Changes in blood microbiota profiles associated with liver fibrosis in obese patients: A pilot analysis.

The early detection of liver fibrosis among patients with nonalcoholic fatty liver disease (NAFLD) is an important clinical need. In view of the suggested role played by bacterial translocation in liver disease and obesity, we sought to investigate the relationship between blood microbiota and liver fibrosis (LF) in European cohorts of patients with severe obesity. We carried out a cross-sectional study of obese patients, well characterized with respect to the severity of the NAFLD, in the cohort FLORINASH. This cohort has been divided into a discovery cohort comprising 50 Spanish patients and then in a validation cohort of 71 Italian patients. Blood bacterial DNA was analyzed both quantitatively by 16S ribosomal DNA (rDNA) quantitative polymerase chain reaction and qualitatively by 16S rDNA targeted metagenomic sequencing and functional metagenome prediction. Spanish plasma bile acid contents were analyzed by liquid chromatography/mass spectrometry. The 16S rDNA concentration was significantly higher in patients of the discovery cohort with LF. By 16S sequencing, we found specific differences in the proportion of several bacterial taxa in both blood and feces that correlate with the presence of LF, thus defining a specific signature of the liver disease. Several secondary/primary bile acid ratios were also decreased with LF in the discovery cohort. We confirmed, in the validation cohort, the correlation between blood 16S rDNA concentration and LF, whereas we did not confirm the specific bacterial taxa signature, despite a similar trend in patients with more-severe fibrosis. CONCLUSION: Changes in blood microbiota are associated with LF in obese patients. Blood microbiota analysis provides potential biomarkers for the detection of LF in this population. (Hepatology 2016;64:2015-2027).

Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing.

BACKGROUND: Recent studies have revealed that the blood of healthy humans is not as sterile as previously supposed. The objective of this study was to provide a comprehensive description of the microbiome present in different fractions of the blood of healthy individuals. STUDY DESIGN AND METHODS: The study was conducted in 30 healthy blood donors to the French national blood collection center (Établissement Français du Sang). We have set up a 16S rDNA quantitative polymerase chain reaction assay as well as a 16S targeted metagenomics sequencing pipeline specifically designed to analyze the blood microbiome, which we have used on whole blood as well as on different blood fractions (buffy coat [BC], red blood cells [RBCs], and plasma). RESULTS: Most of the blood bacterial DNA is located in the BC (93.74%), and RBCs contain more bacterial DNA (6.23%) than the plasma (0.03%). The distribution of 16S DNA is different for each fraction and spreads over a relatively broad range among donors. At the phylum level, blood fractions contain bacterial DNA mostly from the Proteobacteria phylum (more than 80%) but also from Actinobacteria, Firmicutes, and Bacteroidetes. At deeper taxonomic levels, there are striking differences between the bacterial profiles of the different blood fractions. CONCLUSION: We demonstrate that a diversified microbiome exists in healthy blood. This microbiome has most likely an important physiologic role and could be implicated in certain transfusion-transmitted bacterial infections. In this regard, the amount of 16S bacterial DNA or the microbiome profile could be monitored to improve the safety of the blood supply.

The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline.

BACKGROUND: Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. RESULTS: We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). CONCLUSION: The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

Sulfate-reducing bacteria inhabiting natural corrosion deposits from marine steel structures.

In the present study, investigations were conducted on natural corrosion deposits to better understand the role of sulfate-reducing bacteria (SRB) in the accelerated corrosion process of carbon steel sheet piles in port environments. We describe the abundance and diversity of total and metabolically active SRB within five natural corrosion deposits located within tidal or low water zone and showing either normal or accelerated corrosion. By using molecular techniques, such as quantitative real-time polymerase chain reaction, denaturing gel gradient electrophoresis, and sequence cloning based on 16S rRNA, dsrB genes, and their transcripts, we demonstrated a clear distinction between SRB population structure inhabiting normal or accelerated low-water corrosion deposits. Although SRB were present in both normal and accelerated low-water corrosion deposits, they dominated and were exclusively active in the inner and intermediate layers of accelerated corrosion deposits. We also highlighted that some of these SRB populations are specific to the accelerated low-water corrosion deposit environment in which they probably play a dominant role in the sulfured corrosion product enrichment.

Impact of a simulated oil spill on benthic phototrophs and nitrogen-fixing bacteria in mudflat mesocosms.

Coastal and estuarine ecosystems are highly susceptible to crude oil pollution. Therefore, in order to examine the resilience of benthic phototrophs that are pivotal to coastal ecosystem functioning, we simulated an oil spill in tidal mesocosms consisting of intact sediment cores from a mudflat at the mouth of the Colne Estuary, UK. At day 21, fluorescence imaging revealed a bloom of cyanobacteria on the surface of oiled sediment cores, and the upper 1.5 cm thick sediment had 7.2 times more cyanobacterial and 1.7 times more diatom rRNA sequences when treated with oil. Photosystem II operating efficiency (Fq'/Fm') was significantly reduced in oiled sediments at day 7, implying that the initial diatom-dominated community was negatively affected by oil, but this was no longer apparent by day 21. Oil addition significantly reduced numbers of the key deposit feeders, and the decreased grazing pressure is likely to be a major factor in the increased abundance of both diatoms and cyanobacteria. By day 5 concentrations of dissolved inorganic nitrogen were significantly lower in oiled mesocosms, likely resulting in the observed increase in nifH-containing, and therefore potentially dinitrogen-fixing, cyanobacteria. Thus, indirect effects of oil, rather than direct inhibition, are primarily responsible for altering the microphytobenthos.

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel.

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.

Central role of dynamic tidal biofilms dominated by aerobic hydrocarbonoclastic bacteria and diatoms in the biodegradation of hydrocarbons in coastal mudflats.

Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.

Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution.

The early functional response of a bacterial community from the sediments of a chronically oil-polluted retention basin located at the Etang de Berre (France) was investigated just after petroleum addition. After removing hydrocarbon compounds by natural abiotic and biotic processes, the sediments were maintained in microcosms and Vic Bilh petroleum was added. The diversity and the expression of genes encoding ring-hydroxylating dioxygenases (RHD) were examined just after the petroleum addition until 14 days focussing on the first hours following the contamination. RHD gene copy numbers and diversity were maintained throughout all the incubation period; however, transcripts were detected only during the first 2 days. One dominant RHD gene, immediately and specifically expressed in response to petroleum contamination, was related to RHD gene carried by a plasmid found in Pseudomonas spp. The expression of the RHD genes was correlated with high biodegradation levels observed for low molecular weight PAHs at 7 days of incubation. The study shows that the bacterial metabolism induced just after the oil input is a key stage that could determine the bacterial community structure changes. Monitoring the expression of RHD genes, key genes involved in hydrocarbon degradation, may provide useful information for managing bioremediation processes.

Abundance, diversity and activity of sulfate-reducing prokaryotes in heavy metal-contaminated sediment from a salt marsh in the Medway Estuary (UK).

We investigated the diversity and activity of sulfate-reducing prokaryotes (SRP) in a 3.5-m sediment core taken from a heavy metal-contaminated site in the Medway Estuary, UK. The abundance of SRPs was quantified by qPCR of the dissimilatory sulfite reductase gene ß-subunit (dsrB) and taking into account DNA extraction efficiency. This showed that SRPs were abundant throughout the core with maximum values in the top 50 cm of the sediment core making up 22.4% of the total bacterial community and were 13.6% at 250 cm deep. Gene libraries for dsrA (dissimilatory sulfite reductase α-subunit) were constructed from the heavily contaminated (heavy metals) surface sediment (top 20 cm) and from the less contaminated and sulfate-depleted, deeper zone (250 cm). Certain cloned sequences were similar to dsrA found in members of the Syntrophobacteraceae, Desulfobacteraceae and Desulfovibrionaceae as well as a large fraction (60%) of novel sequences that formed a deep branching dsrA lineage. Phylogenetic analysis of metabolically active SRPs was performed by reverse transcription PCR and single strand conformational polymorphism analysis (RT-PCR-SSCP) of dsrA genes derived from extracted sediment RNA. Subsequent comparative sequence analysis of excised SSCP bands revealed a high transcriptional activity of dsrA belonging to Desulfovibrio species in the surface sediment. These results may suggest that members of the Desulfovibrionaceae are more active than other SRP groups in heavy metal-contaminated surface sediments.

Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?

The diversity of alkB-related alkane hydroxylase sequences and the relationship between alkB gene expression and the hydrocarbon contamination level have been investigated in the chronically polluted Etang-de-Berre sediments. For this purpose, these sediments were maintained in microcosms and submitted to a controlled oil input miming an oil spill. New degenerated PCR primers targeting alkB-related alkane hydroxylase sequences were designed to explore the diversity and the expression of these genes using terminal restriction fragment length polymorphism fingerprinting and gene library analyses. Induction of alkB genes was detected immediately after oil addition and their expression detected only during 2 days, although the n-alkane degradation was observed throughout the 14 days of incubation. The alkB gene expression within triplicate microcosms was heterogeneous probably due to the low level of alkB transcripts. Moreover, the alkB gene expression of dominant OTUs has been observed in unoiled microcosms indicating that the expression of this gene cannot be directly related to the oil contamination. Although the dominant alkB genes and transcripts detected were closely related to the alkB of Marinobacter aquaeolei isolated from an oil-producing well, and to alkB genes related to the obligate alkanotroph Alcanivorax borkumensis, no clear relationship between the oil contamination and the expression of the alkB genes could be established. This finding suggests that in such coastal environments, alkB gene expression is not a function relevant enough to monitor bacterial response to oil contamination.

How a bacterial community originating from a contaminated coastal sediment responds to an oil input.

Bacterial communities inhabiting coastal sediments are subjected to oil spills. In order to examine the early structural response of a complex bacterial community to oil pollution, a kinetic study of the crude oil impact on bacterial communities inhabiting sediments from the contaminated Etang-de-Berre lagoon was performed. The sediments were maintained in slurries in presence or absence of crude oil and the kinetic study was carried out 14 days. During this period, 54% of crude oil was biodegraded showing the importance of the early degradation step. The metabolically active community (16S rRNA transcript analysis) was immediately impacted by the oil input, observed as an apparent decrease of species richness in the first hour of incubation. Nevertheless, this shift was quickly reversed, highlighting a fast, adaptative and efficient response of the metabolically active bacterial population. The high proportion of sequences related to hydrocarbonoclastic strains or petroleum-associated clones in active oiled community was consistent with significant increasing numbers of cultivable hydrocarbonoclastic bacteria at the end of the experiment. We concluded that "Etang-de-Berre" bacterial communities inhabiting oiled sediments for decades adopted a specific structure depending on oil presence and were able to face hydrocarbon contamination quickly and efficiently.

Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment.

The bacterial diversity of a chronically oil-polluted retention basin sediment located in the Berre lagoon (Etang-de-Berre, France) was investigated. This study combines chemical and molecular approaches in order to define how the in situ petroleum hydrocarbon contamination level affects the bacterial community structure of a subsurface sediment. Hydrocarbon content analysis clearly revealed a gradient of hydrocarbon contamination in both the water and the sediment following the basin periphery from the pollution input to the lagoon water. The nC17 and pristane concentrations suggested alkane biodegradation in the sediments. These results, combined with those of terminal-restriction fragment length polymorphism analysis of the 16S rRNA genes, indicated that bacterial community structure was obviously associated with the gradient of oil contamination. The analysis of bacterial community composition revealed dominance of bacteria related to the Proteobacteria phylum (Gamma-, Delta-, Alpha-, Epsilon- and Betaproteobacteria), Bacteroidetes and Verrucomicrobium groups and Spirochaetes, Actinobacteria and Cyanobacteria phyla. The adaptation of the bacterial community to oil contamination was not characterized by dominance of known oil-degrading bacteria, because a predominance of populations associated to the sulphur cycle was observed. The input station presented particular bacterial community composition associated with a low oil concentration in the sediment, indicating the adaptation of this community to the oil contamination.