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USGS2004

Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10 6 cells filter -1 . In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10 5 , the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.

Applied and Environmental Microbiology↗

USGS2005

Comparison of Bacteroides-Prevotella 16S rRNA genetic markers for fecal samples from different animal species

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

Applied and Environmental Microbiology↗

USGS2005

Chloroethene biodegradation in sediments at 4°C

Microbial reductive dechlorination of [1,2- 14 C]trichloroethene to [ 14 C] cis -dichloroethene and [ 14 C]vinyl chloride was observed at 4°C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2- 14 C] cis -dichloroethene and [1,2- 14 C]vinyl chloride to 14 CO 2 also was observed under these conditions.

Applied and Environmental Microbiology↗

USGS2005

A method adapting microarray technology for signature tagged mutagenesis of Dusulfovibrio dusulfuricans G20 and Shewanella oneidensis MR-1 in anaerobic sediment survival experiments

Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn 10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.

Applied and Environmental Microbiology↗

USGS2005

Effect of the oxidation rate and Fe(II) state on microbial nitrate-dependent Fe(III) mineral formation

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to Klebsiella oxytoca. FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II) sol ] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II) sol yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II) sol and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.

Applied and Environmental Microbiology↗

USGS2010

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

Letters in Applied Microbiology↗

USGS2011

The fecal bacteria

The Fecal Bacteria offers a balanced, integrated discussion of fecal bacteria and their presence and ecology in the intestinal tract of mammals, in the environment, and in the food supply. This new volume covers their use in examining and assessing water quality in order to offer protection from illnesses related to swimming in or ingesting contaminated water, in addition to discussing their use in engineering considerations of water quality, modeling, monitoring, and regulations. Fecal bacteria are additionally used as indicators of contamination of ready-to-eat foods and fresh produce. The intestinal environment, the microbial community structure of the gut microbiota, and the physiology and genomics of this broad group of microorganisms are explored in the book. With contributions from an internationally recognized group of experts, the book integrates medicine, public health, environmental, and microbiological topics in order to provide a unique, holistic understanding of fecal bacteria. Moreover, it shows how the latest basic science and applied research findings are helping to solve problems and develop effective management strategies. For example, readers will discover how the latest tools and molecular approaches have led to our current understanding of fecal bacteria and enabled us to improve human health and water quality. The Fecal Bacteria is recommended for microbiologists, clinicians, animal scientists, engineers, environmental scientists, food safety experts, water quality managers, and students. It will help them better understand fecal bacteria and use their knowledge to protect human and environmental health. They can also apply many of the techniques and molecular tools discussed in this book to the study of a broad range of microorganisms in a variety of habitats.

Book↗

USGS2006

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. ?? 2005 Society for Applied Microbiology and Blackwell Publishing Ltd.

Environmental Microbiology↗

USGS2007

Molecular analysis of fungal communities and laccase genes in decomposing litter reveals differences among forest types but no impact of nitrogen deposition

The fungal community of the forest floor was examined as the cause of previously reported increases in soil organic matter due to experimental N deposition in ecosystems producing predominantly high-lignin litter, and the opposite response in ecosystems producing low-lignin litter. The mechanism proposed to explain this phenomenon was that white-rot basidiomycetes are more important in the degradation of high-lignin litter than of low-lignin litter, and that their activity is suppressed by N deposition. We found that forest floor mass in the low-lignin sugar-maple dominated system decreased in October due to experimental N deposition, whereas forest floor mass of high-lignin oak-dominated ecosystems was unaffected by N deposition. Increased relative abundance of basidiomycetes in high-lignin forest floor was confirmed by denaturing gradient gel electrophoresis (DGGE) and sequencing. Abundance of basidiomycete laccase genes, encoding an enzyme used by white-rot basidiomycetes in the degradation of lignin, was 5-10 times greater in high-lignin forest floor than in low-lignin forest floor. While the differences between the fungal communities in different ecosystems were consistent with the proposed mechanism, no significant effects of N deposition were detected on DGGE profiles, laccase gene abundance, laccase length heterogeneity profiles, or phenol oxidase activity. Our observations indicate that the previously detected accumulation of soil organic matter in the high-lignin system may be driven by effects of N deposition on organisms in the mineral soil, rather than on organisms residing in the forest floor. However, studies of in situ gene expression and temporal and spatial variability within forest floor communities will be necessary to further relate the ecosystem dynamics of organic carbon to microbial communities and atmospheric N deposition. ?? 2007 The Authors; Journal compilation ?? 2007 Society for Applied Microbiology and Blackwell Publishing Ltd.

Environmental Microbiology↗

USGS2017

Environmental Escherichia coli : Ecology and public health implications - A review

Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae . The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia . The impact of environmental E. coli on water quality and public health is also discussed.

Journal of Applied Microbiology↗

USGS2016

Developing integrated methods to address complex resource and environmental issues

Introduction This circular provides an overview of selected activities that were conducted within the U.S. Geological Survey (USGS) Integrated Methods Development Project, an interdisciplinary project designed to develop new tools and conduct innovative research requiring integration of geologic, geophysical, geochemical, and remote-sensing expertise. The project was supported by the USGS Mineral Resources Program, and its products and acquired capabilities have broad applications to missions throughout the USGS and beyond. In addressing challenges associated with understanding the location, quantity, and quality of mineral resources, and in investigating the potential environmental consequences of resource development, a number of field and laboratory capabilities and interpretative methodologies evolved from the project that have applications to traditional resource studies as well as to studies related to ecosystem health, human health, disaster and hazard assessment, and planetary science. New or improved tools and research findings developed within the project have been applied to other projects and activities. Specifically, geophysical equipment and techniques have been applied to a variety of traditional and nontraditional mineral- and energy-resource studies, military applications, environmental investigations, and applied research activities that involve climate change, mapping techniques, and monitoring capabilities. Diverse applied geochemistry activities provide a process-level understanding of the mobility, chemical speciation, and bioavailability of elements, particularly metals and metalloids, in a variety of environmental settings. Imaging spectroscopy capabilities maintained and developed within the project have been applied to traditional resource studies as well as to studies related to ecosystem health, human health, disaster assessment, and planetary science. Brief descriptions of capabilities and laboratory facilities and summaries of some applications of project products and research findings are included in this circular. The work helped support the USGS mission to “provide reliable scientific information to describe and understand the Earth; minimize loss of life and property from natural disasters; manage water, biological, energy, and mineral resources; and enhance and protect our quality of life.” Activities within the project include the following: Spanned scales from microscopic to planetary; Demonstrated broad applications across disciplines; Included life-cycle studies of mineral resources; Incorporated specialized areas of expertise in applied geochemistry including mineralogy, hydrogeology, analytical chemistry, aqueous geochemistry, biogeochemistry, microbiology, aquatic toxicology, and public health; and Incorporated specialized areas of expertise in geophysics including magnetics, gravity, radiometrics, electromagnetics, seismic, ground-penetrating radar, borehole radar, and imaging spectroscopy. This circular consists of eight sections that contain summaries of various activities under the project. The eight sections are listed below: Laboratory Facilities and Capabilities, which includes brief descriptions of the various types of laboratories and capabilities used for the project; Method and Software Development, which includes summaries of remote-sensing, geophysical, and mineralogical methods developed or enhanced by the project; Instrument Development, which includes descriptions of geophysical instruments developed under the project; Minerals, Energy, and Climate, which includes summaries of research that applies to mineral or energy resources, environmental processes and monitoring, and carbon sequestration by earth materials; Element Cycling, Toxicity, and Health, which includes summaries of several process-oriented geochemical and biogeochemical studies and health-related research activities; Hydrogeology and Water Quality, which includes descriptions of innovative geophysical, remote-sensing, and geochemical research pertaining to hydrogeology and water-quality applications; Hazards and Disaster Assessment, which includes summaries of research and method development that were applied to natural hazards, human-caused hazards, and disaster assessments; and Databases and Framework Studies, which includes descriptions of fundamental applications of geophysical studies and of the importance of archived data.

Circular↗

USGS2023

Identifying sources of antibiotic resistance genes in the environment using the microbial Find, Inform, and Test framework

Introduction: Antimicrobial resistance (AMR) is an increasing public health concern for humans, animals, and the environment. However, the contributions of spatially distributed sources of AMR in the environment are not well defined. Methods: To identify the sources of environmental AMR, the novel microbial Find, Inform, and Test (FIT) model was applied to a panel of five antibiotic resistance-associated genes (ARGs), namely, erm(B), tet(W), qnrA, sul1, and intI1, quantified from riverbed sediment and surface water from a mixed-use region. Results: A one standard deviation increase in the modeled contributions of elevated AMR from bovine sources or land-applied waste sources [land application of biosolids, sludge, and industrial wastewater (i.e., food processing) and domestic (i.e., municipal and septage)] was associated with 34–80% and 33–77% increases in the relative abundances of the ARGs in riverbed sediment and surface water, respectively. Sources influenced environmental AMR at overland distances of up to 13 km. Discussion: Our study corroborates previous evidence of offsite migration of microbial pollution from bovine sources and newly suggests offsite migration from land-applied waste. With FIT, we estimated the distance-based influence range overland and downstream around sources to model the impact these sources may have on AMR at unsampled sites. This modeling supports targeted monitoring of AMR from sources for future exposure and risk mitigation efforts.

Wisconsin↗

USGS2018

Understanding how microbiomes influence the systems they inhabit

Translating the ever-increasing wealth of information on microbiomes (environment, host, or built environment) to advance the understanding of system-level processes is proving to be an exceptional research challenge. One reason for this challenge is that relationships between characteristics of microbiomes and the system-level processes they influence are often evaluated in the absence of a robust conceptual framework and reported without elucidating the underlying causal mechanisms. The reliance on correlative approaches limits the potential to expand the inference of a single relationship to additional systems and advance the field. We propose that research focused on how microbiomes influence the systems they inhabit should work within a common framework and target known microbial processes that contribute to the system-level processes of interest. Here we identify three distinct categories of microbiome characteristics (microbial processes, microbial community properties, and microbial membership) and propose a framework to empirically link each of these categories to each other and the broader system level processes they affect. We posit that it is particularly important to distinguish microbial community properties that can be predicted from constituent taxa (community aggregated traits) from and those properties that are currently unable to be predicted from constituent taxa (emergent properties). Existing methods in microbial ecology can be applied to more explicitly elucidate properties within each of these categories and connect these three categories of microbial characteristics with each other. We view this proposed framework, gleaned from a breadth of research on environmental microbiomes and ecosystem processes, as a promising pathway with the potential to advance discovery and understanding across a broad range of microbiome science.

Nature Microbiology↗

USGS2010

Linking microbial and ecosystem ecology using ecological stoichiometry: a synthesis of conceptual and empirical approaches

Currently, one of the biggest challenges in microbial and ecosystem ecology is to develop conceptual models that organize the growing body of information on environmental microbiology into a clear mechanistic framework with a direct link to ecosystem processes. Doing so will enable development of testable hypotheses to better direct future research and increase understanding of key constraints on biogeochemical networks. Although the understanding of phenotypic and genotypic diversity of microorganisms in the environment is rapidly accumulating, how controls on microbial physiology ultimately affect biogeochemical fluxes remains poorly understood. We propose that insight into constraints on biogeochemical cycles can be achieved by a more rigorous evaluation of microbial community biomass composition within the context of ecological stoichiometry. Multiple recent studies have pointed to microbial biomass stoichiometry as an important determinant of when microorganisms retain or recycle mineral nutrients. We identify the relevant cellular components that most likely drive changes in microbial biomass stoichiometry by defining a conceptual model rooted in ecological stoichiometry. More importantly, we show how X-ray microanalysis (XRMA), nanoscale secondary ion mass spectroscopy (NanoSIMS), Raman microspectroscopy, and in situ hybridization techniques (for example, FISH) can be applied in concert to allow for direct empirical evaluation of the proposed conceptual framework. This approach links an important piece of the ecological literature, ecological stoichiometry, with the molecular front of the microbial revolution, in an attempt to provide new insight into how microbial physiology could constrain ecosystem processes.

Ecosystems↗

USGS2015

Concentration and retention of Toxoplasma gondii oocysts by marine snails demonstrate a novel mechanism for transmission of terrestrial zoonotic pathogens in coastal ecosystems

The parasite T oxoplasma gondii is an environmentally persistent pathogen that can cause fatal disease in humans, terrestrial warm-blooded animals and aquatic mammals. Although an association between T . gondii exposure and prey specialization on marine snails was identified in threatened California sea otters, the ability of kelp-dwelling snails to transmit terrestrially derived pathogens has not been previously investigated. The objective of this study was to measure concentration and retention of T . gondii by marine snails in laboratory aquaria, and to test for natural T . gondii contamination in field-collected snails. Following exposure to T . gondii -containing seawater, oocysts were detected by microscopy in snail faeces and tissues for 10 and 3 days respectively. Nested polymerase chain reaction was also applied as a method for confirming putative T . gondii oocysts detected in snail faeces and tissues by microscopy. T oxoplasma gondii was not detected in field-collected snails. Results suggest that turban snails are competent transport hosts for T . gondii . By concentrating oocysts in faecal pellets, snails may facilitate entry of T . gondii into the nearshore marine food web. This novel mechanism also represents a general pathway by which marine transmission of terrestrially derived microorganisms can be mediated via pathogen concentration and retention by benthic invertebrates.

Environmental Microbiology↗

USGS2020

Mapping metabolic activity at single cell resolution in intact volcanic fumarole soil

Interactions among microorganisms and their mineralogical substrates govern the structure, function, and emergent properties of microbial communities. These interactions are predicated on spatial relationships, which dictate metabolite exchange and access to key substrates. To quantitatively assess links between spatial relationships and metabolic activity, this study presents a novel approach to map all organisms, the metabolically active subset, and associated mineral grains, all while maintaining spatial integrity of an environmental microbiome. We applied this method at an outgassing fumarole of Vanuatu’s Marum Crater, one of the largest point sources of several environmentally relevant gaseous compounds, including H2O, CO2, and SO2. With increasing distance from the soil-air surface and from mineral grain outer boundaries, organism abundance decreased but the proportion of metabolically active organisms often increased. These protected niches may provide more stable conditions that promote consistent metabolic activity of a streamlined community. Conversely, mineral exteriors accumulate more organisms that may cover a wider range of preferred conditions, implying that only a subset of the community will be active under any particular environmental regime. More broadly, the approach presented here allows investigators to see microbial communities “as they really are” and explore determinants of metabolic activity across a range of microbiomes.

FEMS Microbiology Letters↗

USGS2005

Microbial biogeochemistry of uranium mill tailings

Uranium mill tailings (UMT) are the crushed ore residues from the extraction of uranium (U) from ores. Among the radioactive wastes associated with the nuclear fuel cycle, UMT are unique in terms of their volume and their limited isolation from the surficial environment. For this latter reason, their management and long-term fate has many interfaces with environmental microbial communities and processes. The interactions of microorganisms with UMT have been shown to be diverse and with significant consequences for radionuclide mobility and bioremediation. These radionuclides are associated with the U-decay series. The addition of organic carbon and phosphate is required to initiate the reduction of the U present in the groundwater down gradient of the mills. Investigations on sediment and water from the U-contaminated aquifer, indicates that the addition of a carbon source stimulates the rate of U removal by microbial reduction. Moreover, most attention with respect to passive or engineered removal of U from groundwaters focuses on iron-reducing and sulfate-reducing bacteria.

Advances in Applied Microbiology↗

USGS2015

Hydrogeochemistry and microbiology of mine drainage: An update

The extraction of mineral resources requires access through underground workings, or open pit operations, or through drillholes for solution mining. Additionally, mineral processing can generate large quantities of waste, including mill tailings, waste rock and refinery wastes, heap leach pads, and slag. Thus, through mining and mineral processing activities, large surface areas of sulfide minerals can be exposed to oxygen, water, and microbes, resulting in accelerated oxidation of sulfide and other minerals and the potential for the generation of low-quality drainage. The oxidation of sulfide minerals in mine wastes is accelerated by microbial catalysis of the oxidation of aqueous ferrous iron and sulfide. These reactions, particularly when combined with evaporation, can lead to extremely acidic drainage and very high concentrations of dissolved constituents. Although acid mine drainage is the most prevalent and damaging environmental concern associated with mining activities, generation of saline, basic and neutral drainage containing elevated concentrations of dissolved metals, non-metals, and metalloids has recently been recognized as a potential environmental concern. Acid neutralization reactions through the dissolution of carbonate, hydroxide, and silicate minerals and formation of secondary aluminum and ferric hydroxide phases can moderate the effects of acid generation and enhance the formation of secondary hydrated iron and aluminum minerals which may lessen the concentration of dissolved metals. Numerical models provide powerful tools for assessing impacts of these reactions on water quality.

Applied Geochemistry↗