Bacterial Fraction Research Articles

Microbial players and processes involved in phytoplankton bloom utilization in the water column of a fast-flowing, river-dominated estuary.

Fueled by seasonal phytoplankton blooms, the Columbia River estuary is a natural bioreactor for organic matter transformations. Prior metagenome analyses indicated high abundances of diverse Bacteroidetes taxa in estuarine samples containing phytoplankton. To examine the hypothesis that Bacteroidetes taxa have important roles in phytoplankton turnover, we further analyzed metagenomes from water collected along a salinity gradient at 0, 5, 15, 25, and 33 PSU during bloom events. Size fractions were obtained by using a 3‐μm prefilter and 0.2‐μm collection filter. Although this approach targeted bacteria by removing comparatively large eukaryotic cells, the metagenome from the ES‐5 sample (5 PSU) nevertheless contained an abundance of diatom DNA. Biogeochemical measurements and prior studies indicated that this finding resulted from the leakage of cellular material due to freshwater diatom lysis at low salinity. Relative to the other metagenomes, the bacterial fraction of ES‐5 was dramatically depleted of genes annotated as Bacteroidetes and lysogenic bacteriophages, but was overrepresented in DNA of protists and Myxococcales bacterivores. We suggest the following equally plausible scenarios for the microbial response to phytoplankton lysis: (1) Bacteroidetes depletion in the free‐living fraction may at least in part be caused by their attachment to fluvial diatoms as the latter are lysed upon contact with low‐salinity estuarine waters; (2) diatom particle colonization is likely followed by rapid bacterial growth and lytic phage infection, resulting in depletion of lysogenic bacteriophages and host bacteria; and (3) the subsequent availability of labile organic matter attracted both grazers and predators to feed in this estuarine biogeochemical “hotspot,” which may have additionally depleted Bacteroidetes populations. These results represent the first detailed molecular analysis of the microbial response to phytoplankton lysis at the freshwater–brackish water interface in the fast‐flowing Columbia River estuary.

Metabolic adaptation of Chlamydia trachomatis to mammalian host cells.

Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco-2 cells with 13 C-marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC-MS-based isotopologue analysis of protein-derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT-ICR-MS analyses also demonstrated that label from exogenous 13 C-glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6-phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous 13 C-malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co-substrate usage of intracellular C. trachomatis in a stream-lined bipartite metabolism with host cell-supplied amino acids for protein biosynthesis, host cell-provided glucose 6-phosphate for cell wall biosynthesis, and, to some extent, one or more host cell-derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso-2,6-diaminopimelate required for the formation of chlamydial peptidoglycan.

The occurrence of antibiotic resistance genes in a Mediterranean river and their persistence in the riverbed sediment

The spread of antibiotic resistance genes (ARGs) in the environment is a serious concern. Bacterial ARGs can spread via different mobile genetic elements as phage particles, which thereby emerge as novel vectors for environmental dissemination. To assess how climate events, such as heavy rains or water scarcity, could affect the spread of ARGs, it is necessary to know their prevalence and abundance in aquatic environments as well as the potential reservoirs from which they could become mobile. This study evaluates the occurrence of ARGs in the water and sediment of a Mediterranean river. Six clinically relevant ARGs (blaTEM, blaCTX-M, qnrA, qnrS, mecA and sul1) were quantified by qPCR in the bacterial and phage fractions of 69 water and 70 sediment samples from the River Llobregat (NE Spain), collected during both dry and rainy periods. blaTEM and sul1 were the most prevalent and abundant ARGs; the others were more variable. Significant seasonal differences in ARG prevalences and abundances were observed. Since ARGs were detected in the sediment, the persistence of the most abundant ARGs naturally occurring in that sediment (blaTEM and sul1) was evaluated under three conditions. No ARG inactivation occurred in fresh sediment over 14 days; while the ARGs declined by less than 2 log10 units over 35 days in semi-dry and dry sediment. The occurrence of ARGs in water and sediment is influenced by seasonal conditions and they can be mobilized by bacteria and phage particles. In sediment, ARGs persist for long periods and hence sediment can be a natural reservoir of ARGs, from where they can spread and cause the emergence of new resistant strains.

Phenotypic and Genetic Evaluation of the Influence of Pseudomonas aeruginosa Culture Fractions on the Human Mesenchymal Stem Cells Viability, Apoptotic Pathways and Cytokine Profile.

The objective of this study was to investigate the effects of P. aeruginosa PAO1 cellular and soluble culture fractions on human mesenchymal stem cells (MSCs) death signaling pathways and cytokine profile. The bone marrow isolated MSCs, incubated for different periods of time with one of the three P. aeruginosa PAO1 culture fractions, i.e. low density whole cultures, heat inactivated bacterial cultures sediments and sterile supernatants, were submitted to the following assays: i) fluorescence microscopy evaluation of cellular morphology and viability; ii) bax, caspase 9, relA and bcl-2 genes expression analysis by qRT-PCR; and iii) quantification of the level of IL-1β, IL-6, IL-8 and IL-10 cytokines released in the MSCs supernatants determined by ELISA. Results were statistically analyzed using the GraphPad In Stat software. The PAO1 whole cultures exhibited the most relevant influences, impacting on MSCs morphology and viability, interfering with apoptotic pathways and significantly stimulating the production of IL-1β and IL-10, while decreasing the production of IL-6 and IL-8. The culture supernatants increased the production of IL-1β and reduced the secretion of all other tested cytokines, while heat-inactivated bacterial cells significantly stimulated both IL-1β and IL-10 production. These data could suggest that in vivo, the fate of P. aeruginosa infection depends on the proportion between different bacterial culture fractions (i.e. the number of viable bacterial cells, the number of dead cells and the amount of bacterial soluble products accumulated locally) that could be influenced by the initial infective dose, by the host defense mechanisms, and also by the administered antimicrobial treatment that may thus interfere with the evolution and magnitude of the induced lesions.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome.

The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.

Microbial diversity in pitted sweet cherries (Prunus avium L.) as affected by High-Hydrostatic Pressure treatment

Sweet cherries are a highly appreciated seasonal fruit rich in anthocyanins. The purpose of the present study was to determine the effect of High-Hydrostatic Pressure (HHP) processing on the microbiological quality and bacterial biodiversity of sweet cherries. Pitted cherries inoculated with their own epiphyte microbiota to simulate a worst-case scenario of contamination during preparation and processing were treated or not by HHP (600MPa, 8min) and stored at 4°C for 60days. HHP treatment reduced total viable counts by 4.65 log cycles. The surviving bacterial fraction did not increase significantly (p<0.05) for the first 15days of storage. Concentrations of yeasts and molds were reduced below detectable levels. Upon prolonged storage (60days), microbial growth was observed. Bacterial biodiversity studied by high-throughput sequencing of the 16S rRNA gene revealed that Proteobacteria had highest relative abundance (88.70%) in the spiked cherries followed by Firmicutes (11.04%). Gluconobacter and Enterobacteriaceae together with Leuconostoc were the most abundant Operational Taxonomic Units (OTUs). Upon application of HHP treatment, 97.62% of OTUs from the surviving fraction belonged to Proteobacteria. The relative abundance of Enterobacteriaceae also decreased markedly while Acetobacteraceae (represented mainly by Gluconobacter) increased to 89.18%. Gluconobacter dominated during storage. Results from the present study provide insights on the microbiota of sweet cherries and the dynamics of the bacterial populations surviving HHP treatments that may be useful to improve the non-thermal preservation of cherries.

Surface complexation modeling of Cd(II) sorption to montmorillonite, bacteria, and their composite

Abstract. Surface complexation modeling (SCM) has emerged as a powerful tool for simulating heavy metal adsorption processes on the surface of soil solid components under different geochemical conditions. The component additivity (CA) approach is one of the strategies that have been widely used in multicomponent systems. In this study, potentiometric titration, isothermal adsorption, zeta potential measurement, and extended X-ray absorption fine-structure (EXAFS) spectra analysis were conducted to investigate Cd adsorption on 2 : 1 clay mineral montmorillonite, on Gram-positive bacteria Bacillus subtilis, and their mineral–organic composite. We developed constant capacitance models of Cd adsorption on montmorillonite, bacterial cells, and mineral–organic composite. The adsorption behavior of Cd on the surface of the composite was well explained by CA-SCM. Some deviations were observed from the model simulations at pH &lt; 5, where the values predicted by the model were lower than the experimental results. The Cd complexes of X2Cd, SOCd+, R-COOCd+, and R-POCd+ were the predominant species on the composite surface over the pH range of 3 to 8. The distribution ratio of the adsorbed Cd between montmorillonite and bacterial fractions in the composite as predicted by CA-SCM closely coincided with the estimated value of EXAFS at pH 6. The model could be useful for the prediction of heavy metal distribution at the interface of multicomponents and their risk evaluation in soils and associated environments.

Competitive adsorption of Pb and Cd on bacteria–montmorillonite composite

The characteristics and mechanisms of competitive adsorption of trace metals on bacteria-associated clay mineral composites have never been studied, despite their being among the most common organic–mineral complexes in geological systems. Herein, competitive adsorption of Pb and Cd on Pseudomonas putida–montmorillonite composite was investigated through adsorption–desorption experiment, isothermal titration calorimetry (ITC), and synchrotron micro X-ray fluorescence (μ-XRF). From the experiment, stronger competition was observed on clay mineral than on bacteria–clay composite because more non-specific sites accounted for heavy metal adsorption on clay mineral surface at the studied pH 5. Both competing heavy metals tended to react with bacterial fractions in the composite, which was verified by the higher correlation of Cd (and Pb) with Zn (R2 = 0.41) elemental distribution than with Si (R2 = 0.10). ITC results showed that competitive adsorption exhibited a lower entropy change (ΔS) at the metal-sorbent interfaces compared with single-metal adsorption, revealing that Cd and Pb are bound to the same types of adsorption sites on the sorbent. The competitive effect on bacteria–clay composite was found to be helpful for a better understanding on the fixation, remobilization and subsequent migration of heavy metals in multi-metal contaminated environments.

Mesophilic biogenic H2 production using galactose in a fixed bed reactor

This study investigated hydrogen fermentation from galactose in a fixed bed reactor (FBR) under a mesophilic temperature of 37 °C. The fixed bed reactor was packed with Lantec HD-Q-PAC material to support biomass growth and microbial assisted granule formation over the time course of the reactor operation. Hydraulic retention times (HRT) of 1.5–12 h over 79 days of bioreactor operation were assessed to determine the optimal hydrogen production efficiency of the system. The maximum hydrogen production rate and hydrogen yield of 65.5 L/L/d and 2.60 mol/mol hexoseadded were achieved at a hydraulic retention time (HRT) of 2 h, which is higher than the reported maximum hydrogen production performance of reactors fed with galactose using other reactor configuration. The major volatile fatty acids formed were butyric and acetic acids. Microbial community analysis by quantitative real time polymerase chain reaction revealed that population changes greatly affected hydrogen production performance. The increase in the bacterial fraction of Lactobacillus spp. over 21.5% at a 1.5 h HRT led to increased lactic acid production up to 1865 mg/L, which deteriorated the reactor performances. Conversely, a bacterial fraction of Clostridium butyricum over 98% was observed when the H2 production performance and organic acid distribution were recovered using a 2 h HRT. FBR would be a promising reactor configuration to achieve high hydrogen productivity while preventing the wash out of active biomass.

Schizoxylon as an experimental model for studying interkingdom symbiosis.

Experiments to re-synthesise lichens so far focused on co-cultures of fungal and algal partners. However, recent studies have revealed that bacterial communities colonise lichens in a stable and host-specific manner. We were therefore interested in testing how lichenised fungi and algae interact with selected bacteria in an experimental setup. We selected the symbiotic system of Schizoxylon albescens and the algal genera Coccomyxa and Trebouxia as a suitable model. We isolated bacterial strains from the naturally occurring bacterial fraction of freshly collected specimens and established tripartite associations under mixed culture experiments. The bacteria belong to Actinobacteria, Firmicutes and Proteobacteria and corresponded to groups already found associated with fungi including lichens. In mixed cultures with Coccomyxa, the fungus formed a characteristic filamentous matrix and tightly contacted the algae; the bacteria distributed in small patches between the algal cells and attached to the cell walls. In mixed cultures with Trebouxia, the fungus did not develop the filamentous matrix, but bacterial cells were observed to be tightly adhering to the fungal hyphae. Our experiments show that this tripartite fungal-algal-bacterial model system can be maintained in culture and can offer multiple opportunities for functional studies based on experiments under controlled conditions in the laboratory.

Fractionation of Hydrogen Isotopes by Sulfate- and Nitrate-Reducing Bacteria.

Hydrogen atoms from water and food are incorporated into biomass during cellular metabolism and biosynthesis, fractionating the isotopes of hydrogen—protium and deuterium—that are recorded in biomolecules. While these fractionations are often relatively constant in plants, large variations in the magnitude of fractionation are observed for many heterotrophic microbes utilizing different central metabolic pathways. The correlation between metabolism and lipid δ2H provides a potential basis for reconstructing environmental and ecological parameters, but the calibration dataset has thus far been limited mainly to aerobes. Here we report on the hydrogen isotopic fractionations of lipids produced by nitrate-respiring and sulfate-reducing bacteria. We observe only small differences in fractionation between oxygen- and nitrate-respiring growth conditions, with a typical pattern of variation between substrates that is broadly consistent with previously described trends. In contrast, fractionation by sulfate-reducing bacteria does not vary significantly between different substrates, even when autotrophic and heterotrophic growth conditions are compared. This result is in marked contrast to previously published observations and has significant implications for the interpretation of environmental hydrogen isotope data. We evaluate these trends in light of metabolic gene content of each strain, growth rate, and potential flux and reservoir-size effects of cellular hydrogen, but find no single variable that can account for the differences between nitrate- and sulfate-respiring bacteria. The emerging picture of bacterial hydrogen isotope fractionation is therefore more complex than the simple correspondence between δ2H and metabolic pathway previously understood from aerobes. Despite the complexity, the large signals and rich variability of observed lipid δ2H suggest much potential as an environmental recorder of metabolism.

Effects of Different Dietary Selenium Sources on Antioxidant Status and Blood Phagocytic Activity in Sheep.

The objective of this experiment was to investigate the effects of feed supplementation with equivalent doses of selenium from sodium selenite (SS) or selenized yeast (SY) on Se deposition, selenoenzyme activity and lipid peroxidation in tissues as well as in bacterial and protozoal fractions of rumen contents in sheep. The phagocytic activity of monocytes and neutrophils in whole blood was also assessed after 3months of dietary treatment. While animals in the control group were fed with unsupplemented basal diet (BD) containing only background Se (0.16mg/kg DM), the diet of the other two groups (n=6) consisted of identical BD enriched with 0.4mg Se/kg DM either from SS or SY. Concentrations of Se in blood and tissues were found to be significantly increased in both supplemented groups. No response in Se deposition was recorded in the musculus longissimus dorsi of sheep given dietary SS. The intake of SY resulted in a significantly higher Se level in the blood, kidney medulla, skeletal muscles, heart, intestinal and ruminal mucosa than in the case of SS supplementation. No differences appeared between tissue Se contents in the liver and kidney cortex due to the source of added Se. Regardless of source, Se supplementation to feeds significantly increased the glutathione peroxidase (GPx) activity in blood and tissues except the kidney medulla and jejunal mucosa. Supplementation with SY resulted in significantly higher activity of thioredoxin reductase in the liver and ileal mucosa, and also reduced malondialdehyde content in the liver and duodenal mucosa. Dietary Se intake increased Se concentrations in the total rumen contents and bacterial and protozoal fractions. The accumulation of Se in rumen microbiota was associated with increased GPx activity. Phagocytic cell activity was enhanced by Se supplementation. Our results indicate that Se from both sources has beneficial effects on antioxidant status in sheep and can be utilized by rumen microflora.

A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples.

BackgroundStudies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary.ResultsWe developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels.ConclusionThis study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0166-1) contains supplementary material, which is available to authorized users.

Effects of silver nanoparticles on bacterioplankton in a boreal lake

Summary Silver nanoparticles (AgNPs) are widely used antimicrobial agents and a growing body of evidence suggests that their release into aquatic environments threatens natural bacterial communities and whole ecosystems. However, a knowledge gap exists between the toxic effects of AgNPs found in laboratory studies and their potential impacts in natural environments. In an enclosure experiment conducted in a boreal lake, we exposed natural bacterial communities to AgNPs with two common types of coatings (polyvinylpyrrolidone (PVP) and citrate) under two different exposure regimes, a one‐time (pulse) and a continuous (chronic) addition. AgNP additions increased Ag concentrations to nearly 50 μg L−1 in the highest treatments. We examined bacterial responses (abundance, biomass, production, chlorophyll‐a content and nutrient stoichiometry) over the course of 6 weeks in the summer of 2012. Bacterioplankton exposed to AgNPs initially accumulated Ag over the experimental period regardless of AgNP concentration or coating. After the initial period of increase, Ag in the bacterial size fraction changed largely in concert with bacterial biomass. We found no toxic effects of AgNPs on bacterioplankton abundance, biomass, production or chlorophyll‐a content throughout the experiment. Bacterial production was greater after the pulse addition of PVP‐coated AgNPs and in the chronic addition of PVP‐coated AgNPs at the highest concentrations. Furthermore, AgNPs produced no significant changes in nutrient stoichiometry of the bacterioplankton size fraction. This lack of effects of AgNPs on lake bacterioplankton observed under the natural conditions studied here differs from results of short‐term and laboratory studies of single‐species bacterial cultures. Our results thus indicate AgNP effects in lakes may be less than expected based on standard laboratory experiments, and that additional studies are needed to understand AgNP toxicity under realistic natural conditions in lakes and other ecosystems.

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC-eseE Operon.

Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ΔeseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish.

Metagenomic analysis reveals that bacteriophages are reservoirs of antibiotic resistance genes

A metagenomics approach was applied to explore the presence of antibiotic resistance genes (ARGs) in bacteriophages from hospital wastewater. Metagenomic analysis showed that most phage sequences affiliated to the order Caudovirales, comprising the tailed phage families Podoviridae, Siphoviridae and Myoviridae. Moreover, the relative abundance of ARGs in the phage DNA fraction (0.26%) was higher than in the bacterial DNA fraction (0.18%). These differences were particularly evident for genes encoding ATP-binding cassette (ABC) and resistance–nodulation–cell division (RND) proteins, phosphotransferases, β-lactamases and plasmid-mediated quinolone resistance. Analysis of assembled contigs also revealed that blaOXA-10, blaOXA-58 and blaOXA-24 genes belonging to class D β-lactamases as well as a novel blaTEM (98.9% sequence similarity to the blaTEM-1 gene) belonging to class A β-lactamases were detected in a higher proportion in phage DNA. Although preliminary, these findings corroborate the role of bacteriophages as reservoirs of resistance genes and thus highlight the necessity to include them in future studies on the emergence and spread of antibiotic resistance in the environment.

Active and Secretory IgA-Coated Bacterial Fractions Elucidate Dysbiosis in Clostridium difficile Infection.

The onset of Clostridiumdifficile infection (CDI) has been associated with treatment with wide-spectrum antibiotics. Antibiotic treatment alters the activity of gut commensals and may result in modified patterns of immune responses to pathogens. To study these mechanisms during CDI, we separated bacteria with high cellular RNA content (the active bacteria) and their inactive counterparts by fluorescence-activated cell sorting (FACS) of the fecal bacterial suspension. The gut dysbiosis due to the antibiotic treatment may result in modification of immune recognition of intestinal bacteria. The immune recognition patterns were assessed by FACS of bacterial fractions either coated or not with intestinal secretory immunoglobulin A (SIgA). We described the taxonomic distributions of these four bacterial fractions (active versus inactive and SIgA coated versus non-SIgA coated) by massive 16S rRNA gene amplicon sequencing and quantified the proportion of C.difficile toxin genes in the samples. The overall gut microbiome composition was more robustly influenced by antibiotics than by the C.difficile toxins. Bayesian networks revealed that the C.difficile cluster was preferentially SIgA coated during CDI. In contrast, in the CDI-negative group Fusobacterium was the characteristic genus of the SIgA-opsonized fraction. Lactobacillales and Clostridium cluster IV were mostly inactive in CDI-positive patients. In conclusion, although the proportion of C.difficile in the gut is very low, it is able to initiate infection during the gut dysbiosis caused by environmental stress (antibiotic treatment) as a consequence of decreased activity of the protective bacteria. IMPORTANCE C.difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin A (SIgA) separated from the whole bacterial community by FACS elucidated how the gut dysbiosis promotes C.difficile infection (CDI). Bacterial groups with inhibitory effects on C.difficile growth, such as Lactobacillales, were mostly inactive in the CDI patients. C.difficile was typical for the bacterial fraction opsonized by SIgA in patients with CDI, while Fusobacterium was characteristic for the SIgA-opsonized fraction of the controls. The study demonstrates that sequencing of specific bacterial fractions provides additional information about dysbiotic processes in the gut. The detected patterns have been confirmed with the whole patient cohort independently of the taxonomic differences detected in the nonfractionated microbiomes.

Effects Due to Rhizospheric Soil Application of an Antagonistic Bacterial Endophyte on Native Bacterial Community and Its Survival in Soil: A Case Study with Pseudomonas aeruginosa from Banana.

Effective translation of research findings from laboratory to agricultural fields is essential for the success of biocontrol or growth promotion trials employing beneficial microorganisms. The rhizosphere is to be viewed holistically as a dynamic ecological niche comprising of diverse microorganisms including competitors and noxious antagonists to the bio-inoculant. This study was undertaken to assess the effects due to the soil application of an endophytic bacterium with multiple pathogen antagonistic potential on native bacterial community and its sustenance in agricultural soil. Pseudomonas aeruginosa was employed as a model system considering its frequent isolation as an endophyte, wide antagonistic effects reported against different phytopathogens and soil pests, and that the species is a known human pathogen which makes its usage in agriculture precarious. Employing the strain ‘GNS.13.2a’ from banana, its survival in field soil and the effects upon soil inoculation were investigated by monitoring total culturable bacterial fraction as the representative indicator of soil microbial community. Serial dilution plating of uninoculated control versus P. aeruginosa inoculated soil from banana rhizosphere indicated a significant reduction in native bacterial cfu soon after inoculation compared with control soil as assessed on cetrimide- nalidixic acid selective medium against nutrient agar. Sampling on day-4 showed a significant reduction in P. aeruginosa cfu in inoculated soil and a continuous dip thereafter registering >99% reduction within 1 week while the native bacterial population resurged with cfu restoration on par with control. This was validated in contained trials with banana plants. Conversely, P. aeruginosa showed static cfu or proliferation in axenic-soil. Lateral introduction of soil microbiome in P. aeruginosa established soil under axenic conditions or its co-incubation with soil microbiota in suspension indicated significant adverse effects by native microbial community. Direct agar-plate challenge assays with individual environmental bacterial isolates displayed varying interactive or antagonistic effects. In effect, the application of P. aeruginosa in rhizospheric soil did not serve any net benefit in terms of sustained survival. Conversely, it caused a disturbance to the native soil bacterial community. The findings highlight the need for monitoring the bio-inoculant(s) in field-soil and assessing the interactive effects with native microbial community before commercial recommendation. varying interactive or antagonistic effects. In effect, the application of P. aeruginosa in rhizospheric soil did not serve any net benefit in terms of sustained survival. Conversely, it caused a disturbance to the native soil bacterial community. The findings highlight the need for monitoring the bio-inoculant(s) in field-soil and assessing the interactive effects with native microbial community before commercial recommendation.

Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P=0·005, n=20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P=0·054, n=24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods.