Bacterial and viral fecal indicator predictive modeling at three Great Lakes recreational beach sites.

Coliphage are viruses that infect Escherichia coli (E. coli) and may indicate the presence of enteric viral pathogens in recreational waters. There is an increasing interest in using these viruses for water quality monitoring and forecasting; however, the ability to use statistical models to predict the concentrations of coliphage, as often done for cultured fecal indicator bacteria (FIB) such as enterococci and E. coli, has not been widely assessed. The same can be said for FIB genetic markers measured using quantitative polymerase chain reaction (qPCR) methods. Here we institute least-angle regression (LARS) modeling of previously published concentrations of cultured FIB (E. coli, enterococci) and coliphage (F+, somatic), along with newly reported genetic concentrations measured via qPCR for E. coli, enterococci, and general Bacteroidales. We develop site-specific models from measures taken at three beach sites on the Great Lakes (Grant Park, South Milwaukee, WI; Edgewater Beach, Cleveland, OH; Washington Park, Michigan City, IN) to investigate the efficacy of a statistical predictive modeling approach. Microbial indicator concentrations were measured in composite water samples collected five days per week over a beach season (∼15 weeks). Model predictive performance (cross-validated standardized root mean squared error of prediction [SRMSEP] and R2PRED) were examined for seven microbial indicators (using log10 concentrations) and water/beach parameters collected concurrently with water samples. Highest predictive performance was seen for qPCR-based enterococci and Bacteroidales models, with F+ coliphage consistently yielding poor performing models. Influential covariates varied by microbial indicator and site. Antecedent rainfall, bird abundance, wave height, and wind speed/direction were most influential across all models. Findings suggest that some fecal indicators may be more suitable for water quality forecasting than others at Great Lakes beaches.

Persistence and Decay of Fecal Microbiota in Aquatic Habitats.

Fecal microorganisms can enter water bodies in diverse ways, including runoff, sewage discharge, and direct fecal deposition. Once in water, the microorganisms experience conditions that are very different from intestinal habitats. The transition from host to aquatic environment may lead to rapid inactivation, some degree of persistence, or growth. Microorganisms may remain planktonic, be deposited in sediment, wash up on beaches, or attach to aquatic vegetation. Each of these habitats offers a panoply of different stressors or advantages, including UV light exposure, temperature fluctuations, salinity, nutrient availability, and biotic interactions with the indigenous microbiota (e.g., predation and/or competition). The host sources of fecal microorganisms are likewise numerous, including wildlife, pets, livestock, and humans. Most of these microorganisms are unlikely to affect human health, but certain taxa can cause waterborne disease. Others signal increased probability of pathogen presence, e.g., the fecal indicator bacteria Escherichia coli and enterococci and bacteriophages, or act as fecal source identifiers (microbial source tracking markers). The effects of environmental factors on decay are frequently inconsistent across microbial species, fecal sources, and measurement strategies (e.g., culture versus molecular). Therefore, broad generalizations about the fate of fecal microorganisms in aquatic environments are problematic, compromising efforts to predict microbial decay and health risk from contamination events. This review summarizes the recent literature on decay of fecal microorganisms in aquatic environments, recognizes defensible generalizations, and identifies knowledge gaps that may provide particularly fruitful avenues for obtaining a better understanding of the fates of these organisms in aquatic environments.

Comparison of somatic and F+ coliphage enumeration methods with large volume surface water samples.

Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique. Overall, D-HFUF-SAL outperformed other methods as it yielded the lowest frequency of non-detects [(ND); 10.8%] and the highest average concentrations of recovered coliphage for positive samples (2.51 ± 1.02 [standard deviation, SD] log10 plaque forming unit/liter (PFU/L) and 0.79 ± 0.71 (SD) log10 PFU/L for somatic and F+, respectively). M-SAL yielded 29.7% ND and average concentrations of 2.26 ± 1.15 (SD) log10 PFU/L (somatic) and 0.59 ± 0.82 (SD) log10 PFU/L (F+). DMF performance was inferior to D-HFUF-SAL and M-SAL methods (ND of 65.6%; average somatic coliphage concentration 1.52 ± 1.32 [SD] log10 PFU/L, no F+ detected), indicating this procedure is unsuitable for 1 L surface water sample volumes. This study represents an important step toward the use of a coliphage method for recreational water quality criteria purposes.

Incidence of somatic and F+ coliphage in Great Lake Basin recreational waters.

There is a growing interest for the use of coliphage as an alternative indicator to assess fecal pollution in recreational waters. Coliphage are a group of viruses that infect Escherichia coli and are considered as potential surrogates to infer the likely presence of enteric viral pathogens. We report the use of a dead-end hollow fiber ultrafiltration single agar layer method to enumerate F+ and somatic coliphage from surface waters collected from three Great Lake areas. At each location, three sites (two beaches; one river) were sampled five days a week over the 2015 beach season (n = 609 total samples). In addition, culturable E. coli and enterococci concentrations, as well as 16 water quality and recreational area parameters were assessed such as rainfall, turbidity, dissolved oxygen, pH, and ultra violet absorbance. Overall, somatic coliphage levels ranged from non-detectable to 4.39 log10 plaque forming units per liter and were consistently higher compared to F+ (non-detectable to 3.15 log10 PFU/L), regardless of sampling site. Coliphage concentrations weakly correlated with cultivated fecal indicator bacteria levels (E. coli and enterococci) at 75% of beach sites tested in study (r = 0.28 to 0.40). In addition, ultraviolet light absorption and water temperature were closely associated with coliphage concentrations, but not fecal indicator bacteria levels suggesting different persistence trends in Great Lake waters between indicator types (bacteria versus virus). Finally, implications for coliphage water quality management and future research directions are discussed.

Proteomic and Mutant Analysis of the CO2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena.

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.

Differential decomposition of bacterial and viral fecal indicators in common human pollution types.

Understanding the decomposition of microorganisms associated with different human fecal pollution types is necessary for proper implementation of many water quality management practices, as well as predicting associated public health risks. Here, the decomposition of select cultivated and molecular indicators of fecal pollution originating from fresh human feces, septage, and primary effluent sewage in a subtropical marine environment was assessed over a six day period with an emphasis on the influence of ambient sunlight and indigenous microbiota. Ambient water mixed with each fecal pollution type was placed in dialysis bags and incubated in situ in a submersible aquatic mesocosm. Genetic and cultivated fecal indicators including fecal indicator bacteria (enterococci, E. coli, and Bacteroidales), coliphage (somatic and F+), Bacteroides fragilis phage (GB-124), and human-associated genetic indicators (HF183/BacR287 and HumM2) were measured in each sample. Simple linear regression assessing treatment trends in each pollution type over time showed significant decay (p ≤ 0.05) in most treatments for feces and sewage (27/28 and 32/40, respectively), compared to septage (6/26). A two-way analysis of variance of log10 reduction values for sewage and feces experiments indicated that treatments differentially impact survival of cultivated bacteria, cultivated phage, and genetic indicators. Findings suggest that sunlight is critical for phage decay, and indigenous microbiota play a lesser role. For bacterial cultivated and genetic indicators, the influence of indigenous microbiota varied by pollution type. This study offers new insights on the decomposition of common human fecal pollution types in a subtropical marine environment with important implications for water quality management applications.

Effects of field storage method on E. coli concentrations measured in storm water runoff.

Storm water runoff is increasingly assessed for fecal indicator organisms (e.g., Escherichia coli, E. coli) and its impact on contact recreation. Concurrently, use of autosamplers along with logistic, economic, technical, and personnel barriers is challenging conventional protocols for sample holding times and storage conditions in the field. A common holding time limit for E. coli is 8 h with a 10 °C storage temperature, but several research studies support longer hold time thresholds. The use of autosamplers to collect E. coli water samples has received little field research attention; thus, this study was implemented to compare refrigerated and unrefrigerated autosamplers and evaluate potential E. coli concentration differences due to field storage temperature (storms with holding times ≤24 h) and due to field storage time and temperature (storms >24 h). Data from 85 runoff events on four diverse watersheds showed that field storage times and temperatures had minor effects on mean and median E. coli concentrations. Graphs and error values did, however, indicate a weak tendency for higher concentrations in the refrigerated samplers, but it is unknown to what extent differing die-off and/or regrowth rates, heterogeneity in concentrations within samples, and laboratory analysis uncertainty contributed to the results. The minimal differences in measured E. coli concentrations cast doubt on the need for utilizing the rigid conventional protocols for field holding time and storage temperature. This is not to say that proper quality assurance and quality control is not important but to emphasize the need to consider the balance between data quality and practical constraints related to logistics, funding, travel time, and autosampler use in storm water studies.

Protozoan predation is differentially affected by motility of enteric pathogens in water vs. sediments.

Survival of enteric bacteria in aquatic habitats varies depending upon species, strain, and environmental pressures, but the mechanisms governing their fate are poorly understood. Although predation by protozoa is a known, top-down control mechanism on bacterial populations, its influence on the survival of fecal-derived pathogens has not been systematically studied. We hypothesized that motility, a variable trait among pathogens, can influence predation rates and bacterial survival. We compared the survival of two motile pathogens of fecal origin by culturing Escherichia coli O157 and Salmonella enterica Typhimurium. Each species had a motile and non-motile counterpart and was cultured in outdoor microcosms with protozoan predators (Tetrahymena pyriformis) present or absent. Motility had a significant, positive effect on S. enterica levels in water and sediment in the presence or absence of predators. In contrast, motility had a significant negative effect on E. coli O157 levels in sediment, but did not affect water column levels. The presence/absence of protozoa consistently accounted for a greater proportion of the variability in bacterial levels (>95 %) than in bacterial motility (<4 %) in the water column. In sediments, however, motility was more important than predation for both bacteria. Calculations of total CFU/microcosm showed decreasing bacterial concentrations over time under all conditions except for S. enterica in the absence of predation, which increased ∼0.5-1.0 log over 5 days. These findings underscore the complexity of predicting the survival of enteric microorganisms in aquatic habitats, which has implications for the accuracy of risk assessment and modeling of water quality.

Indigenous microbiota and habitat influence Escherichia coli survival more than sunlight in simulated aquatic environments.

The reported fate of Escherichia coli in the environment ranges from extended persistence to rapid decline. Incomplete understanding of factors that influence survival hinders risk assessment and modeling of the fate of fecal indicator bacteria (FIB) and pathogens. FIB persistence in subtropical aquatic environments was explored in outdoor mesocosms inoculated with five E. coli strains. The manipulated environmental factors were (i) presence or absence of indigenous microbiota (attained by natural, disinfected, and cycloheximide treatments), (ii) freshwater versus seawater, and (iii) water column versus sediment matrices. When indigenous microbes were removed (disinfected), E. coli concentrations decreased little despite exposure to sunlight. Conversely, under conditions that included the indigenous microbiota (natural), significantly greater declines in E. coli occurred regardless of the habitat. The presence of indigenous microbiota and matrix significantly influenced E. coli decline, but their relative importance differed in freshwater versus seawater. Cycloheximide, which inhibits protein synthesis in eukaryotes, significantly diminished the magnitude of E. coli decline in water but not in sediments. The inactivation of protozoa and bacterial competitors (disinfected) caused a greater decline in E. coli than cycloheximide alone in water and sediments. These results indicate that the autochthonous microbiota are an important contributor to the decline of E. coli in fresh and seawater subtropical systems, but their relative contribution is habitat dependent. This work advances our understanding of how interactions with autochthonous microbiota influence the fate of E. coli in aquatic environments and provides the framework for studies of the ecology of enteric pathogens and other allochthonous bacteria in similar environments.

The influence of predation and competition on the survival of commensal and pathogenic fecal bacteria in aquatic habitats.

The role of fecal indicator bacteria (FIB) in water quality assessment is to provide a warning of the increased risk of pathogen presence. An effective surrogate for waterborne pathogens would have similar survival characteristics in aquatic environments. Although the effect of abiotic factors such as sunlight and salinity on the survival of FIB and pathogens are becoming better understood, the effect of the indigenous microbiota is not well characterized. The influence of biotic factors on the survival of non-pathogenic Escherichia coli, Enterococcus faecalis, and E. coli O157:H7 were compared in fresh (river) water and sediments over 5 days. Treatments were (i) disinfection (filtration of water and baking of sediments) to remove indigenous protozoa (predators) and bacteria (competitors), and (ii) kanamycin treatment to reduce competition from indigenous bacteria. The disinfection treatment significantly increased survival of E. coli, E. coli O157:H7 and Ent. faecalis in the water column. In sediments, survival of FIB but not that of E. coli O157:H7 increased in disinfected treatments, indicating that the pathogen's survival was unaffected by the natural microbiota. Location (water or sediment) influenced bacterial survival more than species/type in the disinfection experiment. In the competition experiments where only the natural bacterial flora was manipulated, the addition of kanamycin did not affect the survival of Ent. faecalis, but resulted in greater survival of E. coli in water and sediment. Species/type influenced survival more than the level of competition in this experiment. This study demonstrates the complexity of interactions of FIB and pathogens with indigenous microbiota and location in aquatic habitats, and argues against over-generalizing conclusions derived from experiments restricted to a particular organism or habitat.

Characterization of Fe(II) oxidizing bacterial activities and communities at two acidic Appalachian coalmine drainage-impacted sites.

We characterized the microbiologically mediated oxidative precipitation of Fe(II) from coalmine-derived acidic mine drainage (AMD) along flow-paths at two sites in northern Pennsylvania. At the Gum Boot site, dissolved Fe(II) was efficiently removed from AMD whereas minimal Fe(II) removal occurred at the Fridays-2 site. Neither site received human intervention to treat the AMD. Culturable Fe(II) oxidizing bacteria were most abundant at sampling locations along the AMD flow path corresponding to greatest Fe(II) removal and where overlying water contained abundant dissolved O(2). Rates of Fe(II) oxidation determined in laboratory-based sediment incubations were also greatest at these sampling locations. Ribosomal RNA intergenic spacer analysis and sequencing of partial 16S rRNA genes recovered from sediment bacterial communities revealed similarities among populations at points receiving regular inputs of Fe(II)-rich AMD and provided evidence for the presence of bacterial lineages capable of Fe(II) oxidation. A notable difference between bacterial communities at the two sites was the abundance of Chloroflexi-affiliated 16S rRNA gene sequences in clone libraries derived from the Gum Boot sediments. Our results suggest that inexpensive and reliable AMD treatment strategies can be implemented by mimicking the conditions present at the Gum Boot field site.