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Background: The Enterobacterales are a group of Gram-negative bacteria frequently exhibiting extended antimicrobial resistance (AMR) and involved in the transmission of resistance genes to other bacterial species present in the same environment. Due to their impact on human health and the paucity of new antibiotics, the World Health Organization (WHO) categorized carbapenem resistant and ESBL-producing as critical. Enterobacterales are ubiquitous and the role of the environment in the transmission of AMR organisms or antimicrobial resistance genes (ARGs) must be examined in tackling AMR in both humans and animals under the one health approach. Animal manure is recognized as an important source of AMR bacteria entering the environment, in which resistant genes can accumulate., Methods: To gain a better understanding of the dissemination of third generation cephalosporin and fluoroquinolone resistance genes between isolates in the environment, we applied whole genome sequencing (WGS) to Enterobacterales (79 E. coli , 1 Enterobacter cloacae , 1 Klebsiella pneumoniae , and 1 Citrobacter gillenii ) isolated from farm effluents in Ireland before ( n  = 72) and after ( n  = 10) treatment by integrated constructed wetlands (ICWs). DNA was extracted using the MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) followed by WGS on a MiSeq platform (Illumina, Eindhoven, Netherlands) using v3 chemistry as 300-cycle paired-end runs. AMR genes and point mutations were identified and compared to the phenotypic results for better understanding of the mechanisms of resistance and resistance transmission., Results: A wide variety of cephalosporin and fluoroquinolone resistance genes (mobile genetic elements (MGEs) and chromosomal mutations) were identified among isolates that mostly explained the phenotypic AMR patterns. A total of 31 plasmid replicon types were identified among the 82 isolates, with a subset of them ( n  = 24), identified in E. coli isolates. Five plasmid replicons were confined to the Enterobacter cloacae isolate and two were confined to the Klebsiella pneumoniae isolate. Virulence genes associated with functions including stress, survival, regulation, iron uptake secretion systems, invasion, adherence and toxin production were identified., Conclusion: Our study showed that antimicrobial resistant organisms (AROs) can persist even following wastewater treatment and could transmit AMR of clinical relevance to the environment and ultimately pose a risk to human or animal health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Prendergast, Slowey, Burgess, Murphy, Johnston, Morris, O’ Doherty, Moriarty and Gutierrez.)

The ruminal microbial community is an important element in health, nutrition, livestock productivity, and climate impact. Despite the historic and current efforts to characterize this microbial diversity, many of its members remain unidentified, making it challenging to associate microbial groups with functions. Here we present a low-cost methodology for rumen sample treatment that separates the microbial community based on cell size, allowing for the identification of subtle compositional changes. In brief, the sample is centrifuged through a series of sucrose density gradients, and cells migrate to their corresponding density fraction. From each fraction, DNA is extracted and 16S rRNA gene amplicons are sequenced. We tested our methodology on four animals under two different conditions, fasting, and post-feeding. Each fraction was examined by confocal microscopy showing that the same sucrose fraction consistently separated similar cell-sized microorganisms independent of the animal or treatment. Microbial composition analysis using metabarcoding showed that our methodology detected low abundance bacterial families and population changes between fasting and post-feeding treatments that could not be observed by bulk DNA analysis. In conclusion, the sucrose-based method is a powerful low-cost approximation to untwine, enrich, and potentially isolate uncharacterized members of the ruminal microbiome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hernández, Jimenez, Vargas-Garcia, Caro-Quintero and Reyes.)

Poly-extreme terrestrial habitats are often used as analogs to extra-terrestrial environments. Understanding the adaptive strategies allowing bacteria to thrive and survive under these conditions could help in our quest for extra-terrestrial planets suitable for life and understanding how life evolved in the harsh early earth conditions. A prime example of such a survival strategy is the modification of vegetative cells into resistant resting structures. These differentiated cells are often observed in response to harsh environmental conditions. The environmental strain (strain Lr5/4) belonging to Serratia ureilytica was isolated from a geothermal spring in Lirima, Atacama Desert, Chile. The Atacama Desert is the driest habitat on Earth and furthermore, due to its high altitude, it is exposed to an increased amount of UV radiation. The geothermal spring from which the strain was isolated is oligotrophic and the temperature of 54°C exceeds mesophilic conditions (15 to 45°C). Although the vegetative cells were tolerant to various environmental insults (desiccation, extreme pH, glycerol), a modified cell type was formed in response to nutrient deprivation, UV radiation and thermal shock. Scanning (SEM) and Transmission Electron Microscopy (TEM) analyses of vegetative cells and the modified cell structures were performed. In SEM, a change toward a circular shape with reduced size was observed. These circular cells possessed what appears as extra coating layers under TEM. The resistance of the modified cells was also investigated, they were resistant to wet heat, UV radiation and desiccation, while vegetative cells did not withstand any of those conditions. A phylogenomic analysis was undertaken to investigate the presence of known genes involved in dormancy in other bacterial clades. Genes related to spore-formation in Myxococcus and Firmicutes were found in S. ureilytica Lr5/4 genome; however, these genes were not enough for a full sporulation pathway that resembles either group. Although, the molecular pathway of cell differentiation in S. ureilytica Lr5/4 is not fully defined, the identified genes may contribute to the modified phenotype in the Serratia genus. Here, we show that a modified cell structure can occur as a response to extremity in a species that was previously not known to deploy this strategy. This strategy may be widely spread in bacteria, but only expressed under poly-extreme environmental conditions.

CL500-11 (phylum Chloroflexi ) is one of the most ubiquitous and abundant bacterioplankton lineages in deep freshwater lakes inhabiting the oxygenated hypolimnion. While metagenomics predicted possible eco-physiological characteristics of this uncultured lineage, no consensus on their ecology has so far been reached, partly because their niche is not clearly understood due to a limited number of quantitative field observations. This study investigated the abundance and distribution of CL500-11 in seven deep perialpine lakes using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken vertically (5-12 depths in each lake) and temporally (in two lakes) at the deepest point of the lakes located in Switzerland, Italy, and Austria with varying depth, trophic state, mixing regime, and water retention time. The results showed a dominance of CL500-11 in all the lakes; their proportion to total prokaryotes ranged from 4.3% (Mondsee) to 24.3% (Lake Garda) and their abundance ranged from 0.65 × 10 5 (Mondsee) to 1.77 × 10 5 (Lake Garda) cells mL -1 . By summarizing available information on CL500-11 occurrence to date, we demonstrated their broad habitat spectrum, ranging from ultra-oligotrophic to meso-eutrophic lakes, while low abundances or complete absence was observed in lakes with shallow depth, low pH, and/or short water retention time (<1 year). Together with available metagenomic and geochemical evidences from literatures, here we reviewed potential substrates supporting growth of CL500-11. Overall, the present study further endorsed ubiquity and quantitative significance of CL500-11 in deep freshwater systems and narrowed the focus on their physiological characteristics and ecological importance.

In many parts of the world, agricultural ecosystems are increasingly exposed to severe drought, and rainfall events due to climate changes. This coincides with a higher vulnerability of crops to soil-borne diseases, which is mostly ascribed to decreased resistance to pathogen attacks. However, loss of the natural capacity of soil microbes to suppress soil-borne plant pathogens may also contribute to increased disease outbreaks. In this perspectives paper, we will discuss the effect of extreme weather events on pathogen-antagonist interactions during drought and rainfall events and upon recovery. We will focus on diseases caused by root-infecting fungi and oomycetes. In addition, we will explore factors that affect restoration of the balance between pathogens and other soil microbes. Finally, we will indicate potential future avenues to improve the resistance and/or recovery of natural biocontrol during, and after water stresses. As such, our perspective paper will highlight a knowledge gap that needs to be bridged to adapt agricultural ecosystems to changing climate scenarios.

The objective was to examine effects of treating commercial beef feedlot cattle with therapeutic doses of tulathromycin, a macrolide antimicrobial drug, on changes in the fecal resistome and microbiome using shotgun metagenomic sequencing. Two pens of cattle were used, with all cattle in one pen receiving metaphylaxis treatment (800 mg subcutaneous tulathromycin) at arrival to the feedlot, and all cattle in the other pen remaining unexposed to parenteral antibiotics throughout the study period. Fecal samples were collected from 15 selected cattle in each group just prior to treatment (Day 1), and again 11 days later (Day 11). Shotgun sequencing was performed on isolated metagenomic DNA, and reads were aligned to a resistance and a taxonomic database to identify alignments to antimicrobial resistance (AMR) gene accessions and microbiome content. Overall, we identified AMR genes accessions encompassing 9 classes of AMR drugs and encoding 24 unique AMR mechanisms. Statistical analysis was used to identify differences in the resistome and microbiome between the untreated and treated groups at both timepoints, as well as over time. Based on composition and ordination analyses, the resistome and microbiome were not significantly different between the two groups on Day 1 or on Day 11. However, both the resistome and microbiome changed significantly between these two sampling dates. These results indicate that the transition into the feedlot-and associated changes in diet, geography, conspecific exposure, and environment-may exert a greater influence over the fecal resistome and microbiome of feedlot cattle than common metaphylactic antimicrobial drug treatment.

Diet and lifestyle have a strong influence on gut microbiota, which in turn has important implications on a variety of health-related aspects. Despite great advances in the field, it remains unclear to which extent the composition of the gut microbiota is modulated by the intake of animal derived products, compared to a vegetable based diet. Here the specific impact of vegan, vegetarian, and omnivore feeding type on the composition of gut microbiota of 101 adults was investigated among groups homogeneous for variables known to have a role in modulating gut microbial composition such as age, anthropometric variables, ethnicity, and geographic area. The results displayed a picture where the three different dietetic profiles could be well distinguished on the basis of participant's dietetic regimen. Regarding the gut microbiota; vegetarians had a significantly greater richness compared to omnivorous. Moreover, counts of Bacteroidetes related operational taxonomic units (OTUs) were greater in vegans and vegetarians compared to omnivores. Interestingly considering the whole bacterial community composition the three cohorts were unexpectedly similar, which is probably due to their common intake in terms of nutrients rather than food, e.g., high fat content and reduced protein and carbohydrate intake. This finding suggests that fundamental nutritional choices such as vegan, vegetarian, or omnivore do influence the microbiota but do not allow to infer conclusions on gut microbial composition, and suggested the possibility for a preferential impact of other variables, probably related to the general life style on shaping human gut microbial community in spite of dietary influence. Consequently, research were individuals are categorized on the basis of their claimed feeding types is of limited use for scientific studies, since it appears to be oversimplified.

Biofilms formed on rubber seals in dishwashers harbor diverse microbiota. In this study, we focussed on the microbial composition of bacteria and fungi, isolated from a defined area of one square centimeter of rubber from four domestic dishwashers and assessed their abilities to in vitro multispecies biofilm formation. A total of 80 isolates (64 bacterial and 16 fungal) were analyzed. Multiple combinations of bacterial isolates from each dishwasher were screened for synergistic interactions. 32 out of 140 tested (23%) four-species bacterial combinations displayed consistent synergism leading to an overall increase in biomass, in all experimental trails. Bacterial isolates from two of the four dishwashers generated a high number of synergistically interacting four-species consortia. Network based correlation analyses also showed higher co-occurrence patterns observed between bacterial members in the same two dishwasher samples, indicating cooperative effects. Furthermore, two synergistic four-species bacterial consortia were tested for their abilities to incorporate an opportunistic fungal pathogen, Exophiala dermatitidis and their establishment as biofilms on sterile ethylene propylene diene monomer M-class (EPDM) rubber and polypropylene (PP) surfaces. When the bacterial consortia included E. dermatitidis , the overall cell numbers of both bacteria and fungi increased and a substantial increase in biofilm biomass was observed. These results indicate a novel phenomenon of cross kingdom synergy in biofilm formation and these observations could have potential implications for human health.

Infections caused by staphylococci represent a medical concern, especially when related to biofilms located in implanted medical devices, such as prostheses and catheters. Unfortunately, their frequent resistance to high doses of antibiotics makes the treatment of these infections a difficult task. Moreover, biofilms represent a hot spot for horizontal gene transfer (HGT) by bacterial conjugation. In this work, 25 biofilm-forming clinical staphylococcal isolates were studied. We found that Staphylococcus epidermidis isolates showed a higher biofilm-forming capacity than Staphylococcus aureus isolates. Additionally, horizontal transfer and relaxase genes of two common staphylococcal plasmids, pSK41 and pT181, were detected in all isolates. In terms of antibiotic resistance genes, aac6-aph2a, ermC , and tetK genes, which confer resistance to gentamicin, erythromycin, and tetracycline, respectively, were the most prevalent. The horizontal transfer and antibiotic resistance genes harbored on these staphylococcal clinical strains isolated from biofilms located in implanted medical devices points to the potential risk of the development and dissemination of multiresistant bacteria.

Bioaugmentation, or the inoculation of microorganisms (e.g., bacteria harboring the required catabolic genes) into soil to enhance the rate of contaminant degradation, has great potential for the bioremediation of soils contaminated with organic compounds. Regrettably, cell bioaugmentation frequently turns into an unsuccessful initiative, owing to the rapid decrease of bacterial viability and abundance after inoculation, as well as the limited dispersal of the inoculated bacteria in the soil matrix. Genes that encode the degradation of organic compounds are often located on plasmids and, consequently, they can be spread by horizontal gene transfer into well-established, ecologically competitive, indigenous bacterial populations. Plasmid-mediated bioaugmentation aims to stimulate the spread of contaminant degradation genes among indigenous soil bacteria by the introduction of plasmids, located in donor cells, harboring such genes. But the acquisition of plasmids by recipient cells can affect the host's fitness, a crucial aspect for the success of plasmid-mediated bioaugmentation. Besides, environmental factors (e.g., soil moisture, temperature, organic matter content) can play important roles for the transfer efficiency of catabolic plasmids, the expression of horizontally acquired genes and, finally, the contaminant degradation activity. For plasmid-mediated bioaugmentation to be reproducible, much more research is needed for a better selection of donor bacterial strains and accompanying plasmids, together with an in-depth understanding of indigenous soil bacterial populations and the environmental conditions that affect plasmid acquisition and the expression and functioning of the catabolic genes of interest.

The type and frequency of disturbances experienced by soil microbiomes is expected to increase given predicted global climate change scenarios and intensified anthropogenic pressures on ecosystems. While the direct effect of multiple disturbances to soil microbes has been explored in terms of function, their effect on the recovery of microbial community composition remains unclear. Here, we used soil microcosm experiments and multiple model disturbances to explore their short-term effect on the recovery of soil microbiota after identical or novel stresses. Soil microcosms were exposed to a heat shock to create an initial effect. Upon initial community recovery (25 days after stress), they were subjected to a second stress, either a heat or a cold shock, and they were monitored for additional 25 days. To carefully verify the bacterial response to the disturbances, we monitored changes in community composition throughout the experiment using 16S rRNA gene transcript amplicon sequencing. The application of a heat shock to soils with or without the initial heat shock resulted in similar successional dynamics, but these dynamics were faster in soils with a prior heat shock. The application of a cold shock had negligible effects on previously undisturbed soils but, in combination with an initial heat shock, caused the largest shift in the community composition. Our findings show that compounded perturbation affects bacterial community recovery by altering community structure and thus, the community's response during succession. By altering dominance patterns, disturbance legacy affects the microbiome's ability to recover from further perturbation within the 25 days studied. Our results highlight the need to consider the soil's disturbance history in the development of soil management practices in order to maintain the system's resilience.

Lactic acid bacteria (LAB) differ in their ability to colonize food and animal-associated habitats: while some species are specialized and colonize a limited number of habitats, other are generalist and are able to colonize multiple animal-linked habitats. In the current study, Carnobacterium was used as a model genus to elucidate the genetic basis of these colonization differences. Analyses of 16S rRNA gene meta-barcoding data showed that C. maltaromaticum followed by C. divergens are the most prevalent species in foods derived from animals (meat, fish, dairy products), and in the gut. According to phylogenetic analyses, these two animal-adapted species belong to one of two deeply branched lineages. The second lineage contains species isolated from habitats where contact with animal is rare. Genome analyses revealed that members of the animal-adapted lineage harbor a larger secretome than members of the other lineage. The predicted cell-surface proteome is highly diversified in C. maltaromaticum and C. divergens with genes involved in adaptation to the animal milieu such as those encoding biopolymer hydrolytic enzymes, a heme uptake system, and biopolymer-binding adhesins. These species also exhibit genes for gut adaptation and respiration. In contrast, Carnobacterium species belonging to the second lineage encode a poorly diversified cell-surface proteome, lack genes for gut adaptation and are unable to respire. These results shed light on the important genomics traits required for adaptation to animal-linked habitats in generalist Carnobacterium .

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes , isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes ( lde and mdrL ) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains.

Interspecies interactions are essential for the persistence and development of any kind of complex community, and microbial biofilms are no exception. Multispecies biofilms are structured and spatially defined communities that have received much attention due to their omnipresence in natural environments. Species residing in these complex bacterial communities usually interact both intra- and interspecifically. Such interactions are considered to not only be fundamental in shaping overall biomass and the spatial distribution of cells residing in multispecies biofilms, but also to result in coordinated regulation of gene expression in the different species present. These communal interactions often lead to emergent properties in biofilms, such as enhanced tolerance against antibiotics, host immune responses, and other stresses, which have been shown to provide benefits to all biofilm members not only the enabling sub-populations. However, the specific molecular mechanisms of cellular processes affecting spatial organization, and vice versa, are poorly understood and very complex to unravel. Therefore, detailed description of the spatial organization of individual bacterial cells in multispecies communities can be an alternative strategy to reveal the nature of interspecies interactions of constituent species. Closing the gap between visual observation and biological processes may become crucial for resolving biofilm related problems, which is of utmost importance to environmental, industrial, and clinical implications. This review briefly presents the state of the art of studying interspecies interactions and spatial organization of multispecies communities, aiming to support theoretical and practical arguments for further advancement of this field.

Coastal ecosystems are considered buffer zones for the discharge of land-derived nutrients without accounting for potential negative side effects. Hence, there is an urgent need to better understand the ecological assembly and dynamics of the microorganisms that are involved in nitrogen (N) cycling in such systems. Here, we employed two complementary methodological approaches (i.e., shotgun metagenomics and quantitative PCR) to examine the distribution and abundance of selected microbial genes involved in N transformations. We used soil samples collected along a well-established pristine salt marsh soil chronosequence that spans over a century of ecosystem development at the island of Schiermonnikoog, The Netherlands. Across the examined soil successional stages, the structure of the populations of genes involved in N cycling processes was strongly related to (shifts in the) soil nitrogen levels (i.e., [Formula: see text], [Formula: see text]), salinity and pH (explaining 73.8% of the total variation, R (2) = 0.71). Quantification of the genes used as proxies for N fixation, nitrification and denitrification revealed clear successional signatures that corroborated the taxonomic assignments obtained by metagenomics. Notably, we found strong evidence for niche partitioning, as revealed by the abundance and distribution of marker genes for nitrification (ammonia-oxidizing bacteria and archaea) and denitrification (nitrite reductase nirK, nirS and nitrous oxide reductase nosZ clades I and II). This was supported by a distinct correlation between these genes and soil physico-chemical properties, such as soil physical structure, pH, salinity, organic matter, total N, [Formula: see text], [Formula: see text] and [Formula: see text], across four seasonal samplings. Overall, this study sheds light on the successional trajectories of microbial N cycle genes along a naturally developing salt marsh ecosystem. The data obtained serve as a foundation to guide the formulation of ecological models that aim to effectively monitor and manage pristine and impacted salt marsh areas. Such models should account for the ecology as well as the historical contingency of N cycling communities.

Trophic interactions are highly complex and modern sequencing techniques reveal enormous biodiversity across multiple scales in marine microbial communities. Within the chemically and physically relatively homogeneous pelagic environment, this calls for an explanation beyond spatial and temporal heterogeneity. Based on observations of simple parasite-host and predator-prey interactions occurring at different trophic levels and levels of phylogenetic resolution, we present a theoretical perspective on this enormous biodiversity, discussing in particular self-similar aspects of pelagic microbial food web organization. Fractal methods have been used to describe a variety of natural phenomena, with studies of habitat structures being an application in ecology. In contrast to mathematical fractals where pattern generating rules are readily known, however, identifying mechanisms that lead to natural fractals is not straight-forward. Here we put forward the hypothesis that trophic interactions between pelagic microbes may be organized in a fractal-like manner, with the emergent network resembling the structure of the Sierpinski triangle. We discuss a mechanism that could be underlying the formation of repeated patterns at different trophic levels and discuss how this may help understand characteristic biomass size-spectra that hint at scale-invariant properties of the pelagic environment. If the idea of simple underlying principles leading to a fractal-like organization of the pelagic food web could be formalized, this would extend an ecologists mindset on how biological complexity could be accounted for. It may furthermore benefit ecosystem modeling by facilitating adequate model resolution across multiple scales.

Traditionally, conservation and management of mountain pastures has been managed solely on the basis of visible biota. However, microorganisms play a vital role for the functioning of the soil ecosystem and, hence, pasture sustainability. Here, we studied the links between soil microbial (belowground) community structure (using amplicon sequencing of prokaryotes and fungi), other soil physicochemical and biological properties and, finally, a variety of pasture management practices. To this aim, during two consecutive years, we studied 104 environmental sites characterized by contrasting elevation, habitats, bedrock, and pasture management; located in or near Gorbeia Natural Park (Basque Country/Spain). Soil pH was found to be one of the most important factors in structuring soil microbial diversity. Interestingly, we observed a striking correlation between prokaryotic, fungal and macrofauna diversity, likely caused by interactions between these life forms. Further studies are needed to better understand such interactions and target the influence of different management practices on the soil microbial community, in face of the significant heterogeneity present. However, clearing of bushes altered microbial community structure, and in sites with calcareous bedrock also the use of herbicide vs. mechanical clearing of ferns.

The annually recurrent spring phytoplankton blooms in freshwater lakes initiate pronounced successions of planktonic ciliate species. Although there is considerable knowledge on the taxonomic diversity of these ciliates, their species-specific interactions with other microorganisms are still not well understood. Here we present the succession patterns of 20 morphotypes of ciliates during spring in Lake Zurich, Switzerland, and we relate their abundances to phytoplankton genera, flagellates, heterotrophic bacteria, and abiotic parameters. Interspecific relationships were analyzed by contemporaneous correlations and time-lagged co-occurrence and visualized as association networks. The contemporaneous network pointed to the pivotal role of distinct ciliate species (e.g., Balanion planctonicum, Rimostrombidium humile) as primary consumers of cryptomonads, revealed a clear overclustering of mixotrophic/omnivorous species, and highlighted the role of Halteria/Pelagohalteria as important bacterivores. By contrast, time-lagged statistical approaches (like local similarity analyses, LSA) proved to be inadequate for the evaluation of high-frequency sampling data. LSA led to a conspicuous inflation of significant associations, making it difficult to establish ecologically plausible interactions between ciliates and other microorganisms. Nevertheless, if adequate statistical procedures are selected, association networks can be powerful tools to formulate testable hypotheses about the autecology of only recently described ciliate species.

Populations of genetically identical microorganisms residing in the same environment can display marked variability in their phenotypic traits; this phenomenon is termed phenotypic heterogeneity. The relevance of such heterogeneity in natural habitats is unknown, because phenotypic characterization of a sufficient number of single cells of the same species in complex microbial communities is technically difficult. We report a procedure that allows to measure phenotypic heterogeneity in bacterial populations from natural environments, and use it to analyze N2 and CO2 fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with (15)N2 and (13)CO2 under in situ conditions with and without NH4 (+). Subsequently, we used flow cell sorting with auto-fluorescence gating based on a pure culture isolate to concentrate C. phaeobacteroides from its natural abundance of 0.2% to now 26.5% of total bacteria. C. phaeobacteroides cells were identified using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting the 16S rRNA in the sorted population with a species-specific probe. In a last step, we used nanometer-scale secondary ion mass spectrometry to measure the incorporation (15)N and (13)C stable isotopes in more than 252 cells. We found that C. phaeobacteroides fixes N2 in the absence of NH4 (+), but not in the presence of NH4 (+) as has previously been suggested. N2 and CO2 fixation were heterogeneous among cells and positively correlated indicating that N2 and CO2 fixation activity interact and positively facilitate each other in individual cells. However, because CARD-FISH identification cannot detect genetic variability among cells of the same species, we cannot exclude genetic variability as a source for phenotypic heterogeneity in this natural population. Our study demonstrates the technical feasibility of measuring phenotypic heterogeneity in a rare bacterial species in its natural habitat, thus opening the door to study the occurrence and relevance of phenotypic heterogeneity in nature.

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.

We imaged reflectance and variable fluorescence in 25 cyanobacterial mats from four distant sites around the globe to assess, at different scales of resolution, spatial variabilities in the physiological parameters characterizing their photosynthetic capacity, including the absorptivity by chlorophyll a (A chl), maximum quantum yield of photosynthesis (Y max), and light acclimation irradiance (I k). Generally, these parameters significantly varied within individual mats on a sub-millimeter scale, with about 2-fold higher variability in the vertical than in the horizontal direction. The average vertical profiles of Ymax and I k decreased with depth in the mat, while A chl exhibited a sub-surface maximum. The within-mat variability was comparable to, but often larger than, the between-sites variability, whereas the within-site variabilities (i.e., between samples from the same site) were generally lowest. When compared based on averaged values of their photosynthetic parameters, mats clustered according to their site of origin. Similar clustering was found when the community composition of the mats' cyanobacterial layers were compared by automated ribosomal intergenic spacer analysis (ARISA), indicating a significant link between the microbial community composition and function. Although this link is likely the result of community adaptation to the prevailing site-specific environmental conditions, our present data is insufficient to identify the main factors determining these patterns. Nevertheless, this study demonstrates that the spatial variability in the photosynthetic capacity and light acclimation of benthic phototrophic microbial communities is at least as large on a sub-millimeter scale as it is on a global scale, and suggests that this pattern of variability scaling is similar for the microbial community composition.

The release of antibiotics (AB) into the environment poses several threats for human health due to potential development of AB-resistant natural bacteria. Even though the use of low-dose antibiotics has been promoted in health care and farming, significant amounts of AB are observed in aquatic environments. Knowledge on the impact of AB on natural bacterial communities is missing both in terms of spread and evolution of resistance mechanisms, and of modifications of community composition and productivity. New approaches are required to study the response of microbial communities rather than individual resistance genes. In this study a chemostat-based experiment with 4 coexisting bacterial strains has been performed to mimicking the response of a freshwater bacterial community to the presence of antibiotics in low and high doses. Bacterial abundance rapidly decreased by 75% in the presence of AB, independently of their concentration, and remained constant until the end of the experiment. The bacterial community was mainly dominated by Aeromonas hydrophila and Brevundimonas intermedia while the other two strains, Micrococcus luteus and Rhodococcus sp. never exceed 10%. Interestingly, the bacterial strains, which were isolated at the end of the experiment, were not AB-resistant, while reassembled communities composed of the 4 strains, isolated from treatments under AB stress, significantly raised their performance (growth rate, abundance) in the presence of AB compared to the communities reassembled with strains isolated from the treatment without AB. By investigating the phenotypic adaptations of the communities subjected to the different treatments, we found that the presence of AB significantly increased co-aggregation by 5-6 fold. These results represent the first observation of co-aggregation as a successful strategy of AB resistance based on phenotype in aquatic bacterial communities, and can represent a fundamental step in the understanding of the effects of AB in aquatic ecosystems.