Antimicrobial Resistance Linked to Septic System Contamination in the Indiana Lake Michigan Watershed.

Extended-spectrum ß-lactamases confer resistance to a variety of ß-lactam antimicrobials, and the genes for these enzymes are often found on plasmids that include additional antimicrobial resistance genes (ARG). We surveyed aquatic environments in the Indiana Lake Michigan watershed in proximity to areas with high densities of residential septic systems to determine if human fecal contamination from septic effluent correlated with the presence of antimicrobial resistance genes and phenotypically resistant bacteria. Of the 269 E. coli isolated from environmental samples and one septic source, 97 isolates were resistant to cefotaxime, a third-generation cephalosporin. A subset of those isolates showed phenotypic resistance to other ß-lactams, fluoroquinolones, sulfonamides, and tetracyclines. Quantitative PCR was used to quantify human-associated Bacteroides dorei gene copies (Human Bacteroides) from water samples and to identify the presence of ARG harbored on plasmids from E. coli isolates or in environmental DNA. We found a strong correlation between the presence of ARG and human fecal concentrations, which supports our hypothesis that septic effluent is a source of ARG and resistant organisms. The observed plasmid-based resistance adds an additional level of risk, as human-associated bacteria from septic systems may expand the environmental resistome by acting as a reservoir of transmissible resistance genes.

Subsurface microbial communities as a tool for characterizing regional-scale groundwater flow.

Subsurface microbial community distribution patterns are influenced by biogeochemical and groundwater fluxes and may inform hydraulic connections along groundwater-flow paths. This study examined the regional-scale microbial community of the Death Valley Regional Flow System and evaluated whether subsurface communities can be used to identify groundwater-flow paths between recharge and discharge areas. Samples were collected from 36 sites in three groundwater basins: Pahute Mesa-Oasis Valley (PMOV), Ash Meadows (AM), and Alkali Flat-Furnace Creek Ranch (AFFCR). Microbial diversity within and between communities varied by location, and communities were separated into two overall groups that affiliated with the AM and PMOV/AFFCR basins. Network analysis revealed patterns between clusters of common microbes that represented groundwaters with similar geochemical conditions and largely corroborated hydraulic connections between recharge and discharge areas. Null model analyses identified deterministic and stochastic ecological processes contributing to microbial community assemblages. Most communities were more different than expected and governed by dispersal limitation, geochemical differences, or undominating processes. However, certain communities from sites located within or near the Nevada National Security Site were more similar than expected and dominated by homogeneous dispersal or selection. Overall, the (dis)similarities between the microbial communities of DVRFS recharge and discharge areas supported previously documented hydraulic connections between: (1) Spring Mountains and Ash Meadows; (2) Frenchman and Yucca Flat and Amargosa Desert; and (3) Amargosa Desert and Death Valley. However, only a portion of the flow path between Pahute Mesa and Oasis Valley could be supported by microbial community analyses, likely due to well-associated artifacts in samples from the two Oasis Valley sites. This study demonstrates the utility of combining microbial data with hydrologic, geologic, and water-chemistry information to comprehensively characterize groundwater systems, highlighting both strengths and limitations of this approach.

Microbial Communities Associated with Farmed Genypterus chilensis: Detection in Water Prior to Bacterial Outbreaks Using Culturing and High-Throughput Sequencing.

The red conger eel (Genypterus chilensis, Guichenot) is a native species included in the Chilean Aquaculture Diversification Program due to high commercial demand. In the context of intensified farming, prior reports link two disease outbreaks with emerging pathogens in the Vibrio and Tenacibaculum genera. However, the roles remain unclear for the bacterial community and each specific bacterium is associated with the rearing environment for healthy specimens. The success of red conger eel farming therefore warrants research into the bacterial composition of aquaculture conditions and the antimicrobial susceptibilities thereof. This study used culturing methods and high-throughput sequencing to describe the bacterial community associated with water in which G. chilensis was farmed. With culturing methods, the predominant genera were Vibrio (21.6%), Pseudolteromonas (15.7%), Aliivibrio (13.7%), and Shewanella (7.8%). Only a few bacterial isolates showed amylase, gelatinase, or lipase activity, and almost all showed inhibition zones to commonly-used antibiotics in aquaculture. By contrast, high-throughput sequencing established Paraperlucidibaca, Colwellia, Polaribacter, Saprospiraceae, and Tenacibaculum as the predominant genera, with Vibrio ranking twenty-seventh in abundance. High-throughput sequencing also established a link between previous outbreaks with increased relative abundances of Vibrio and Tenacibaculum. Therefore, monitoring the presence and abundance of these potential pathogens could be useful in providing prophylactic measures to prevent future outbreaks.

Identification of Specialists and Abundance-Occupancy Relationships among Intestinal Bacteria of Aves, Mammalia, and Actinopterygii.

The coalescence of next-generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineation of host-microbe associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macroorganisms, but it remains unclear how the specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA genes amplified from 106 fecal samples spanning Aves, Mammalia, and Actinopterygii (ray-finned fish). We investigated the interspecific abundance-occupancy relationship, where widespread taxa tend to be more abundant than narrowly distributed taxa, among operational taxonomic units (OTUs) within and among host species. In a separate analysis, we identified specialist OTUs that were highly abundant in a single host and rare in all other hosts by using a multinomial model without excluding undersampled OTUs a priori. We show that intestinal microbes in humans and other vertebrates display abundance-occupancy relationships, but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest that intestinal bacteria can be shared among diverse hosts in ways that resemble the distribution of "free-living" bacteria in the extraintestinal environment.

The microbiome of urban waters

More than 50% of the world’s population lives in urban centers. As collection basins for landscape activity, urban waters are an interface between human activity and the natural environment. The microbiome of urban waters could provide insight into the impacts of pollution, the presence of human health risks, or the potential for long-term consequences for these ecosystems and the people who depend upon them. An integral part of the urban water cycle is sewer infrastructure. Thousands of miles of pipes line cities as part of wastewater and stormwater systems. As stormwater and sewage are released into natural waterways, traces of human and animal microbiomes reflect the sources and magnitude of fecal pollution and indicate the presence of pollutants, such as nutrients, pathogens, and chemicals. Non-fecal organisms are also released as part of these systems. Runoff from impervious surfaces delivers microbes from soils, plants and the built environment to stormwater systems. Further, urban sewer infrastructure contains its own unique microbial community seemingly adapted to this relatively new artificial habitat. High microbial densities are conveyed via pipes to waterways, and these organisms can be found as an urban microbial signature imprinted on the natural community of rivers and urban coastal waters. The potential consequences of mass releases of non-indigenous microorganisms into natural waters include creation of reservoirs for emerging human pathogens, altered nutrient flows into aquatic food webs, and increased genetic exchange between two distinct gene pools. This review highlights the recent characterization of the microbiome of urban sewer and stormwater infrastructure and its connection to and potential impact upon freshwater systems (AU)

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Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups.

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.

The microbiome of urban waters.

More than 50% of the world's population lives in urban centers. As collection basins for landscape activity, urban waters are an interface between human activity and the natural environment. The microbiome of urban waters could provide insight into the impacts of pollution, the presence of human health risks, or the potential for long-term consequences for these ecosystems and the people who depend upon them. An integral part of the urban water cycle is sewer infrastructure. Thousands of miles of pipes line cities as part of wastewater and stormwater systems. As stormwater and sewage are released into natural waterways, traces of human and animal microbiomes reflect the sources and magnitude of fecal pollution and indicate the presence of pollutants, such as nutrients, pathogens, and chemicals. Non-fecal organisms are also released as part of these systems. Runoff from impervious surfaces delivers microbes from soils, plants and the built environment to stormwater systems. Further, urban sewer infrastructure contains its own unique microbial community seemingly adapted to this relatively new artificial habitat. High microbial densities are conveyed via pipes to waterways, and these organisms can be found as an urban microbial signature imprinted on the natural community of rivers and urban coastal waters. The potential consequences of mass releases of non-indigenous microorganisms into natural waters include creation of reservoirs for emerging human pathogens, altered nutrient flows into aquatic food webs, and increased genetic exchange between two distinct gene pools. This review highlights the recent characterization of the microbiome of urban sewer and stormwater infrastructure and its connection to and potential impact upon freshwater systems.

Urban microbial ecology of a freshwater estuary of Lake Michigan.

Freshwater estuaries throughout the Great Lakes region receive stormwater runoff and riverine inputs from heavily urbanized population centers. While human and animal feces contained in this runoff are often the focus of source tracking investigations, non-fecal bacterial loads from soil, aerosols, urban infrastructure, and other sources are also transported to estuaries and lakes. We quantified and characterized this non-fecal urban microbial component using bacterial 16S rRNA gene sequences from sewage, stormwater, rivers, harbor/estuary, and the lake surrounding Milwaukee, WI, USA. Bacterial communities from each of these environments had a distinctive composition, but some community members were shared among environments. We used a statistical biomarker discovery tool to identify the components of the microbial community that were most strongly associated with stormwater and sewage to describe an "urban microbial signature," and measured the presence and relative abundance of these organisms in the rivers, estuary, and lake. This urban signature increased in magnitude in the estuary and harbor with increasing rainfall levels, and was more apparent in lake samples with closest proximity to the Milwaukee estuary. The dominant bacterial taxa in the urban signature were Acinetobacter, Aeromonas, and Pseudomonas, which are organisms associated with pipe infrastructure and soil and not typically found in pelagic freshwater environments. These taxa were highly abundant in stormwater and sewage, but sewage also contained a high abundance of Arcobacter and Trichococcus that appeared in lower abundance in stormwater outfalls and in trace amounts in aquatic environments. Urban signature organisms comprised 1.7% of estuary and harbor communities under baseflow conditions, 3.5% after rain, and >10% after a combined sewer overflow. With predicted increases in urbanization across the Great Lakes, further alteration of freshwater communities is likely to occur with potential long term impacts on the function of estuarine and nearshore ecosystems.

A single genus in the gut microbiome reflects host preference and specificity.

Delineating differences in gut microbiomes of human and animal hosts contributes towards understanding human health and enables new strategies for detecting reservoirs of waterborne human pathogens. We focused upon Blautia, a single microbial genus that is important for nutrient assimilation as preliminary work suggested host-related patterns within members of this genus. In our dataset of 57 M sequence reads of the V6 region of the 16S ribosomal RNA gene in samples collected from seven host species, we identified 200 high-resolution taxonomic units within Blautia using oligotyping. Our analysis revealed 13 host-specific oligotypes that occurred exclusively in fecal samples of humans (three oligotypes), swine (six oligotypes), cows (one oligotype), deer (one oligotype), or chickens (two oligotypes). We identified an additional 171 oligotypes that exhibited differential abundance patterns among all the host species. Blautia oligotypes in the human population obtained from sewage and fecal samples displayed remarkable continuity. Oligotypes from only 10 Brazilian human fecal samples collected from individuals in a rural village encompassed 97% of all Blautia oligotypes found in a Brazilian sewage sample from a city of three million people. Further, 75% of the oligotypes in Brazilian human fecal samples matched those in US sewage samples, implying that a universal set of Blautia strains may be shared among culturally and geographically distinct human populations. Such strains can serve as universal markers to assess human fecal contamination in environmental samples. Our results indicate that host-specificity and host-preference patterns of organisms within this genus are driven by host physiology more than dietary habits.

Blautia and Prevotella sequences distinguish human and animal fecal pollution in Brazil surface waters.

Untreated sewage discharges and limited agricultural manure management practices contribute to fecal pollution in rural Brazilian waterways. Most microbial source tracking studies have focused on Bacteroidales, and few have tested host-specific indicators in underdeveloped regions. Sequencing of sewage and human and animal feces with Illumina HiSeq revealed Prevotellaceae as the most abundant family in humans, with Lachnospiraceae and Ruminococcaceae also comprising a large proportion of the microbiome. These same families were also dominant in animals. Bacteroides, the genus containing the most commonly utilized human-specific marker in the United States was present in very low abundance. We used oligotyping to identify Prevotella and Blautia sequences that can distinguish human fecal contamination. Thirty-five of 61 Blautia oligotypes and 13 of 108 Prevotella oligotypes in humans were host-specific or highly abundant (i.e. host-preferred) compared to pig, dog, horse and cow sources. Certain human Prevotella and Blautia oligotypes increased more than an order of magnitude along a polluted river transect in rural Brazil, but traditional fecal indicator levels followed a steady or even decreasing trend. While both Prevotella and Blautia oligotypes distinguished human and animal fecal pollution in Brazil surface waters, Blautia appears to contain more discriminatory and globally applicable markers for tracking sources of fecal pollution.

Population dynamics and ecology of Arcobacter in sewage.

Arcobacter species are highly abundant in sewage where they often comprise approximately 5-11% of the bacterial community. Oligotyping of sequences amplified from the V4V5 region of the 16S rRNA gene revealed Arcobacter populations from different cities were similar and dominated by 1-3 members, with extremely high microdiversity in the minor members. Overall, nine subgroups within the Arcobacter genus accounted for >80% of the total Arcobacter sequences in all samples analyzed. The distribution of oligotypes varied by both sample site and temperature, with samples from the same site generally being more similar to each other than other sites. Seven oligotypes matched with 100% identity to characterized Arcobacter species, but the remaining 19 abundant oligotypes appear to be unknown species. Sequences representing the two most abundant oligotypes matched exactly to the reference strains for A. cryaerophilus group 1B (CCUG 17802) and group 1A (CCUG 17801(T)), respectively. Oligotype 1 showed generally lower relative abundance in colder samples and higher relative abundance in warmer samples; the converse was true for Oligotype 2. Ten other oligotypes had significant positive or negative correlations between temperature and proportion in samples as well. The oligotype that corresponded to A. butzleri, the Arcobacter species most commonly isolated by culturing in sewage studies, was only the eleventh most abundant oligotype. This work suggests that Arcobacter populations within sewer infrastructure are modulated by temperature. Furthermore, current culturing methods used for identification of Arcobacter fail to identify some abundant members of the community and may underestimate the presence of species with affinities for growth at lower temperatures. Understanding the ecological factors that affect the survival and growth of Arcobacter spp. in sewer infrastructure may better inform the risks associated with these emerging pathogens.

Analysis of the gull fecal microbial community reveals the dominance of Catellicoccus marimammalium in relation to culturable Enterococci.

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-ß-D-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.

Pyrosequencing reveals high-temperature cellulolytic microbial consortia in Great Boiling Spring after in situ lignocellulose enrichment.

To characterize high-temperature cellulolytic microbial communities, two lignocellulosic substrates, ammonia fiber-explosion-treated corn stover and aspen shavings, were incubated at average temperatures of 77 and 85°C in the sediment and water column of Great Boiling Spring, Nevada. Comparison of 109,941 quality-filtered 16S rRNA gene pyrosequences (pyrotags) from eight enrichments to 37,057 quality-filtered pyrotags from corresponding natural samples revealed distinct enriched communities dominated by phylotypes related to cellulolytic and hemicellulolytic Thermotoga and Dictyoglomus, cellulolytic and sugar-fermenting Desulfurococcales, and sugar-fermenting and hydrogenotrophic Archaeoglobales. Minor enriched populations included close relatives of hydrogenotrophic Thermodesulfobacteria, the candidate bacterial phylum OP9, and candidate archaeal groups C2 and DHVE3. Enrichment temperature was the major factor influencing community composition, with a negative correlation between temperature and richness, followed by lignocellulosic substrate composition. This study establishes the importance of these groups in the natural degradation of lignocellulose at high temperatures and suggests that a substantial portion of the diversity of thermophiles contributing to consortial cellulolysis may be contained within lineages that have representatives in pure culture.

Microbially mediated aerobic and anaerobic degradation of acrylamide in a western United States irrigation canal.

Acrylamide (AMD), a neurotoxin and suspected carcinogen, is present at concentrations of up to 0.05% in linear anionic polyacrylamide, which is under evaluation as a temporary sealant in unlined irrigation canal systems across the United States. We examined the microbially mediated degradation of AMD and diversity of AMD-degrading microbial physiotypes in the Rocky Ford Highline Canal, Colorado to better constrain the potential fate ofAMD in a canal environment. Microorganisms able to use AMD (500 mg L(-1)) as a sole nitrogen source were relatively abundant (2.3 x 10(3) to 9.4 x 10(3) cells mL(-1) in water and 4.2 x 10(3) to 2.3 x 10(5) cells g(-1) in sediment). Only sediment samples contained microorganisms able to use AMD as a sole carbon source. Acrylamide (up to 100 mg L(-1)) was efficiently removed from amended canal water and sediment slurries under aerobic conditions, but no AMD degradation was observed in abiotic controls. Anaerobic degradation of AMD by nitrate-, sulfate-, and iron-reducing microorganisms was also tested, with nitrate reducers affecting the highest amounts of AMD removal (70.3-85%) after 60 d. All representatives (n=15) from a collection of 256 AMD-degrading microbial isolates from Rocky Ford Highline Canal were closely related to well characterized environmental bacteria capable of facultative nitrate respiration. Our results demonstrate that natural microbial populations within this canal are capable of AMD degradation under aerobic and anaerobic conditions and that this degradation is performed by naturally abundant bacteria likely to be present in other freshwater irrigation canals or similar lotic habitats.

A new role for sulfur in arsenic cycling.

Sulfur and arsenic often coexist in the environment and share similar microbial redox transformations. We examined the effects of sulfide on aerobic arsenite oxidation in alkaline lake water samples and in laboratory enrichment cultures. Significant arsenite oxidation occurred only in treatments with bacteria present, and production of arsenate was greatly enhanced by the addition of sulfide or thiosulfate. IC-ICP-MS analysis of samples showed that mono- and dithioarsenate formed in arsenite + sulfide amended lake water. Our data indicate that these two thioarsenic compounds are fairly stable in sterile alkaline solutions, but are transformed predominantly to arsenate when bacteria are present. Enrichment culture experiments suggest that sulfur-oxidizing bacteria use free or arsenic-bound sulfur as a growth substrate and directly or indirectly transform arsenite and thioarsenates to arsenate during growth. Increases in cell density resulted in more rapid conversion of arsenite and thioarsenates. The rate and extent of these processes appearto be controlled bythe concentration of bacteria and the ratio of reduced sulfur to arsenite present. Sulfur-driven arsenite oxidation and microbial thioarsenate transformation may be important biogeochemical processes in the arsenic cycle of our study site (Mono Lake, CA, USA) and other alkaline environments as well.

Selenate-dependent anaerobic arsenite oxidation by a bacterium from Mono Lake, California.

Arsenate was produced when anoxic Mono Lake water samples were amended with arsenite and either selenate or nitrate. Arsenite oxidation did not occur in killed control samples or live samples with no added terminal electron acceptor. Potential rates of anaerobic arsenite oxidation with selenate were comparable to those with nitrate ( approximately 12 to 15 mumol.liter(-1) h(-1)). A pure culture capable of selenate-dependent anaerobic arsenite oxidation (strain ML-SRAO) was isolated from Mono Lake water into a defined salts medium with selenate, arsenite, and yeast extract. This strain does not grow chemoautotrophically, but it catalyzes the oxidation of arsenite during growth on an organic carbon source with selenate. No arsenate was produced in pure cultures amended with arsenite and nitrate or oxygen, indicating that the process is selenate dependent. Experiments with washed cells in mineral medium demonstrated that the oxidation of arsenite is tightly coupled to the reduction of selenate. Strain ML-SRAO grows optimally on lactate with selenate or arsenate as the electron acceptor. The amino acid sequences deduced from the respiratory arsenate reductase gene (arrA) from strain ML-SRAO are highly similar (89 to 94%) to those from two previously isolated Mono Lake arsenate reducers. The 16S rRNA gene sequence of strain ML-SRAO places it within the Bacillus RNA group 6 of gram-positive bacteria having low G+C content.