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Complications of diabetes, such as diabetic foot ulcers (DFUs), are common, multifactorial in origin, and costly to treat. DFUs are the cause of nearly 90% of limb amputations among persons with diabetes. In most chronic infections such as DFU, biofilms are involved. Bacteria in biofilms are 100-1000 times more resistant to antibiotics than their planktonic counterparts. Multidrug-resistant (MDR) Staphylococcus aureus and Pseudomonas aeruginosa infections in DFUs may require alternative therapeutic agents such as bacteriophages ("phages"). This study describes the lytic activity of phage cocktails AB-SA01 (3-phage cocktail) and AB-PA01 (4-phage cocktail), which target S. aureus and P. aeruginosa, respectively. The host range and lytic effect of AB-SA01 and AB-PA01 on a planktonic culture, single-species biofilm, and mixed-species biofilm were evaluated. In vitro testing showed that 88.7% of S. aureus and 92.7% of P. aeruginosa isolates were susceptible to AB-SA01 and AB-PA01, respectively, in the planktonic state. The component phages of AB-SA01 and AB-PA01 infected 66% to 94.3% of the bacterial isolates tested. Furthermore, AB-SA01 and AB-PA01 treatment significantly (p < 0.05) reduced the biofilm biomass of their hosts, regardless of the antibiotic-resistant characteristics of the isolates and the presence of a non-susceptible host. In conclusion, the strong lytic activity, broad host range, and significant biofilm biomass reduction of AB-SA01 and AB-PA01 suggest the considerable potential of phages in treating antibiotic-resistant S. aureus and P. aeruginosa infections alone or as coinfections in DFUs.
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Bacteriófagos , Diabetes Mellitus , Pie Diabético , Staphylococcus aureus Resistente a Meticilina , Humanos , Staphylococcus aureus , Pie Diabético/terapia , Antibacterianos/farmacología , BiopelículasRESUMEN
Allometric settings of population dynamics models are appealing due to their parsimonious nature and broad utility when studying system level effects. Here, we parameterise the size-scaled Rosenzweig-MacArthur differential equations to eliminate prey-mass dependency, facilitating an in depth analytic study of the equations which incorporates scaling parameters' contributions to coexistence. We define the functional response term to match empirical findings, and examine situations where metabolic theory derivations and observation diverge. The dynamical properties of the Rosenzweig-MacArthur system, encompassing the distribution of size-abundance equilibria, the scaling of period and amplitude of population cycling, and relationships between predator and prey abundances, are consistent with empirical observation. Our parameterisation is an accurate minimal model across 15+ orders of mass magnitude.
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Ciclismo , Dinámica PoblacionalRESUMEN
Environments shape communities by driving individual interactions and the evolutionary outcome of competition. In static, homogeneous environments a robust, evolutionary stable, outcome is sometimes reachable. However, inherently stochastic, this evolutionary process need not stabilize, resulting in a dynamic ecological state, often observed in microbial communities. We use evolutionary games to study the evolution of phenotypic competition in dynamic environments. Under the assumption that phenotypic expression depends on the environmental shifts, existing periodic relationships may break or result in formation of new periodicity in phenotypic interactions. The exact outcome depends on the environmental shift itself, indicating the importance of understanding how environments influence affected systems. Under periodic environmental fluctuations, a stable state preserving dominant phenotypes may exist. However, rapid environmental shifts can lead to critical shifts in the phenotypic evolutionary balance. This might lead to environmentally favoured phenotypes dominating making the system vulnerable. We suggest that understanding of the robustness of the system's current state is necessary to anticipate when it will shift to a new equilibrium via understanding what level of perturbations the system can take before its equilibrium changes. Our results provide insights in how microbial communities can be steered to states where they are dominated by desired phenotypes.
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BACKGROUND: Due to the ever-expanding gap between the number of proteins being discovered and their functional characterization, protein function inference remains a fundamental challenge in computational biology. Currently, known protein annotations are organized in human-curated ontologies, however, all possible protein functions may not be organized accurately. Meanwhile, recent advancements in natural language processing and machine learning have developed models which embed amino acid sequences as vectors in n-dimensional space. So far, these embeddings have primarily been used to classify protein sequences using manually constructed protein classification schemes. RESULTS: In this work, we describe the use of amino acid sequence embeddings as a systematic framework for studying protein ontologies. Using a sequence embedding, we show that the bacterial carbohydrate metabolism class within the SEED annotation system contains 48 clusters of embedded sequences despite this class containing 29 functional labels. Furthermore, by embedding Bacillus amino acid sequences with unknown functions, we show that these unknown sequences form clusters that are likely to have similar biological roles. CONCLUSIONS: This study demonstrates that amino acid sequence embeddings may be a powerful tool for developing more robust ontologies for annotating protein sequence data. In addition, embeddings may be beneficial for clustering protein sequences with unknown functions and selecting optimal candidate proteins to characterize experimentally.
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Biología Computacional , Proteínas , Secuencia de Aminoácidos , Bacterias , Biología Computacional/métodos , Humanos , Aprendizaje Automático , Anotación de Secuencia Molecular , Proteínas/químicaRESUMEN
The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation; however, this relies on compatibility between yeast and bacterial strains. For the first time, quantitative trait loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny from a hybrid S. cerevisiae strain (SBxGN), co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulphite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results revealed a strong effect of SSU1 haploinsufficiency on O. oeni's ability to complete malolactic fermentation during co-inoculation and pave the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions. KEY POINTS: ⢠For the first time, QTL analysis has been used to study yeast-bacteria interactions. ⢠A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes. ⢠S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.
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Oenococcus , Vino , Fermentación , Determinismo Genético , Malatos , Sitios de Carácter Cuantitativo , Saccharomyces cerevisiae/genética , Vino/análisisRESUMEN
The ability of influenza A virus to evolve, coupled with increasing antimicrobial resistance, could trigger an influenza pandemic with great morbidity and mortality. Much of the 1918 influenza pandemic mortality was likely due to bacterial coinfection, including Staphylococcus aureus pneumonia. S. aureus resists many antibiotics. The lack of new antibiotics suggests alternative antimicrobials, such as bacteriophages, are needed. Potential delivery routes for bacteriophage therapy (BT) include inhalation and intravenous injection. BT has recently been used successfully in compassionate access pulmonary infection cases. Phage lysins, enzymes that hydrolyze bacterial cell walls and which are bactericidal, are efficacious in animal pneumonia models. Clinical trials will be needed to determine whether BT can ameliorate disease in influenza and S. aureus coinfection.
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Bacteriófagos/fisiología , Coinfección/terapia , Virus de la Influenza A/fisiología , Gripe Humana/terapia , Terapia de Fagos , Neumonía Estafilocócica/terapia , Staphylococcus aureus/virología , Animales , Coinfección/microbiología , Coinfección/mortalidad , Coinfección/virología , Humanos , Virus de la Influenza A/genética , Gripe Humana/mortalidad , Gripe Humana/virología , Neumonía Estafilocócica/microbiología , Neumonía Estafilocócica/mortalidad , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologíaRESUMEN
Flow cytometry is a high-throughput tool for determining microbial abundance in a range of medical, environmental, and food-related samples. For wine, determining the abundance of Saccharomyces cerevisiae is well-defined and reliable. However, for the most common wine bacterium, Oenococcus oeni, using flow cytometry to determine cell concentration poses some challenges. O. oeni most often occurs in doublets or chains of varying lengths that can be greater than seven cells. This wine bacterium is also small, at 0.2-0.6 µm and may exhibit a range of morphologies including binary fission and aggregated complexes. This work demonstrates a straightforward approach to determining the suitability of flow cytometry for the chain-forming bacteria, O. oeni, and considerations when using flow cytometry for the enumeration of small microorganisms (<0.5 µm). © 2020 International Society for Advancement of Cytometry.
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Oenococcus , Vino , Fermentación , Saccharomyces cerevisiae , Vino/análisisRESUMEN
High rate algal ponds (HRAPs) are shallow, mixed systems for wastewater treatment, which use sunlight exposure for disinfection. Little is known regarding the relationships between the bacteria and viruses within HRAP systems. Uniquely, flow cytometry permits the rapid identification of bacterial and viral populations in wastewater samples, separating populations based on genome and particle size. Treated wastewater samples were collected from an HRAP at Kingston on Murray, South Australia. Flow cytometry analysis detected bacterial populations and discriminated virus-like particles (VLP) and large VLP (LVLP). Rapid, short term, fluctuations in the abundance of all three populations were observed. Changes in the abundance of these populations was compared; wastewater composition was used as metadata for the comparisons. Linear regression determined relationships in abundances between bacteria and LVLP (R2 0.2985); LVLP and VLP (R2 0.5829) and bacteria and VLP (R2 0.5778) all with p-values of <0.001. Bacterial, LVLP and VLP abundance positively correlated with each other, indicating potential microbial interactions. Overall, the results suggest a parasitic relationship was occurring and driving the abundances of bacteria and viruses within the system.
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Estanques , Eliminación de Residuos Líquidos , Citometría de Flujo , Australia del Sur , Aguas ResidualesRESUMEN
BACKGROUND: Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus. Antibiotic-resistant Staphylococcus aureus is frequently isolated from DFU infections. Bacteriophages (phages) represent an alternative or adjunct treatment to antibiotic therapy. Here we describe the efficacy of AB-SA01, a cocktail of three S. aureus Myoviridae phages, made to current good manufacturing practice (cGMP) standards, and which has undergone two phase I clinical trials, in treatment of multidrug-resistant (MDR) S. aureus infections. RESULTS: Wounds of saline-treated mice showed no healing, but expanded and became inflamed, ulcerated, and suppurating. In contrast, AB-SA01 treatment decreased the bacterial load with efficacy similar or superior to vancomycin treatment. At the end of the treatment period, there was a significant decrease (p < 0.001) in bacterial load and wound size in infected phage- and vancomycin-treated groups compared with infected saline-treated mice. In phage-treated mice, wound healing was seen similar to vancomycin treatment. No mortality was recorded associated with infections, and post-mortem examinations did not show any evident pathological lesions other than the skin wounds. No adverse effects related to the application of phages were observed. CONCLUSION: Topical application of phage cocktail AB-SA01 is effective, as shown by bacterial load reduction and wound closure, in the treatment of diabetic wound infections caused by MDR S. aureus. Our results suggest that topical phage cocktail treatment may be effective in treating antibiotic-resistant S. aureus DFU infections.
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Diabetes Mellitus Experimental/complicaciones , Pie Diabético/microbiología , Terapia de Fagos/métodos , Infecciones Estafilocócicas/terapia , Staphylococcus aureus/crecimiento & desarrollo , Infección de Heridas/microbiología , Animales , Carga Bacteriana/efectos de los fármacos , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple , Masculino , Ratones , Staphylococcus aureus/aislamiento & purificación , Vancomicina/administración & dosificación , Vancomicina/farmacología , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/terapiaRESUMEN
The efficacy of phages in multispecies infections has been poorly examined. The in vitro lytic efficacies of phage cocktails AB-SA01, AB-PA01, which target Staphylococcus aureus and Pseudomonas aeruginosa, respectively, and their combination against their hosts were evaluated in S. aureus and P. aeruginosa mixed-species planktonic and biofilm cultures. Green fluorescent protein (GFP)-labelled P. aeruginosa PAO1 and mCherry-labelled S. aureus KUB7 laboratory strains and clinical isolates were used as target bacteria. During real-time monitoring using fluorescence spectrophotometry, the density of mCherry S. aureus KUB7 and GFP P. aeruginosa PAO1 significantly decreased when treated by their respective phage cocktail, a mixture of phage cocktails, and gentamicin. The decrease in bacterial density measured by relative fluorescence strongly associated with the decline in bacterial cell counts. This microplate-based mixed-species culture treatment monitoring through spectrophotometry combine reproducibility, rapidity, and ease of management. It is amenable to high-throughput screening for phage cocktail efficacy evaluation. Each phage cocktail, the combination of the two phage cocktails, and tetracycline produced significant biofilm biomass reduction in mixed-species biofilms. This study result shows that these phage cocktails lyse their hosts in the presence of non-susceptible bacteria. These data support the use of phage cocktails therapy in infections with multiple bacterial species.
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Bacteriófagos/fisiología , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Antibacterianos/farmacología , Bacteriófagos/clasificación , Biopelículas/efectos de los fármacos , Técnicas de Cocultivo , Recuento de Colonia Microbiana , Farmacorresistencia Bacteriana , Fluorescencia , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/virología , Reproducibilidad de los Resultados , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/virologíaRESUMEN
Objective: Microhabitats in the oral cavity differ in microbial taxonomy. However, abundance variations of bacterial and viral communities within these microhabitats are not fully understood. Aims and Hypothesis: To assess the spatial distribution and dynamics of the microbial abundances within 6 microhabitats of the oral cavity before and after sleep. We hypothesise that the abundance distributions of these microbial communities will differ among oral sites. Methods: Using flow cytometry, bacterial and virus-like particle (VLP) abundances were enumerated for 6 oral microhabitats before and after sleep in 10 healthy paediatric sleepers. Results: Bacterial counts ranged from 7.2 ± 2.8 × 105 at the palate before sleep to 1.3 ± 0.2 × 108 at the back of the tongue after sleep, a difference of 187 times. VLPs ranged from 1.9 ± 1.0 × 106 at the palate before sleep to 9.2 ± 5.0 × 107 at the back of the tongue after sleep, a difference of 48 times. Conclusion: The oral cavity is a dynamic numerically heterogeneous environment where microbial communities can increase by a count of 100 million during sleep. Quantification of the paediatric oral microbiome complements taxonomic diversity information to show how biomass varies and shifts in space and time.
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Bacteria are ubiquitous on the Earth, and many use chemotaxis to colonise favourable microenvironments. The colonisation process is continuous, where animals, plants, protists, viruses and chemical and physical factors frequently remove bacteria from wide volume ranges. Colonisation processes are poorly understood in natural communities. Here, we investigated niche partitioning during colonisation in aquatic microbial communities using bands of bacterial chemotaxis in petri dishes from mixed-species communities. The community partitioned into loiterers, primary and secondary colonisers, each having distinct abundances and taxonomy. Within marine samples, Shewanella dominated the primary colonisers, whilst Enterobacteriaceae dominated this group within the freshwater samples. Whether the success of these specific groups translates to what occurs within natural communities is uncertain, but here we show these taxa have the capacity to colonise new, unexplored environments. A strong negative association existed between the primary colonisers and viral abundance, suggesting that successful colonisers simultaneously move toward areas of heightened resources, which correlated with lower virus-like particles. Here, we show that microbial communities constantly sort themselves into distinct taxonomic groups as they move into new environments. This sorting during colonisation may be fundamental to microbial ecology, industry, technology, and disease development by setting the initial conditions that determine the winners as a community develops.
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Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Agua Dulce/microbiología , Agua de Mar/microbiología , Animales , Bacterias/clasificación , Agua Dulce/virología , Microbiota , Agua de Mar/virología , Virus , Microbiología del AguaRESUMEN
Subsurface environments hold the largest reservoir of microbes in the biosphere. They play essential roles in transforming nutrients, degrading contaminants and recycling organic matter. Here, we propose a previously unrecognised fundamental microbial process that influences aquifer bioremediation dynamics and that applies to all microbial communities. In contrast to previous models, our proposed Piggyback-the-Persistent (PtP) mechanism occurs when viruses become more dominated by those exhibiting temperate rather than lytic lifestyles driven by persistent chemicals (in our case chlorinated-hydrocarbon pollutants) that provide long-term carbon sources and that refocus the aquifer carbon cycle, thus altering the microbial community. In this ultra-oligotrophic system, the virus:microbial ratio (VMR) ranges from below the detection limit of 0.0001 to 0.6, well below the common aquatic range of 3-10. Shortest-average-path network analysis revealed VMR and trichlorethene (TCE) as nodes through which ecosystem information and biomass most efficiently pass. Novel network rearrangement revealed a hierarchy of Kill-the-Winner (KtW), Piggyback-the-Winner (PtW) and PtP nodes. We propose that KtW, PtW and PtP occur simultaneously as competing strategies, with their relative importance depending on conditions at a particular time and location with unusual nutrient sources, such as TCE, appearing to contribute to a shift in this balance between viral mechanisms.
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Bacterias/virología , Agua Subterránea/microbiología , Tricloroetileno/metabolismo , Fenómenos Fisiológicos de los Virus/efectos de los fármacos , Virus/crecimiento & desarrollo , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Biodegradación Ambiental , Biomasa , Ecosistema , Hidrocarburos/metabolismo , Carga ViralRESUMEN
There is potential for phages to prevent and control bacterial biofilms, but few studies have examined the effect of phages on the multispecies biofilms that characterize most bacterial infections. This paper reviews the mechanism of action of phages, the evidence supporting the view that phage therapy will be effective against bacterial targets and the opposite viewpoint, phage application approaches, and the comparative advantage of phage therapy in multispecies biofilms. The few reports measuring the actions of lytic phages against multispecies biofilms are also reviewed. The authors are cautiously optimistic about the application of phages against their targets when in multispecies biofilms because some lysis mechanisms do not require species specificity.
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Bacteriófagos , Biopelículas , HumanosRESUMEN
Groundwater is increasingly used globally for domestic, industrial and agricultural production. While many studies have focused on groundwater as a resource, the diverse ecosystems within are often ignored. Here, we assess 54 Southern South Australian groundwater microbial communities from the populated part of the state to assess their status and dynamics in isolated groundwater systems. We observed a strong site-to-site individuality in groundwater bacterial communities, likely due to the isolated nature of groundwater bodies leading to unique ecosystems. Rank abundance analysis indicates bacterial diversity is maintained even at low abundances and that the distribution fits classical ecological models for strong competition in resource-limited environments. Combined, our data indicates that despite overrepresentation of pollutant-associated bacterial orders in and around the Adelaide metropolitan area, microbial communities remain diverse and show little evidence of converging on a common pollutant-effected community.
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Agua Subterránea/microbiología , Microbiología del Agua , Biodiversidad , Ecosistema , Agua Subterránea/química , Australia del Sur , Contaminantes del Agua/análisisRESUMEN
The spatial distributions of organism abundance and diversity are often heterogeneous. This includes the sub-centimetre distributions of microbes, which have 'hotspots' of high abundance, and 'coldspots' of low abundance. Previously we showed that 300 µl abundance hotspots, coldspots and background regions were distinct at all taxonomic levels. Here we build on these results by showing taxonomic micropatches within these 300 µl microscale hotspots, coldspots and background regions at the 1 µl scale. This heterogeneity among 1 µl subsamples was driven by heightened abundance of specific genera. The micropatches were most pronounced within hotspots. Micropatches were dominated by Pseudomonas, Bacteroides, Parasporobacterium and Lachnospiraceae incertae sedis, with Pseudomonas and Bacteroides being responsible for a shift in the most dominant genera in individual hotspot subsamples, representing up to 80.6% and 47.3% average abundance, respectively. The presence of these micropatches implies the ability these groups have to create, establish themselves in, or exploit heterogeneous microenvironments. These genera are often particle-associated, from which we infer that these micropatches are evidence for sub-millimetre aggregates and the aquatic polymer matrix. These findings support the emerging paradigm that the microscale distributions of planktonic microbes are numerically and taxonomically heterogeneous at scales of millimetres and less. We show that microscale microbial hotspots have internal structure within which specific local nutrient exchanges and cellular interactions might occur.
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Bacterias/clasificación , Bacterias/crecimiento & desarrollo , Evolución Biológica , ARN Ribosómico 16S/genética , Ríos/microbiología , Microbiología del Agua , Bacterias/genética , Biología Computacional/métodos , Ríos/virologíaRESUMEN
The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.
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Pinzones/parasitología , Microbiota , Muscidae/microbiología , Animales , Ecuador , Especies Introducidas , Islas , Larva/microbiología , Parásitos/microbiología , ARN Ribosómico 16S/genéticaRESUMEN
More than 97% of the world's freshwater reserves are found in aquifers, making groundwater one of the most important resources on the planet. Prokaryotic communities in groundwater underpin the turnover of energy and matter while also maintaining groundwater purity. Thus, knowledge of microbial transport in the subsurface is crucial for maintaining groundwater health. Here, we describe for the first time the importance of stygofauna as vectors for prokaryotes. The "hitch-hiking" prokaryotes associated with stygofauna may be up to 5 orders of magnitude higher in abundance and transported up to 34× faster than bulk groundwater flow. We also demonstrate that prokaryotic diversity associated with stygofauna may be higher than that of the surrounding groundwater. Stygofauna are a newly recognized prokaryotic niche in groundwater ecosystems that have the potential to transport remediating, water purifying and pathogenic prokaryotes. Therefore, stygofauna may influence ecosystem dynamics and health at a microbial level, and at a larger scale could be a new source of prokaryotic diversity in groundwater ecosystems.
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Agua Subterránea/microbiología , Células Procariotas/fisiología , Biodiversidad , EcosistemaRESUMEN
Viral communities are important for ecosystem function as they are involved in critical biogeochemical cycles and controlling host abundance. This study investigates riverine viral communities around a small rural town that influences local water inputs. Myoviridae, Siphoviridae, Phycodnaviridae, Mimiviridae, Herpesviridae, and Podoviridae were the most abundant families. Viral species upstream and downstream of the town were similar, with Synechoccocus phage, salinus, Prochlorococcus phage, Mimivirus A, and Human herpes 6A virus most abundant, contributing to 4.9-38.2% of average abundance within the metagenomic profiles, with Synechococcus and Prochlorococcus present in metagenomes as the expected hosts for the phage. Overall, the majority of abundant viral species were or were most similar to those of marine origin. At over 60 km to the river mouth, the presence of marine communities provides some support for the Baas-Becking hypothesis "everything is everywhere, but, the environment selects." We conclude marine microbial species may occur more frequently in freshwater systems than previously assumed, and hence may play important roles in some freshwater ecosystems within tens to a hundred kilometers from the sea.