Brian D. Badgley

Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes

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 allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microorganisms. 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.

Soil bacterial and fungal communities show distinct recovery patterns during forest ecosystem restoration

ABSTRACT Bacteria and fungi are important mediators of biogeochemical processes and play essential roles in the establishment of plant communities, which makes knowledge about their recovery after extreme disturbances valuable for understanding ecosystem development. However, broad ecological differences between bacterial and fungal organisms, such as growth rates, stress tolerance, and substrate utilization, suggest they could follow distinct trajectories and show contrasting dynamics during recovery. In this study, we analyzed both the intra-annual variability and decade-scale recovery of bacterial and fungal communities in a chronosequence of reclaimed mined soils using next-generation sequencing to quantify their abundance, richness, β-diversity, taxonomic composition, and cooccurrence network properties. Bacterial communities shifted gradually, with overlapping β-diversity patterns across chronosequence ages, while shifts in fungal communities were more distinct among different ages. In addition, the magnitude of intra-annual variability in bacterial β-diversity was comparable to the changes across decades of chronosequence age, while fungal communities changed minimally across months. Finally, the complexity of bacterial cooccurrence networks increased with chronosequence age, while fungal networks did not show clear age-related trends. We hypothesize that these contrasting dynamics of bacteria and fungi in the chronosequence result from (i) higher growth rates for bacteria, leading to higher intra-annual variability; (ii) higher tolerance to environmental changes for fungi; and (iii) stronger influence of vegetation on fungal communities. IMPORTANCE Both bacteria and fungi play essential roles in ecosystem functions, and information about their recovery after extreme disturbances is important for understanding whole-ecosystem development. Given their many differences in phenotype, phylogeny, and life history, a comparison of different bacterial and fungal recovery patterns improves the understanding of how different components of the soil microbiota respond to ecosystem recovery. In this study, we highlight key differences between soil bacteria and fungi during the restoration of reclaimed mine soils in the form of long-term diversity patterns, intra-annual variability, and potential interaction networks. Cooccurrence networks revealed increasingly complex bacterial community interactions during recovery, in contrast to much simpler and more isolated fungal network patterns. This study compares bacterial and fungal cooccurrence networks and reveals cooccurrences persisting through successional ages.

Storm loads of culturable and molecular fecal indicators in an inland urban stream.

Elevated concentrations of fecal indicator bacteria in receiving waters during wet-weather flows are a considerable public health concern that is likely to be exacerbated by future climate change and urbanization. Knowledge of factors driving the fate and transport of fecal indicator bacteria in stormwater is limited, and even less is known about molecular fecal indicators, which may eventually supplant traditional culturable indicators. In this study, concentrations and loading rates of both culturable and molecular fecal indicators were quantified throughout six storm events in an instrumented inland urban stream. While both concentrations and loading rates of each fecal indicator increased rapidly during the rising limb of the storm hydrographs, it is the loading rates rather than instantaneous concentrations that provide a better estimate of transport through the stream during the entire storm. Concentrations of general fecal indicators (both culturable and molecular) correlated most highly with each other during storm events but not with the human-associated HF183 Bacteroides marker. Event loads of general fecal indicators most strongly correlated with total runoff volume, maximum discharge, and maximum turbidity, while event loads of HF183 most strongly correlated with the time to peak flow in a hydrograph. These observations suggest that collection of multiple samples during a storm event is critical for accurate predictions of fecal indicator loading rates and total loads during wet-weather flows, which are required for effective watershed management. In addition, existing predictive models based on general fecal indicators may not be sufficient to predict source-specific genetic markers of fecal contamination.

Hydrometeorological and Physicochemical Drivers of Fecal Indicator Bacteria in Urban Stream Bottom Sediments

High levels of fecal indicator bacteria (FIB) are the leading cause of surface water quality impairments in the United States. Watershed-scale models are commonly used to identify relative contributions of watershed sources and to evaluate the effectiveness of remediation strategies. However, most existing models simplify FIB transport behavior as equivalent to that of dissolved-phase contaminants, ignoring the impacts of sediment on the fate and transport of FIB. Implementation of sediment-related processes within existing models is limited by minimal available monitoring data on sediment FIB concentrations for model development, calibration, and validation purposes. The purpose of the present study is to evaluate FIB levels in the streambed sediments as compared to those in the water column and to identify environmental variables that influence water and underlying sediment FIB levels. Concentrations of and enterococci in the water column and sediments of an urban stream were monitored weekly for 1 yr and correlated with a variety of potential hydrometeorological and physicochemical variables. Increased FIB concentrations in both the water column and sediments were most strongly correlated with increased antecedent 24-h rainfall, increased stream water temperature, decreased dissolved oxygen, and decreased specific conductivity. These observations will support future efforts to incorporate sediment-related processes in existing models through the identification of key FIB relationships with other model inputs, and the provision of sediment FIB concentrations for direct model calibration. In addition, identified key variables can be used in quick evaluation of the effectiveness of potential remediation strategies.

Exposure to dairy manure leads to greater antibiotic resistance and increased mass-specific respiration in soil microbial communities

Intensifying livestock production to meet the demands of a growing global population coincides with increases in both the administration of veterinary antibiotics and manure inputs to soils. These trends have the potential to increase antibiotic resistance in soil microbial communities. The effect of maintaining increased antibiotic resistance on soil microbial communities and the ecosystem processes they regulate is unknown. We compare soil microbial communities from paired reference and dairy manure-exposed sites across the USA. Given that manure exposure has been shown to elicit increased antibiotic resistance in soil microbial communities, we expect that manure-exposed sites will exhibit (i) compositionally different soil microbial communities, with shifts toward taxa known to exhibit resistance; (ii) greater abundance of antibiotic resistance genes; and (iii) corresponding maintenance of antibiotic resistance would lead to decreased microbial efficiency. We found that bacterial and fungal communities differed between reference and manure-exposed sites. Additionally, the β-lactam resistance gene ampC was 5.2-fold greater under manure exposure, potentially due to the use of cephalosporin antibiotics in dairy herds. Finally, ampC abundance was positively correlated with indicators of microbial stress, and microbial mass-specific respiration, which increased 2.1-fold under manure exposure. These findings demonstrate that the maintenance of antibiotic resistance associated with manure inputs alters soil microbial communities and ecosystem function.

Long-term performance and microbial community characterization of an osmotic anammox system for removing reverse-fluxed ammonium.

A novel osmotic anammox (OsAMX) system coupling nitritation-anammox with forward osmosis (FO) has been developed for removal of reverse-fluxed ammonium when using NH4HCO3 as a draw solute. In this study, long-term performance and microbial community structure were investigated. The nitritation-anammox reactor maintained an ammonium concentration of 7.0±5.0mgNL(-1) (DO=0.9±0.2mgO2L(-1)), while the FO achieved a water flux of 2.3±0.4LMH (0.5M NH4HCO3 draw). The low water flux was obtained likely due to concentration polarization, reverse salt flux (RSF) and membrane fouling. Sequencing analyses reveled that Candidatus Jettenia was the dominant anammox genus, while Candidatus Brocadia was most abundant in biofilm. The shift of anammox bacterial population indicated possible higher tolerance of Ca. Brocadia for DO or elevated RSF. These results encourage further investigation of OsAMX system optimization, membrane fouling migration strategies, and application with actual wastewater.

Measurement and modeling of denitrification in sand-bed streams under various land uses

Although many studies have measured denitrification in stream sediments, few have utilized these data with local water column and sediment measurements to develop a predictive model for NO uptake. In this study, sediment denitrification was measured from cores in five streams under various land uses in south-central Minnesota using denitrification enzyme activity (DEA) assays and amplification of the gene via real-time, quantitative polymerase chain reaction. Hydraulic and environmental variables were measured in the vicinity of the sediment cores to evaluate the influence of fluid flow and chemical variables on denitrification activity. Potential denitrification rates measured using DEA assays ranged from 0.02 to 10.1 mg N m h, and the abundance of the denitrifier gene was positively correlated with these measurements ( = 0.79, < 0.001) for most of the streams studied. A predictive model to determine NO uptake via denitrification was derived, implementing dimensional analysis of variables that mediate denitrification in sand-bed streams. The proposed model explained 75% of the variability in DEA rates. The results of this study show that denitrification is most dependent on the distribution of sediment organic matter, interstitial pore space, and stream hydraulic characteristics, including shear velocity at the sediment-water interface and stream depth.

Unravelling and Reconstructing the Nexus of Salinity, Electricity and Microbial Ecology for Bioelectrochemical Desalination

Microbial desalination cells (MDCs) are an emerging concept for simultaneous water/wastewater treatment and energy recovery. The key to developing MDCs is to understand fundamental problems, such as the effects of salinity on system performance and the role of microbial community and functional dynamics. Herein, a tubular MDC was operated under a wide range of salt concentrations (0.05-4 M), and the salinity effects were comprehensively examined. The MDC generated higher current with higher salt concentrations in the desalination chamber. When fed with 4 M NaCl, the MDC achieve a current density of 300 A m-3 (anode volume), which was one of the highest among bioelectrochemical system studies. Community analysis and electrochemical measurements suggested that electrochemically active bacteria Pseudomonas and Acinetobacter transferred electrons extracellularly via electron shuttles, and the consequent ion migration led to high anode salinities and conductivity that favored their dominance. Predictive functional dynamics and Bayesian networks implied that the taxa putatively not capable of extracellular electron transfer (e.g., Bacteroidales and Clostridiales) might indirectly contribute to bioelectrochemical desalination. By integrating the Bayesian network with logistic regression, current production was successfully predicted from taxonomic data. This study has demonstrated uncompromised system performance under high salinity and thus has highlighted the potential of MDCs as an energy-efficient technology to address water-energy challenges. The statistical modeling approach developed in this study represents a significant step toward understating microbial communities and predicting system performance in engineered biological systems.

Vancomycin resistant enterococci and bacterial community structure following a sewage spill into an aquatic environment

ABSTRACT Sewage spills can release antibiotic-resistant bacteria into surface waters, contributing to environmental reservoirs and potentially impacting human health. Vancomycin-resistant enterococci (VRE) are nosocomial pathogens that have been detected in environmental habitats, including soil, water, and beach sands, as well as wildlife feces. However, VRE harboring vanA genes that confer high-level resistance have infrequently been found outside clinical settings in the United States. This study found culturable Enterococcus faecium harboring the vanA gene in water and sediment for up to 3 days after a sewage spill, and the quantitative PCR (qPCR) signal for vanA persisted for an additional week. Culturable levels of enterococci in water exceeded recreational water guidelines for 2 weeks following the spill, declining about five orders of magnitude in sediments and two orders of magnitude in the water column over 6 weeks. Analysis of bacterial taxa via 16S rRNA gene sequencing showed changes in community structure through time following the sewage spill in sediment and water. The spread of opportunistic pathogens harboring high-level vancomycin resistance genes beyond hospitals and into the broader community and associated habitats is a potential threat to public health, requiring further studies that examine the persistence, occurrence, and survival of VRE in different environmental matrices. IMPORTANCE Vancomycin-resistant enterococci (VRE) are harmful bacteria that are resistant to the powerful antibiotic vancomycin, which is used as a last resort against many infections. This study followed the release of VRE in a major sewage spill and their persistence over time. Such events can act as a means of spreading vancomycin-resistant bacteria in the environment, which can eventually impact human health.

Tracking the downstream impacts of inadequate sanitation in central Appalachia

Poor sanitation in rural infrastructure is often associated with high levels of fecal contamination in adjacent surface waters, which presents a community health risk. Although microbial source tracking techniques have been widely applied to identify primary remediation needs in urban and/or recreational waters, use of human-specific markers has been more limited in rural watersheds. This study quantified the human source tracking marker Bacteroides-HF183, along with more general fecal indicators (i.e. culturable Escherichia coli and a molecular Enterococcus marker), in two Appalachian streams above and below known discharges of untreated household waste. Although E. coli and Enterococcus were consistently recovered in samples collected from both streams, Bacteroides-HF183 was only detected sporadically in one stream. Multiple linear regression analysis demonstrated a positive correlation between the concentration of E. coli and the proximity and number of known waste discharge points upstream; this correlation was not significant with respect to Bacteroides-HF183, likely due to the low number of quantifiable samples. These findings suggest that, while the application of more advanced source targeting strategies can be useful in confirming the influence of substandard sanitation on surface waters to justify infrastructure improvements, they may be of limited use without concurrent traditional monitoring targets and on-the-ground sanitation surveys.