[show abstract][hide abstract] ABSTRACT: Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.
[show abstract][hide abstract] ABSTRACT: Molecular microbial community analyses provide information on thousands of microorganisms simultaneously, and integrate biotic and abiotic perturbations caused by fecal contamination entering water bodies. A few studies have explored community methods as emerging approaches for microbial source tracking (MST), however, an evaluation of the current state of this approach is lacking. Here, we utilized three types of community-based methods with 64 blind, single- or dual-source, challenge samples generated from 12 sources, including: humans (feces), sewage, septage, dogs, pigs, deer, horses, cows, chickens, gulls, pigeons, and geese. Each source was a composite from multiple donors from four representative geographical regions in California. Methods evaluated included terminal restriction fragment polymorphism (TRFLP), phylogenetic microarray (PhyloChip), and next generation (Illumina) sequencing. These methods correctly identified dominant (or sole) sources in over 90% of the challenge samples, and exhibited excellent specificity regardless of source, rarely detecting a source that was not present in the challenge sample. Sensitivity, however, varied with source and community analysis method. All three methods distinguished septage from human feces and sewage, and identified deer and horse with 100% sensitivity and 100% specificity. Method performance improved if the composition of blind dual-source reference samples were defined by DNA contribution of each single source within the mixture, instead of by Enterococcus colony forming units. Data analysis approach also influenced method performance, indicating the need to standardize data interpretation. Overall, results of this study indicate that community analysis methods hold great promise as they may be used to identify any source, and they are particularly useful for sources that currently do not have, and may never have, a source-specific single marker gene.
[show abstract][hide abstract] ABSTRACT: Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.
[show abstract][hide abstract] ABSTRACT: Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters that rely on the association of certain fecal microorganisms with a particular host. MST is used to assess recreational water quality and associated human health risk, and total maximum daily load (TMDL) allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microbes. Human sewage pollution is among the greatest concerns for human health due to 1) the known risk of exposure to human waste, and 2) the public and regulatory will to reduce sewage pollution; however, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves. Here, we review the performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR). Relationships among human-associated MST markers, fecal indicator bacteria, pathogens, and human health outcomes are presented along with recommendations for future research. An integrated understanding of the advantages and drawbacks of the many MST methods targeting human sources advanced over the past several decades will benefit managers, regulators, researchers, and other users of this rapidly growing area of environmental microbiology. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: In 2012, the U.S. EPA suggested that coastal and Great Lakes states adopt enterococci as an alternative indicator for monitoring recreational water quality. Limited information, however, is known about the presence and persistence of enterococci in Lake Superior. In this study the density, species composition, and persistence of enterococci in sand, sediment, water, and soil samples was examined at two sites in a Lake Superior watershed, from May to September over a 2 year period. The genetic diversity of E. faecalis isolates collected from environmental samples was also studied by using the HFERP DNA fingerprinting technique. Results obtained by MPN analyses indicated that enterococci were present in 149 of 159 samples (94%) and their densities were generally higher in the summer than in the other months examined. Enterococcus species composition displayed spatial and temporal changes, with the dominant species being E. hirae, E. faecalis, E. faecium, E. mundtii, and E. casseliflavus. DNA fingerprint analyses indicated that the E. faecalis population in the watershed was genetically diverse, and changed spatially and temporally. Moreover, some DNA fingerprints re-occurred over multiple sampling events. Taken together, these results suggest that some enterococci are able to persist and grow in the Lake Superior watershed, especially in soils, for a prolonged time after being introduced.
Applied and environmental microbiology 03/2013; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Escherichia coli, an important fecal indicator bacterium, is known to persist and reproduce in association with Cladophora and other macrophytic algae and environmental substrates. Recent increases in the growth of Cladophora and other macrophytic algae in many of the Laurentian Great Lakes result in the accumulation of large amounts of algal biomass along the shoreline and on beaches. While the Cladophora–E. coli association may pose substantial public health risks, detailed laboratory-based studies have not been done to investigate the bases of the interaction. This is due, in large part, to past inabilities to culture many macrophytic algae under near axenic conditions. Here we describe the development and experimental use of laboratory microcosms to study the synergistic interaction and growth of the green alga Pithophora, a close relative of Cladophora, with environmental E. coli. In the absence of exogenous organic carbon supply, the E. coli population attached to algal filaments increased approximately three orders of magnitude within 72 h of inoculation. Growth of E. coli on Pithophora appeared to be limited by dissolved nitrogen with a concentration of ≥ 66 μg/mL N allowing maximal bacterial growth. In contrast, an environmental strain of Salmonella did not grow under identical conditions in the microcosms, suggesting that this bacterium requires additional growth factors not provided by Cladophora. Since the alga can be maintained in the laboratory for long periods of time, this system allows for further experimentation and understanding of macroalga–microbe interactions.
Journal of Great Lakes Research 06/2012; 38(2):390–395. · 2.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Water Research 01/2011; 45(2):721-31. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Elevated concentrations of fecal indicator bacteria (FIB) in aquatic sediments and vegetation have prompted concern that environmental reservoirs of FIB disrupt the correlation between indicator organisms, pathogens and human health risks. FIB numbers, however, are typically normalized to volume of water or mass of substrate. Because these reservoirs tend to differ greatly in magnitude within and between water bodies, direct comparison between water column and benthic population sizes can be problematic. Normalization to a set volume of water or mass of substrate, e.g. cfu (100 ml)(-1) or cfu(100 g)(-1), can give a false picture of the relative contributions of various reservoirs to FIB numbers across the ecosystem, and of the potential for FIBs to trigger health advisories as they pass from one reservoir to another. Here, we normalized enterococci concentrations from water, sediment and submerged aquatic vegetation (SAV) to land surface area (m(2) ) to compare their relative importance in the entire system. SAV-associated enterococci comprised only 0-18% of the entire population, even though they displayed the highest concentrations of enterococci per unit mass. The largest proportion of the enterococci population was in the water column (4-77%) or sediments (20-95%), depending on the volume of each substrate available at a site and FIB concentrations within them. Models indicated that large shifts in the relative size of FIB populations in each substrate can result from changes in per cent SAV cover, water depth and depth of sediment colonization. It follows that high concentrations of FIB in sediments or SAV do not necessarily signify large environmental reservoirs of FIB that can affect the water column. Comprehensive analyses that include FIB measurements from water, SAV and sediment normalized to land surface area offer a more balanced perspective on total FIB numbers contained in various matrices of an aquatic system.
[show abstract][hide abstract] ABSTRACT: Recent evidence of extended survival of fecal indicator bacteria in sediments and submerged aquatic vegetation (SAV) has raised concerns about using indicator bacteria to reliably detect fecal contamination. We monitored enterococci densities and population structure in water, sediment and SAV simultaneously at sites across a subtropical watershed (Tampa Bay, FL, USA) over one year to determine the extent to which each matrix serves as a potential reservoir of enterococci. SAV harbored significantly higher mean densities of enterococci than sediments, which harbored higher densities than water. Mean enterococci densities were also greater at sites located further upstream in the watershed. The population structure assessed by BOX-PCR genotyping was relatively dissimilar in each sample, although some similarities among samples suggested grouping by location. Strain diversity ranged from very high to negligible, with lowest overall diversity in lake samples taken during the summer. Several strains were highly abundant and cosmopolitan (found across sites, seasons, and matrices) and were identified by 16S rRNA gene sequencing as the Enterococcus species casseliflavus, faecalis, faecium, hirae, and mundtii. The proportional dominance of certain strains suggests the existence of persistent and possibly naturalized indicator bacteria populations that are not directly related to pollution events.
Water Research 12/2010; 44(20):5857-66. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Enterococcus spp. are utilized worldwide as faecal indicator bacteria, but certain strains exhibit extended survival in environmental habitats and the factors influencing their persistence are poorly understood. We used flowing freshwater mesocosms to explore the effect of submerged aquatic vegetation (SAV) on the persistence of natural enterococci populations from a subtropical lake. The highest mean densities of culturable enterococci over 2 weeks occurred in SAV [8.6 x 10(2) colony-forming units (cfu) per 100 g wet weight], followed by sediments (1.3 x 10(2) cfu per 100 g) and water (18 cfu per 100 ml). However, due to relative differences in the total mass of each substrate in the entire system (water > sediments > SAV), SAV-associated enterococci represented only a minor proportion of the total population. Vegetated mesocosms harboured significantly higher mean cfu per mesocosm and cfu densities in sediments compared with their unvegetated counterparts, suggesting that SAV indirectly facilitates persistence in aquatic habitats. Populations were dominated (> 96%) by a single Enterococcus casseliflavus strain according to BOX-PCR genotyping, which did not change over the 10-month study and strongly suggests bacterial replication in the lake. The presence of such strains in the environment may represent highly competitive, naturalized and reproducing indicator bacteria populations that are not directly related to pollution events.