae. In contrast, adult SLs were enriched with Aeromonas, Iodobacter, Shewanella, and Flavobacterium. Collectively, our findings show that bacterial communities in the SL gut are dramatically different among its life stages. Understanding how these communities change over time within and among SL life stages may shed more light on the role that these gut microbes play in host growth and fitness.

Influence of Filter Pore Size on Composition and Relative Abundance of Bacterial Communities and Select Host-Specific MST Markers in Coastal Waters of Southern Lake Michigan.

Water clarity is often the primary guiding factor in determining whether a prefiltration step is needed to increase volumes processed for a range of microbial endpoints. In this study, we evaluate the effect of filter pore size on the bacterial communities detected by 16S rRNA gene sequencing and incidence of two host-specific microbial source tracking (MST) markers in a range of coastal waters from southern Lake Michigan, using two independent data sets collected in 2015 (bacterial communities) and 2016-2017 (MST markers). Water samples were collected from river, shoreline, and offshore areas. For bacterial communities, each sample was filtered through a 5.0-µm filter, followed by filtration through a 0.22-µm filter, resulting in 70 and 143 filter pairs for bacterial communities and MST markers, respectively. Following DNA extraction, the bacterial communities were compared using 16S rRNA gene amplicons of the V3-V4 region sequenced on a MiSeq Illumina platform. Presence of human (Bacteroides HF183) and gull (Gull2, Catellicoccus marimammalium) host-specific MST markers were detected by qPCR. Actinobacteriota, Bacteroidota, and Proteobacteria, collectively represented 96.9% and 93.9% of the relative proportion of all phyla in the 0.22- and 5.0-µm pore size filters, respectively. There were more families detected in the 5.0-µm pore size filter (368) than the 0.22-µm (228). There were significant differences in the number of taxa between the two filter sizes at all levels of taxonomic classification according to linear discriminant analysis (LDA) effect size (LEfSe) with as many as 986 taxa from both filter sizes at LDA effect sizes greater than 2.0. Overall, the Gull2 marker was found in higher abundance on the 5.0-µm filter than 0.22 µm with the reverse pattern for the HF183 marker. This discrepancy could lead to problems with identifying microbial sources of contamination. Collectively, these results highlight the importance of analyzing pre- and final filters for a wide range of microbial endpoints, including host-specific MST markers routinely used in water quality monitoring programs. Analysis of both filters may increase costs but provides more complete genomic data via increased sample volume for characterizing microbial communities in coastal waters.

Evaluating the impacts of foreshore sand and birds on microbiological contamination at a freshwater beach.

Beaches along the Great Lakes shorelines are important recreational and economic resources. However, contamination at the beaches can threaten their usage during the swimming season, potentially resulting in beach closures and/or advisories. Thus, understanding the dynamics that control nearshore water quality is integral to effective beach management. There have been significant improvements in this effort, including incorporating modeling (empirical, mechanistic) in recent years. Mechanistic modeling frameworks can contribute to this understanding of dynamics by determining sources and interactions that substantially impact fecal indicator bacteria concentrations, an index routinely used in water quality monitoring programs. To simulate E. coli concentrations at Jeorse Park beaches in southwest Lake Michigan, a coupled hydrodynamic and wave-current interaction model was developed that progressively added contaminant sources from river inputs, avian presence, bacteria-sediment interactions, and bacteria-sand-sediment interactions. Results indicated that riverine inputs affected E. coli concentrations at Jeorse Park beaches only marginally, while avian, shoreline sand, and sediment sources were much more substantial drivers of E. coli contamination at the beach. By including avian and riverine inputs, as well as bacteria-sand-sediment interactions at the beach, models can reasonably capture the variability in observed E. coli concentrations in nearshore water and bed sediments at Jeorse Park beaches. Consequently, it will be crucial to consider avian contamination sources and water-sand-sediment interactions in effective management of the beach for public health and as a recreational resource and to extend these findings to similar beaches affected by shoreline embayment.

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.

Bacterial Community 16S rRNA Gene Sequencing Characterizes Riverine Microbial Impact on Lake Michigan.

Restoration of degraded aquatic habitats is critical to preserve and maintain ecosystem processes and economic viability. Effective restoration requires contaminant sources identification. Microbial communities are increasingly used to characterize fecal contamination sources. The objective was to determine whether nearshore and adjacent beach bacterial contamination originated from the Grand Calumet River, a highly urbanized aquatic ecosystem, and to determine if there were correlations between pathogens/feces associated bacteria in any of the samples to counts of the pathogen indicator species Escherichia coli. Water samples were collected from the river, river mouth, nearshore, and offshore sites along southern Lake Michigan. Comparisons among communities were made using beta diversity distances (weighted and unweighted Unifrac, and Bray Curtis) and Principal Coordinate Analysis of 16S rRNA gene Illumina sequence data that indicated river bacterial communities differed significantly from the river mouth, nearshore lake, and offshore lake samples. These differences were further supported using Source Tracker software that indicated nearshore lake communities differed significantly from river and offshore samples. Among locations, there was separation by sampling date that was associated with environmental factors (e.g., water and air temperature, water turbidity). Although about half the genera (48.1%) were common to all sampling sites, linear discriminant analysis effect size indicated there were several taxa that differed significantly among sites; there were significant positive correlations of feces-associated genera with E. coli most probable numbers. Results collectively highlight that understanding microbial communities, rather than relying solely on select fecal indicators with uncertain origin, are more useful for developing strategies to restore degraded aquatic habitats.

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.

Virulence and biodegradation potential of dynamic microbial communities associated with decaying Cladophora in Great Lakes.

Cladophora mats that accumulate and decompose along shorelines of the Great Lakes create potential threats to the health of humans and wildlife. The decaying algae create a low oxygen and redox potential environment favoring growth and persistence of anaerobic microbial populations, including Clostridium botulinum, the causal agent of botulism in humans, birds, and other wildlife. In addition to the diverse population of microbes, a dynamic chemical environment is generated, which involves production of numerous organic and inorganic substances, many of which are believed to be toxic to the sand and aquatic biotic communities. In this study, we used 16S-rDNA-based-amplicon sequencing and microfluidic-based quantitative PCR approaches to characterize the bacterial community structure and the abundances of human pathogens associated with Cladophora at different stages (up to 90days) of algal decay in laboratory microcosms. Oxygen levels were largely depleted after a few hours of incubation. As Cladophora decayed, the algal microbial biodiversity decreased within 24h, and the mat transitioned from an aerobic to anaerobic environment. There were increasing abundances of enteric and pathogenic bacteria during decomposition of Cladophora, including Acinetobacter, Enterobacter, Kluyvera, Cedecea, and others. In contrast, there were no or very few sequences (<0.07%) assigned to such groups in fresh Cladophora samples. Principal coordinate analysis indicated that the bacterial community structure was dynamic and changed significantly with decay time. Knowledge of microbial communities and chemical composition of decaying algal mats is critical to our further understanding of the role that Cladophora plays in a beach ecosystem's structure and function, including the algal role in trophic interactions. Based on these findings, public and environmental health concerns should be considered when decaying Cladophora mats accumulate Great Lakes shorelines.

U.S. Recreational Water Quality Criteria: A Vision for the Future.

This manuscript evaluates the U.S. Recreational Water Quality Criteria (RWQC) of 2012, based upon discussions during a conference held 11-13 March 2013, in Honolulu, Hawaii. The RWQC of 2012 did not meet expectations among the research community because key recommended studies were not completed, new data to assess risks to bathers exposed to non-point sources of fecal indicator bacteria (FIB) were not developed, and the 2012 RWQC did not show marked improvements in strategies for assessing health risks for bathers using all types of recreational waters. The development of the 2012 RWQC was limited in scope because the epidemiologic studies at beach sites were restricted to beaches with point sources of pollution and water samples were monitored for only enterococci. The vision for the future is development of effective RWQC guidelines based on epidemiologic and quantitative microbial risk assessment (QMRA) studies for sewage specific markers, as well as human enteric pathogens so that health risks for bathers at all recreational waters can be determined. The 2012 RWQC introduced a program for states and tribes to develop site-specific water quality criteria, and in theory this approach can be used to address the limitations associated with the measurements of the traditional FIB.

Prevalence of toxin-producing Clostridium botulinum associated with the macroalga Cladophora in three Great Lakes: growth and management.

The reemergence of avian botulism caused by Clostridium botulinum type E has been observed across the Great Lakes in recent years. Evidence suggests an association between the nuisance algae, Cladophora spp., and C. botulinum in nearshore areas of the Great Lakes. However, the nature of the association between Cladophora and C. botulinum is not fully understood due, in part, to the complex food web interactions in this disease etiology. In this study, we extensively evaluated their association by quantitatively examining population size and serotypes of C. botulinum in algal mats collected from wide geographic areas in lakes Michigan, Ontario, and Erie in 2011-2012 and comparing them with frequencies in other matrices such as sand and water. A high prevalence (96%) of C. botulinum type E was observed in Cladophora mats collected from shorelines of the Great Lakes in 2012. Among the algae samples containing detectable C. botulinum, the population size of C. Botulinum type E was 10(0)-10(4) MPN/g dried algae, which was much greater (up to 10(3) fold) than that found in sand or the water column, indicating that Cladophora mats are sources of this pathogen. Mouse toxinantitoxin bioassays confirmed that the putative C. botulinum belonged to the type E serotype. Steam treatment was effective in reducing or eliminating C. botulinum type E viable cells in Cladophora mats, thereby breaking the potential transmission route of toxin up to the food chain. Consequently, our data suggest that steam treatment incorporated with a beach cleaning machine may be an effective treatment of Cladophora-borne C. botulinum and may reduce bird mortality and human health risks.

Probabilistic analysis showing that a combination of Bacteroides and Methanobrevibacter source tracking markers is effective for identifying waters contaminated by human fecal pollution.

Microbial source tracking assays to identify sources of waterborne contamination typically target genetic markers of host-specific microorganisms. However, no bacterial marker has been shown to be 100% host-specific, and cross-reactivity has been noted in studies evaluating known source samples. Using 485 challenge samples from 20 different human and animal fecal sources, this study evaluated microbial source tracking markers including the Bacteroides HF183 16S rRNA, M. smithii nifH, and Enterococcus esp gene targets that have been proposed as potential indicators of human fecal contamination. Bayes' Theorem was used to calculate the conditional probability that these markers or a combination of markers can correctly identify human sources of fecal pollution. All three human-associated markers were detected in 100% of the sewage samples analyzed. Bacteroides HF183 was the most effective marker for determining whether contamination was specifically from a human source, and greater than 98% certainty that contamination was from a human source was shown when both Bacteroides HF183 and M. smithii nifH markers were present. A high degree of certainty was attained even in cases where the prior probability of human fecal contamination was as low as 8.5%. The combination of Bacteroides HF183 and M. smithii nifH source tracking markers can help identify surface waters impacted by human fecal contamination, information useful for prioritizing restoration activities or assessing health risks from exposure to contaminated waters.

Choices in recreational water quality monitoring: new opportunities and health risk trade-offs.

With the recent release of new recreational water quality monitoring criteria, there are more options for regulatory agencies seeking to protect beachgoers from waterborne pathogens. Included are methods that can reduce analytical time, providing timelier estimates of water quality, but the application of these methods has not been examined at most beaches for expectation of health risk and management decisions. In this analysis, we explore health and monitoring outcomes expected at Lake Michigan beaches using protocols for indicator bacteria including culturable Escherichia coli (E. coli; EC), culturable enterococci (ENT), and enterococci as analyzed by qPCR (QENT). Correlations between method results were generally high, except at beaches with historically high concentrations of EC. The "beach action value" was exceeded most often when using EC or ENT as the target indicator; QENT exceeded the limit far less frequently. Measured water quality between years was varied. Although methods with equivalent health expectation have been established, the lack of relationship among method outcomes and annual changes in mean indicator bacteria concentrations complicates the decision-making process. The monitoring approach selected by beach managers may be a combination of available tools that maximizes timely health protection, cost efficiency, and collaboration among beach jurisdictions.

Association of toxin-producing Clostridium botulinum with the macroalga Cladophora in the Great Lakes.

Avian botulism, a paralytic disease of birds, often occurs on a yearly cycle and is increasingly becoming more common in the Great Lakes. Outbreaks are caused by bird ingestion of neurotoxins produced by Clostridium botulinum, a spore-forming, gram-positive, anaerobe. The nuisance, macrophytic, green alga Cladophora (Chlorophyta; mostly Cladophora glomerata L.) is a potential habitat for the growth of C. botulinum. A high incidence of botulism in shoreline birds at Sleeping Bear Dunes National Lakeshore (SLBE) in Lake Michigan coincides with increasingly massive accumulations of Cladophora in nearshore waters. In this study, free-floating algal mats were collected from SLBE and other shorelines of the Great Lakes between June and October 2011. The abundance of C. botulinum in algal mats was quantified and the type of botulism neurotoxin (bont) genes associated with this organism were determined by using most-probable-number PCR (MPN-PCR) and five distinct bont gene-specific primers (A, B, C, E, and F). The MPN-PCR results showed that 16 of 22 (73%) algal mats from the SLBE and 23 of 31(74%) algal mats from other shorelines of the Great Lakes contained the bont type E (bont/E) gene. C. botulinum was present up to 15000 MPN per gram dried algae based on gene copies of bont/E. In addition, genes for bont/A and bont/B, which are commonly associated with human diseases, were detected in a few algal samples. Moreover, C. botulinum was present as vegetative cells rather than as dormant spores in Cladophora mats. Mouse toxin assays done using supernatants from enrichment of Cladophora containing high densities of C. botulinum (>1000 MPN/g dried algae) showed that Cladophora-borne C. botulinum were toxin-producing species (BoNT/E). Our results indicate that Cladophora provides a habitat for C. botulinum, warranting additional studies to better understand the relationship between this bacterium and the alga, and how this interaction potentially contributes to botulism outbreaks in birds.

Enterococci in the environment.

Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.

Evidence for occurrence, persistence, and growth potential of Escherichia coli and enterococci in Hawaii's soil environments.

High densities of Escherichia coli and enterococci are common in freshwaters on Oahu and other Hawaiian Islands. Soil along stream banks has long been suspected as the likely source of these bacteria; however, the extent of their occurrence and distribution in a wide range of soils remained unknown until the current investigation. Soil samples representing the seven major soil associations were collected on the island of Oahu and analyzed for fecal coliforms, E. coli, and enterococci by the most probable number method. Fecal coliforms, E. coli, and enterococci were found in most of the samples analyzed; log mean densities (MPN ± SE g soil⁻¹) were 1.96 ± 0.18, n=61; 1.21 ± 0.17, n=57; and 2.99 ± 0.12, n=62, respectively. Representative, presumptive cultures of E. coli and enterococci collected from the various soils were identified and further speciated using the API scheme; at least six species of Enterococcus, including Enterococcus faecalis and Enterococcus faecium, were identified. In mesocosm studies, E. coli and enterococci increased by 100-fold in 4 days, after mixing sewage-spiked soil (one part) with autoclaved soil (nine parts). E. coli remained metabolically active in the soil and readily responded to nutrients, as evidenced by increased dehydrogenase activity. Collectively, these findings indicate that populations of E. coli and enterococci are part of the natural soil microflora, potentially influencing the quality of nearby water bodies.

The population structure of Escherichia coli isolated from subtropical and temperate soils.

While genotypically-distinct naturalized Escherichia coli strains have been shown to occur in riparian soils of Lake Michigan and Lake Superior watersheds, comparative analyses of E. coli populations in diverse soils across a range of geographic and climatic conditions have not been investigated. The main objectives of this study were to: (a) examine the population structure and genetic relatedness of E. coli isolates collected from different soil types on a tropical island (Hawaii), and (b) determine if E. coli populations from Hawaii and temperate soils (Indiana, Minnesota) shared similar genotypes that may be reflective of biome-related soil conditions. DNA fingerprint and multivariate statistical analyses were used to examine the population structure and genotypic characteristics of the E. coli isolates. About 33% (98 of 293) of the E. coli from different soil types and locations on the island of Oahu, Hawaii, had unique DNA fingerprints, indicating that these bacteria were relatively diverse; the Shannon diversity index for the population was 4.03. Nearly 60% (171 of 293) of the E. coli isolates from Hawaii clustered into two major groups and the rest, with two or more isolates, fell into one of 22 smaller groups, or individual lineages. Multivariate analysis of variance of 89, 21, and 106 unique E. coli DNA fingerprints for Hawaii, Indiana, and Minnesota soils, respectively, showed that isolates formed tight cohesive groups, clustering mainly by location. However, there were several instances of clonal isolates being shared between geographically different locations. Thus, while nearly identical E. coli strains were shared between disparate climatologically- and geographically-distinct locations, a vast majority of the soil E. coli strains were genotypically diverse and were likely derived from separate lineages. This supports the hypothesis that these bacteria are not unique and multiple genotypes can readily adapt to become part of the soil autochthonous microflora.

Multi-scale temporal and spatial variation in genotypic composition of Cladophora-borne Escherichia coli populations in Lake Michigan.

High concentrations of Escherichia coli in mats of Cladophora in the Great Lakes have raised concern over the continued use of this bacterium as an indicator of microbial water quality. Determining the impacts of these environmentally abundant E. coli, however, necessitates a better understanding of their ecology. In this study, the population structure of 4285 Cladophora-borne E. coli isolates, obtained over multiple three day periods from Lake Michigan Cladophora mats in 2007-2009, was examined by using DNA fingerprint analyses. In contrast to previous studies that have been done using isolates from attached Cladophora obtained over large time scales and distances, the extensive sampling done here on free-floating mats over successive days at multiple sites provided a large dataset that allowed for a detailed examination of changes in population structure over a wide range of spatial and temporal scales. While Cladophora-borne E. coli populations were highly diverse and consisted of many unique isolates, multiple clonal groups were also present and accounted for approximately 33% of all isolates examined. Patterns in population structure were also evident. At the broadest scales, E. coli populations showed some temporal clustering when examined by year, but did not show good spatial distinction among sites. E. coli population structure also showed significant patterns at much finer temporal scales. Populations were distinct on an individual mat basis at a given site, and on individual days within a single mat. Results of these studies indicate that Cladophora-borne E. coli populations consist of a mixture of stable, and possibly naturalized, strains that persist during the life of the mat, and more unique, transient strains that can change over rapid time scales. It is clear that further study of microbial processes at fine spatial and temporal scales is needed, and that caution must be taken when interpolating short term microbial dynamics from results obtained from weekly or monthly samples.

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.

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.