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Contaminated water is globally the main vehicle for microbial pathogens in most regions. Teaching future microbiologist and employees in the food industry on the importance of hygienically satisfactory water, microbiological analyses and how to ensure good water quality and safety is highly relevant. This paper presents a complete experimental design for water analyses as a tool to teach students the methods and other key elements in microbiology, including food safety, environmental dissemination and survival of microorganisms, laboratory practices, water legislation and critical evaluation of results. All results from the last 10 classes (2006-2015) in a university course on seafood microbiology have been compiled and are presented here. Questionnaires used with former students reveal that the laboratory course is highly appreciated, and that many students remember important aspects of the water analyses, even after several years. The questionnaire results were consistent with our perception that some students find calculation of dilutions difficult to comprehend., (© FEMS 2015. All rights reserved.)

Over the past 15 years, microbiology has undergone a momentous shift toward molecular methods. New sequences appear daily in the public databases and new computer tools and web servers are published on a regular basis. Major advances in molecular identifications of pathogens have been made because new biotechnology methods have appeared that often require a thorough in silico analysis of sequences. However, significant difficulties partly remain in developing efficient methods because the public databases contain many poorly annotated or partial sequences (often of environmental origin) and also because there are few dedicated web servers and curated databases.

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.

A.J. Kluyver and C.B. van Niel introduced many scientists to the exceptional metabolic capacity of microbes and their remarkable ability to adapt to changing environments in The Microbe's Contribution to Biology. Beyond providing an overview of the physiology and adaptability of microbes, the book outlined many of the basic principles for the emerging discipline of microbial ecology. While the study of pure cultures was highlighted, provided a unifying framework for understanding the vast metabolic potential of microbes and their roles in the global cycling of elements, extrapolation from pure cultures to natural environments has often been overshadowed by microbiologists inability to culture many of the microbes seen in natural environments. A combination of genomic approaches is now providing a culture-independent view of the microbial world, revealing a more diverse and dynamic community of microbes than originally anticipated. As methods for determining the diversity of microbial communities become increasingly accessible, a major challenge to microbial ecologists is to link the structure of natural microbial communities with their functions. This article presents several examples from studies of aquatic and terrestrial microbial communities in which culture and culture-independent methods are providing an enhanced appreciation for the microbe's contribution to the evolution and maintenance of life on Earth, and offers some thoughts about the graduate-level educational programs needed to enhance the maturing field of microbial ecology.

An experimental approach for investigation of genetically modified microorganisms (GMMO) introduced into model ecosystems to evaluate potential risk of propagation of recombinant plasmids in surrounding medium has been developed. The object of modeling was Escherichia coli Z905 strain with a recombinant plasmid with bacterial luminescence genes, which was introduced into water microcosms of different structure. The approach involves comprehensive investigation of GMMO at four hierarchical levels: molecular (retaining the structure of the plasmid and expression of cloned genes); cellular (variation of metabolic activity); population (competitive power and metabolic interactions of GMMO with indigenous microflora, migration of recombinant and natural plasmids); ecosystem (effect of GMMO and cloned genes on ecosystem parameters). The experimental evidence and theoretical estimates are intended to form grounds to develop a basic version of an ecological certificate for different GMMO variants.

This memoir describes the professional life and times of Thomas D. Brock, with an emphasis on those aspects of his career relating to research in microbial ecology, and how this work led to field research in Yellowstone. The first discovery of extremely thermophilic bacteria is described, followed by a discussion of some of the consequences of this discovery for biotechnology and microbiology. Also covered briefly in this memoir are Brock's activities in textbook writing, publishing, computers, and the history of science.

Of the nine packets of specimens sent by van Leeuwenhoek to London in the seventeenth century, and which were recently rediscovered in their original condition, three contained dried aquatic microorganisms. The author has reconstituted portions of this material. Several recognizable organisms have been noted, including portions of this material. Several recognizable organisms have been noted, including water-fleas, chlorophyte and cyanophyte algae, desmids, diatoms and rotifers. These provide evidence for the formation of the 'heavenly paper', which was believed at the time to represent charred notepaper dropped from the skies, but which Leeuwenhoek rightly assumed to be dried algal felts. Photomicrographs reveal many cytological details in the material, and this has been correlated with Leeuwenhoek's descriptions, extracted from the original correspondence.

Photodieldrin, previously reported as the major conversion product of dieldrin by sunlight, was found among the metabolic products of dieldrin among microorganisms isolated from various environments including soil, water (Lake Michigan), rat intestines, and rumen stomach contents of a cow.

AimsThe DNA marker HF183 is a partial 16S rRNA gene sequence highly specific to human-associated Bacteroides including Bacteroides dorei. While HF183 is used to assess human faecal contamination in aquatic environments worldwide, little is known about the existence of HF183 and B. dorei in human microbiomes outside of the human gastrointestinal tract and faeces.Methods and resultsPreviously published human skin and urine microbiome data sets from five independent human body skin studies, the Human Microbiome Project (HMP) and three independent human urine studies were analysed. The HF183 gene sequence was detected in all skin data sets, with the ratios of positive samples ranging from 0.5% to 36.3%. Popliteal fossa (knee), volar forearm and inguinal (groin) creases were identified as hot spots. HF183 was detected in two of three urine data sets, with ratios of positive samples ranging from 0% to 37.5%. All HF183-containing sequences from these data sets were classified as associated with B. dorei.ConclusionsHF183 is widespread on human skin and present in urine.Significance and impact of studySkin and urine microbiomes could be sources of HF183 to environmental waters. Such non-faecal sources of HF183 might explain low concentrations of HF183 in recreational waters when swimmers are present.

Violacein has different bioactive properties conferring distinct selective advantages, such as defense from predation and interspecific competition. Adaptation of Janthinobacterium to diverse habitats likely leads to variation in violacein production among phylogenetically closely related species inhabiting different environments, yet genomic mechanisms and the influence of adaptive evolution underpinning violacein biosynthesis in Janthinobacterium are not clear. In this study, we performed genome sequencing, comparative genomic analysis, and phenotypic characterization to investigate genomic factors regulating violacein production in nine Janthinobacterium strains, including a type strain from soil and eight strains we isolated from terrestrial subsurface sediment and groundwater. Results show that although all nine Janthinobacterium strains are phylogenetically closely related and contain genes essential for violacein biosynthesis, they vary in carbon usage and violacein production. Sediment and groundwater strains are weak violacein producers and possess far fewer secondary metabolite biosynthesis genes, indicating genome adaptation compared to soil strains. Further examination suggests that quorum sensing (QS) may play an important role in regulating violacein in Janthinobacterium: the strains exhibiting strong potential in violacein production possess both N-acyl-homoserine lactone (AHL) QS and Janthinobacterium QS (JQS) systems in their genomes, while weaker violacein-producing strains harbor only the JQS system. Preliminary tests of spent media of two Janthinobacterium strains possessing both AHL QS and JQS systems support the potential role of AHLs in inducing violacein production in Janthinobacterium. Overall, results from this study reveal potential genomic mechanisms involved in violacein biosynthesis in Janthinobacterium and provide insights into evolution of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment. IMPORTANCE Phylogenetically closely related bacteria can thrive in diverse environmental habitats due to adaptive evolution. Genomic changes resulting from adaptive evolution lead to variations in cellular function, metabolism, and secondary metabolite biosynthesis. The most well-known secondary metabolite produced by Janthinobacterium is the purple-violet pigment violacein. To date, the mechanisms of induction of violacein biosynthesis in Janthinobacterium is not clear. Comparative genome analysis of closely related Janthinobacterium strains isolated from different environmental habitats not only reveals potential mechanisms involved in induction of violacein production by Janthinobacterium but also provides insights into the survival strategy of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment.

The World Health Organization (WHO) estimated that pathogens like Escherichia coli, primarily linked to food and water contamination, are associated with 485,000 deaths from diarrheal diseases annually, translating to a staggering worldwide economic loss of nearly 12 billion USD per annum. International organizations like the WHO and United Nations Children’s Fund (UNICEF) have established related guidelines and criteria for pathogenic detection technologies and driving the search for innovative and efficient detection methods. This comprehensive review examines the trajectory of waterborne pathogenic bacteria detection technologies from traditional techniques, i.e., culture-based methods, to current detection methods including various forms of polymerase chain reaction (PCR) techniques [qualitative real-time PCR, digital PCR, ELISA, loop-mediated isothermal amplification, next-generation sequencing (NGS)] and to emerging techniques, i.e., biosensors and artificial intelligence (AI). The scope of the review paper focuses on waterborne pathogenic bacteria that are recognized as human pathogens, posing tangible threats to public health through waterborne. The detection techniques’ merits, constraints, research gaps and future perspectives are critically discussed. Advancements in digital droplet PCR, NGS and biosensors have significantly improved sensitivity and specificity, revolutionizing pathogen detection. Additionally, the integration of artificial intelligence (AI) with these technologies has enhanced detection accuracy, enabling real-time analysis of large datasets. Molecular-based methods and biosensors show promise for efficient water quality monitoring, especially in resource-constrained settings, but on-site practical implementation remains a challenge. The pairwise comparison metrics used in this review also offer valuable insights into quick evaluation on the advantages, limitations and research gaps of various techniques, focusing on their applicability in field settings and timely analyses. Future research efforts should focus on developing robust, cost-effective and user-friendly techniques for routine waterborne bacteria monitoring, ultimately safeguarding global water supplies and public health, with AI and data analysis playing a crucial role in advancing these methods for a safer environment.

AimsSulphate-reducing bacteria (SRB) are ecologically important group of anaerobic micro-organisms that can reduce sulphate to form hydrogen sulphide-a toxic gas causing iron corrosion on metal surfaces. In this work, SRB strains were isolated from aquatic environments in the country of Georgia to determine their lysogenicity and the role of temperate phages in host metabolism.Methods and resultsSRB strains were isolated in samples from the Black Sea coast of Georgia. Based on their genetic, cytological and physiological properties of bacteria, 10 Georgian isolates were assigned to the genus Desulfovibrio. Temperate bacteriophages were induced from three out of ten strains by UV-exposure. Comparison of metal (Fe and Cr) reduction and utilization of various carbon sources by the wild-type (lysogenic) bacterial strains and their UV-irradiated counterparts was done.ConclusionsTemperate phage in the cells of SRB could alter significant functions of bacteria and may have a contribution in the acquisition of different traits by SRB.Significance and impact of the studyThis article pointed to a significant role for temperate bacteriophages in the metabolism and metabolic potential of host strains of SRB, which were first isolated from the aquatic environment of Georgia.

Enteric viruses (EVs) are the largest contributors to foodborne illnesses and outbreaks globally. Their ability to persist in the environment, coupled with the challenges experienced in environmental monitoring, creates a critical aperture through which agricultural crops may become contaminated. This study involved a 17-month investigation of select human EVs and viral indicators in nontraditional irrigation water sources (surface and reclaimed waters) in the Mid-Atlantic region of the United States. Real-time quantitative PCR was used for detection of Aichi virus, hepatitis A virus, and norovirus genotypes I and II (GI and GII, respectively). Pepper mild mottle virus (PMMoV), a common viral indicator of human fecal contamination, was also evaluated, along with atmospheric (air and water temperature, cloud cover, and precipitation 24 h, 7 days, and 14 days prior to sample collection) and physicochemical (dissolved oxygen, pH, salinity, and turbidity) data, to determine whether there were any associations between EVs and measured parameters. EVs were detected more frequently in reclaimed waters (32% [n = 22]) than in surface waters (4% [n = 49]), similar to PMMoV detection frequency in surface (33% [n = 42]) and reclaimed (67% [n = 21]) waters. Our data show a significant correlation between EV and PMMoV (R2 = 0.628, P

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract.

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P

Successful rearing of fish in hatcheries is critical for conservation, recreational fishing, commercial fishing through wild stock enhancements, and aquaculture production. Flowthrough (FT) hatcheries require more water than recirculating aquaculture systems (RAS), which enable up to 99% of their water to be recycled, thus significantly reducing environmental impacts. Here, we evaluated the biological and physical microbiome interactions of three Atlantic salmon hatcheries (RAS n = 2, FT n = 1). Gill, skin, and digesta from six juvenile fish along with tank biofilms and water were sampled from tanks in each of the hatcheries (60 fish across 10 tanks) to assess the built environment and mucosal microbiota using 16S rRNA gene sequencing. The water and tank biofilm had more microbial richness than fish mucus, while skin and digesta from RAS fish had 2 times the richness of FT fish. Body sites each had unique microbiomes (P

Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.

Benthic microorganisms transported into the water column potentially influence biogeochemical cycles and the pelagic food web structure. In the present study six gas-releasing vent sites in the Coal Oil Point seep field (California) were investigated, and the dislocation of microorganisms from the sediment into the water column via gas bubbles released from the seabed was documented. It was found that the methanotrophs transport efficiency was dependent on the volumetric gas flow, with the highest transport rate of 22.7 × 103 cells mLgas-1 at a volumetric gas flow of 0.07 mLgas s-1, and the lowest rate of 0.2 × 103 cells mLgas-1 at a gas flow of 2.2 mLgas s-1. A simple budget approach showed that this bubble-mediated transport has the potential to maintain a relevant part of the water-column methanotrophs in the seep field. The bubble-mediated link between the benthic and pelagic environment was further supported by genetic analyses, indicating a transportation of methanotrophs of the family Methylomonaceae and oil degrading bacteria of the genus Cycloclasticus from the sediment into the water column. These findings demonstrate that the bubble-mediated transport of microorganisms influences the pelagic microbial abundance and community composition at gas-releasing seep sites.

During the water crisis in Flint, Michigan, USA (2014-2015), 2 outbreaks of Legionnaires' disease occurred in Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella pneumophila isolates submitted to a laboratory in Genesee County during the second outbreak with 103 water isolates collected the following year. We documented a genetically diverse range of L. pneumophila strains across clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates, 3 water isolates from a Flint hospital, 1 water isolate from a Flint residence, and the reference Paris strain) had a high degree of similarity (2-1,062 single-nucleotide polymorphisms), all L. pneumophila sequence type 1, serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were widespread in Flint hospital water samples but bore no resemblance to available clinical isolates. L. pneumophila strains in Flint tap water after the outbreaks were diverse and similar to some disease-causing strains.

Frequent and persistent heavy metal pollution has profound effects on the composition and activity of microbial communities. Heavy metals select for metal resistance but can also co-select for resistance to antibiotics, which is a global health concern. We here document metal concentration, metal resistance and antibiotic resistance along a sediment archive from a pond in the North West of the United Kingdom covering over a century of anthropogenic pollution. We specifically focus on zinc, as it is a ubiquitous and toxic metal contaminant known to co-select for antibiotic resistance, to assess the impact of temporal variation in heavy metal pollution on microbial community diversity and to quantify the selection effects of differential heavy metal exposure on antibiotic resistance. Zinc concentration and bioavailability was found to vary over the core, likely reflecting increased industrialisation around the middle of the 20th century. Zinc concentration had a significant effect on bacterial community composition, as revealed by a positive correlation between the level of zinc tolerance in culturable bacteria and zinc concentration. The proportion of zinc resistant isolates was also positively correlated with resistance to three clinically relevant antibiotics (oxacillin, cefotaxime and trimethoprim). The abundance of the class 1 integron-integrase gene, intI1, marker for anthropogenic pollutants correlated with the prevalence of zinc- and cefotaxime resistance but not with oxacillin and trimethoprim resistance. Our microbial palaeontology approach reveals that metal-contaminated sediments from depths that pre-date the use of antibiotics were enriched in antibiotic resistant bacteria, demonstrating the pervasive effects of metal-antibiotic co-selection in the environment.

Production of unconventional oil and gas continues to rise, but the effects of high-density hydraulic fracturing (HF) activity near aquatic ecosystems are not fully understood. A commonly used biocide in HF, 2,2-dibromo-3-nitrilopropionamide (DBNPA), was studied in microcosms of HF-impacted (HF+) versus HF-unimpacted (HF-) surface water streams to (i) compare the microbial community response, (ii) investigate DBNPA degradation products based on past HF exposure, and (iii) compare the microbial community response differences and similarities between the HF biocides DBNPA and glutaraldehyde. The microbial community responded to DBNPA differently in HF-impacted versus HF-unimpacted microcosms in terms of the number of 16S rRNA gene copies quantified, alpha and beta diversity, and differential abundance analyses of microbial community composition through time. The differences in microbial community changes affected degradation dynamics. HF-impacted microbial communities were more sensitive to DBNPA, causing the biocide and by-products of the degradation to persist for longer than in HF-unimpacted microcosms. A total of 17 DBNPA by-products were detected, many of them not widely known as DBNPA by-products. Many of the brominated by-products detected that are believed to be uncharacterized may pose environmental and health impacts. Similar taxa were able to tolerate glutaraldehyde and DBNPA; however, DBNPA was not as effective for microbial control, as indicated by a smaller overall decrease of 16S rRNA gene copies/ml after exposure to the biocide, and a more diverse set of taxa was able to tolerate it. These findings suggest that past HF activity in streams can affect the microbial community response to environmental perturbation such as that caused by the biocide DBNPA.IMPORTANCE Unconventional oil and gas activity can affect pH, total organic carbon, and microbial communities in surface water, altering their ability to respond to new environmental and/or anthropogenic perturbations. These findings demonstrate that 2,2-dibromo-3-nitrilopropionamide (DBNPA), a common hydraulic fracturing (HF) biocide, affects microbial communities differently as a consequence of past HF exposure, persisting longer in HF-impacted (HF+) waters. These findings also demonstrate that DBNPA has low efficacy in environmental microbial communities regardless of HF impact. These findings are of interest, as understanding microbial responses is key for formulating remediation strategies in unconventional oil and gas (UOG)-impacted environments. Moreover, some DBNPA degradation by-products are even more toxic and recalcitrant than DBNPA itself, and this work identifies novel brominated degradation by-products formed.

Background Many studies consider the contamination of dental unit waterlines (DUWLs), but few of them have studied the possible presence of antibiotic resistant Pseudomonas aeruginosa in the DUWLs.Aims Investigation of the presence of P. aeruginosa and Pseudomonas spp. strains in DUWLs and evaluation of their resistance to six antibiotics (ceftazidime, netilmicin, piperacillin/tazobactam, meropenem, levofloxacin, colistin sulfate) at a public dental clinic in Milan, Italy.Results Dental units were contaminated by P. aeruginosa with loads of 2–1,000 CFU/L and were mainly located on the mezzanine floor, with a range of 46–54%, while Pseudomonas spp. were primarily found on the first and second floors, ranging from 50 to 91%. P. aeruginosa was antibiotic resistant in 30% of the strains tested, andPseudomonas spp. in 31.8% . Cold water from controls was also contaminated by these microorganisms.Conclusion Monitoring antibiotic resistance in the water and adopting disinfection procedures on DUs are suggested within the Water Safety Plan.

To elucidate the importance and mechanisms of particle-attached microorganisms on ammonia oxidation, we conducted a controlled simulation experiment with samples collected from the Shunao River, an ammonia-rich hypereutrophic urban river in eastern China. The effects of particle concentration, ammonia concentration, organic carbon source and concentration, dissolved oxygen concentration, and pH were investigated on ammonia transformation rate (ammonia removal rate and NO2 -  + NO3 - accumulation rate) and abundance of particle-attached ammonia-oxidizing bacteria (AOB) and archaea (AOA). All these factors significantly influenced ammonia transformation rates. Our results provided direct evidence that microorganisms attached on riverine suspended particles were associated with ammonia oxidation. Sequencing revealed that the AOA genus Nitrososphaera, and the AOB genus Nitrosomonas were the most dominant in particle-attached ammonia-oxidizing microbial communities. Further analysis showed that AOB communities had higher species richness and diversity compared with AOA communities. Additionally, AOB amoA genes were ~10-100 times more abundant than AOA amoA genes, and AOB abundance was more strongly correlated with ammonia transformation rates than AOA abundance in most experiments, indicating that particle-attached AOB were more important than AOA in the hypereutrophic urban river. This study adds to our knowledge of particle-attached microorganism oxidation of ammonia.

Marine microbial communities are critical for biogeochemical cycles and the productivity of ocean ecosystems. Primary productivity in the surface ocean is constrained by nutrients which are supplied, in part, by mixing with deeper water. Little is known about the time scales, frequency, or impact of mixing on microbial communities. We combined in situ sampling using the Environmental Sample Processor and a small-scale mixing experiment with lower euphotic zone water to determine how individual populations respond to mixing. Transcriptional responses were measured using the MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories) microarray, which targets all three domains of life and viruses. The experiment showed that mixing substantially affects photosynthetic taxa as expected, but surprisingly also showed that populations respond differently to unfiltered deep water which contains particles (organisms and detritus) compared to filtered deep water that only contains nutrients and viruses, pointing to the impact of biological interactions associated with these events. Comparison between experimental and in situ population transcription patterns indicated that manipulated populations can serve as analogs for natural populations, and that natural populations may be frequently or continuously responding to nutrients from deeper waters. Finally, this study also shows that the microarray approach, which is complementary to metatranscriptomic sequencing, is useful for determining the physiological status of in situ microbial communities.

The globally abundant marine Cyanobacteria Prochlorococcus and Synechococcus share many physiological traits but presumably have different evolutionary histories and associated phylogeography. In Prochlorococcus, there is a clear phylogenetic hierarchy of ecotypes, whereas multiple Synechococcus clades have overlapping physiologies and environmental distributions. However, microbial traits are associated with different phylogenetic depths. Using this principle, we reclassified diversity at different phylogenetic levels and compared the phylogeography. We sequenced the genetic diversity of Prochlorococcus and Synechococcus from 339 samples across the tropical Pacific Ocean and North Atlantic Ocean using a highly variable phylogenetic marker gene (rpoC1). We observed clear parallel niche distributions of ecotypes leading to high Pianka's Index values driven by distinct shifts at two transition points. The first transition point at 6°N distinguished ecotypes adapted to warm waters but separated by macronutrient content. At 39°N, ecotypes adapted to warm, low macronutrient vs. colder, high macronutrient waters shifted. Finally, we detected parallel vertical and regional single-nucleotide polymorphism microdiversity within clades from both Prochlorococcus and Synechococcus, suggesting uniquely adapted populations at very specific depths, as well as between the Atlantic and Pacific Oceans. Overall, this study demonstrates that Prochlorococcus and Synechococcus have shared phylogenetic organization of traits and associated phylogeography.

Sinking particles transport carbon and nutrients from the surface ocean into the deep sea and are considered hot spots for bacterial diversity and activity. In the oligotrophic oceans, nitrogen (N2)-fixing organisms (diazotrophs) are an important source of new N but the extent to which these organisms are present and exported on sinking particles is not well known. Sinking particles were collected every 6 h over a 2-day period using net traps deployed at 150 m in the North Pacific Subtropical Gyre. The bacterial community and composition of diazotrophs associated with individual and bulk sinking particles was assessed using 16S rRNA and nifH gene amplicon sequencing. The bacterial community composition in bulk particles remained remarkably consistent throughout time and space while large variations of individually picked particles were observed. This difference suggests that unique biogeochemical conditions within individual particles may offer distinct ecological niches for specialized bacterial taxa. Compared to surrounding seawater, particle samples were enriched in different size classes of globally significant N2-fixing cyanobacteria including Trichodesmium, symbionts of diatoms, and the unicellular cyanobacteria Crocosphaera and UCYN-A. The particles also contained nifH gene sequences of diverse non-cyanobacterial diazotrophs suggesting that particles could be loci for N2 fixation by heterotrophic bacteria. The results demonstrate that diverse diazotrophs were present on particles and that new N may thereby be directly exported from surface waters on sinking particles.

Knowledge of the ecology of N2-fixing (diazotrophic) plankton is mainly limited to oligotrophic (sub)tropical oceans. However, diazotrophs are widely distributed and active throughout the global ocean. Likewise, relatively little is known about the temporal dynamics of diazotrophs in productive areas. Between February 2014 and December 2015, we carried out 9 one-day samplings in the temperate northwestern Iberian upwelling system to investigate the temporal and vertical variability of the diazotrophic community and its relationship with hydrodynamic forcing. In downwelling conditions, characterized by deeper mixed layers and a homogeneous water column, non-cyanobacterial diazotrophs belonging mainly to nifH clusters 1G (Gammaproteobacteria) and 3 (putative anaerobes) dominated the diazotrophic community. In upwelling and relaxation conditions, affected by enhanced vertical stratification and hydrographic variability, the community was more heterogeneous vertically but less diverse, with prevalence of UCYN-A (unicellular cyanobacteria, subcluster 1B) and non-cyanobacterial diazotrophs from clusters 1G and 3. Oligotyping analysis of UCYN-A phylotype showed that UCYN-A2 sublineage was the most abundant (74%), followed by UCYN-A1 (23%) and UCYN-A4 (2%). UCYN-A1 oligotypes exhibited relatively low frequencies during the three hydrographic conditions, whereas UCYN-A2 showed higher abundances during upwelling and relaxation. Our findings show the presence of a diverse and temporally variable diazotrophic community driven by hydrodynamic forcing in an upwelling system.

Determining the carbon sources for active microbial populations in the subsurface is a challenging but highly informative component of subsurface microbial ecology. This work developed a method to provide ecological insights into groundwater microbial communities by characterizing community RNA through its radiocarbon and ribosomal RNA (rRNA) signatures. RNA was chosen as the biomolecule of interest because rRNA constitutes the majority of RNA in prokaryotes, represents recently active organisms, and yields detailed taxonomic information. The method was applied to a groundwater filter collected from a shallow alluvial aquifer in Colorado. RNA was extracted, radiometrically dated, and the 16S rRNA was analyzed by RNA-Seq. The RNA had a radiocarbon signature (Δ14C) of -193.4 ± 5.6‰. Comparison of the RNA radiocarbon signature to those of potential carbon pools in the aquifer indicated that at least 51% of the RNA was derived from autotrophy, in close agreement with the RNA-Seq data, which documented the prevalence of autotrophic taxa, such as Thiobacillus and Gallionellaceae. Overall, this hybrid method for RNA analysis provided cultivation-independent information on the in-situ carbon sources of active subsurface microbes and reinforced the importance of autotrophy and the preferential utilization of dissolved over sedimentary organic matter in alluvial aquifers.

Microbial communities are critical to ecosystem function. A key objective of metagenomic studies is to analyse organism-specific metabolic pathways and reconstruct community interaction networks. This requires accurate assignment of assembled genome fragments to genomes. Existing binning methods often fail to reconstruct a reasonable number of genomes and report many bins of low quality and completeness. Furthermore, the performance of existing algorithms varies between samples and biotopes. Here, we present a dereplication, aggregation and scoring strategy, DAS Tool, that combines the strengths of a flexible set of established binning algorithms. DAS Tool applied to a constructed community generated more accurate bins than any automated method. Indeed, when applied to environmental and host-associated samples of different complexity, DAS Tool recovered substantially more near-complete genomes, including previously unreported lineages, than any single binning method alone. The ability to reconstruct many near-complete genomes from metagenomics data will greatly advance genome-centric analyses of ecosystems.