Identification of a muropeptide precursor transporter from gut microbiota and its role in preventing intestinal inflammation.

The gut microbiota is a considerable source of biologically active compounds that can promote intestinal homeostasis and improve immune responses. Here, we used large expression libraries of cloned metagenomic DNA to identify compounds able to sustain an anti-inflammatory reaction on host cells. Starting with a screen for NF-κB activation, we have identified overlapping clones harbouring a heterodimeric ATP-binding cassette (ABC)-transporter from a Firmicutes. Extensive purification of the clone's supernatant demonstrates that the ABC-transporter allows for the efficient extracellular accumulation of three muropeptide precursor, with anti-inflammatory properties. They induce IL-10 secretion from human monocyte-derived dendritic cells and proved effective in reducing AIEC LF82 epithelial damage and IL-8 secretion in human intestinal resections. In addition, treatment with supernatants containing the muropeptide precursor reduces body weight loss and improves histological parameters in Dextran Sulfate Sodium (DSS)-treated mice. Until now, the source of peptidoglycan fragments was shown to come from the natural turnover of the peptidoglycan layer by endogenous peptidoglycan hydrolases. This is a report showing an ABC-transporter as a natural source of secreted muropeptide precursor and as an indirect player in epithelial barrier strengthening. The mechanism described here might represent an important component of the host immune homeostasis.

Real-world outcomes of Cook Zilver PTX in femoro-popliteal district from multicenter experience.

BACKGROUND: The purpose is to evaluate the follow-up outcomes after femoro-popliteal stenting with Cook Zilver PTX in a multicenter experience. METHODS: Collected data from four Units were retrospectively joined and analyzed considering Zilver PTX deployed from August 2009 according to the instruction for use. Patient demographics, preoperative comorbidities, Rutherford classification, arterial characteristics and stent data were considered. Target lesion revascularization (TLR) was defined as reintervention performed for ≥50% diameter stenosis after recurrent clinical symptoms. Primary outcome was the freedom from TLR (ffTLR) and its risk factors. Secondary outcomes were primary patency (PP) of the stent, amputation-free survival (AFS) and their risk factors. RESULTS: Considering 203 patients (mean age: 73.5 years ±10.6; male: 66.5%) and 263 stents (median 2 stents/patient, range 1-5stent/patient), chronic limb-threatening ischemia (CLTI) affected 154 patients (75.9%). The length of the treated lesion was <120 mm in 99 (48.8%), ≥120 mm and <200 mm in 65 (32%) and ≥200 mm in 39 (19.2%) cases, respectively; the reference vessel mean diameter was 5.5±0.7 mm; chronic total occlusion was treated in 153 (75.4%) patients, the popliteal artery was involved in 56 (27.6%) cases and prior endovascular intervention was performed in 27 (13.3%) cases. Two or more crural run-off vessels were patent in 124 (61.1%). Mean follow-up was 23.2 months ±21.3. At 1, 2 and 3 years, the ffTLR was 90.6±4.2%, 86.4±6.1% and 80.4±8.3%, respectively, and the PP was 85.6±5.0%, 74.2±7.6% and 72.7±8.2%, respectively. Negative prognostic factor for ffTLR and PP was the reference vessel diameter (P=0.001 and P<0.001, respectively). At 1, 2 and 3 years, the AFS was 81.8±6.0%, 75.5±7.1% and 74.2±7.5% respectively; coronary artery disease (P=0.041) and CLTI (P=0.011) resulted negative prognostic factors. CONCLUSIONS: In the real-world practice, around 3/4 of patients were treated for CLTI. The rate of ffTLR is high, and PP is substantially lower. A small vessel diameter (<5 mm) is a negative factor for both ffTLR and PP. The rate of AFS is about 75% at 2 years and CLTI and coronary artery disease are negative prognostic factors.

Low-dose IL-2 shapes a tolerogenic gut microbiota that improves autoimmunity and gut inflammation.

Gut microbiota dysbiosis is associated with inflammatory bowel diseases and with cardiometabolic, neurological, and autoimmune diseases. Gut microbiota composition has a direct effect on the immune system, and vice versa, and it has a particular effect on Treg homeostasis. Low-dose IL-2 (IL-2LD) stimulates Tregs and is a promising treatment for autoimmune and inflammatory diseases. We aimed to evaluate the impact of IL-2LD on gut microbiota and correlatively on the immune system. We used 16S ribosomal RNA profiling and metagenomics to characterize gut microbiota of mice and humans treated or not with IL-2LD. We performed fecal microbiota transplantation (FMT) from IL-2LD-treated to naive recipient mice and evaluated its effects in models of gut inflammation and diabetes. IL-2LD markedly affected gut microbiota composition in mice and humans. Transfer of an IL-2-tuned microbiota by FMT protected C57BL/6J mice from dextran sulfate sodium-induced colitis and prevented diabetes in NOD mice. Metagenomic analyses highlighted a role for several species affected by IL-2LD and for microbial pathways involved in the biosynthesis of amino acids, short-chain fatty acids, and L-arginine. Our results demonstrate that IL-2LD induced changes in gut microbiota that are involved in the immunoregulatory effects of IL-2LD and suggest a crosstalk between Tregs and gut microbiota. These results provide potentially novel insight for understanding the mode of action of Treg-directed therapies.

Blockage of bacterial FimH prevents mucosal inflammation associated with Crohn's disease.

BACKGROUND: An Escherichia coli (E. coli) pathotype with invasive properties, first reported by Darfeuille-Michaud and termed adherent-invasive E. coli (AIEC), was shown to be prevalent in up to half the individuals with Crohn's Disease (CD), suggesting that these bacteria could be involved in the pathophysiology of CD. Among the genes related to AIEC pathogenicity, fim has the potential to generate an inflammatory reaction from the intestinal epithelial cells and macrophages, as it interacts with TLR4, inducing the production of inflammatory cytokines independently of LPS. Therefore, targeting the bacterial adhesion of FimH-expressing bacteria seems a promising therapeutic approach, consisting of disarming bacteria without killing them, representing a selective strategy to suppress a potentially critical trigger of intestinal inflammation, without disturbing the intestinal microbiota. RESULTS: We analyzed the metagenomic composition of the gut microbiome of 358 patients with CD from two different cohorts and characterized the presence of FimH-expressing bacteria. To assess the pathogenic role of FimH, we used human intestinal explants and tested a specific FimH blocker to prevent bacterial adhesion and associated inflammation. We observed a significant and disease activity-dependent enrichment of Enterobacteriaceae in the gut microbiome of patients with CD. Bacterial FimH expression was functionally confirmed in ileal biopsies from 65% of the patients with CD. Using human intestinal explants, we further show that FimH is essential for adhesion and to trigger inflammation. Finally, a specific FimH-blocker, TAK-018, inhibits bacterial adhesion to the intestinal epithelium and prevents inflammation, thus preserving mucosal integrity. CONCLUSIONS: We propose that TAK-018, which is safe and well tolerated in humans, is a promising candidate for the treatment of CD and in particular in preventing its recurrence. Video abstract.

GeneMark-HM: improving gene prediction in DNA sequences of human microbiome.

Computational reconstruction of nearly complete genomes from metagenomic reads may identify thousands of new uncultured candidate bacterial species. We have shown that reconstructed prokaryotic genomes along with genomes of sequenced microbial isolates can be used to support more accurate gene prediction in novel metagenomic sequences. We have proposed an approach that used three types of gene prediction algorithms and found for all contigs in a metagenome nearly optimal models of protein-coding regions either in libraries of pre-computed models or constructed de novo. The model selection process and gene annotation were done by the new GeneMark-HM pipeline. We have created a database of the species level pan-genomes for the human microbiome. To create a library of models representing each pan-genome we used a self-training algorithm GeneMarkS-2. Genes initially predicted in each contig served as queries for a fast similarity search through the pan-genome database. The best matches led to selection of the model for gene prediction. Contigs not assigned to pan-genomes were analyzed by crude, but still accurate models designed for sequences with particular GC compositions. Tests of GeneMark-HM on simulated metagenomes demonstrated improvement in gene annotation of human metagenomic sequences in comparison with the current state-of-the-art gene prediction tools.

Fecal Microbiota Transplant from Human to Mice Gives Insights into the Role of the Gut Microbiota in Non-Alcoholic Fatty Liver Disease (NAFLD).

Non-alcoholic fatty liver diseases (NAFLD) are associated with changes in the composition and metabolic activities of the gut microbiota. However, the causal role played by the gut microbiota in individual susceptibility to NAFLD and particularly at its early stage is still unclear. In this context, we transplanted the microbiota from a patient with fatty liver (NAFL) and from a healthy individual to two groups of mice. We first showed that the microbiota composition in recipient mice resembled the microbiota composition of their respective human donor. Following administration of a high-fructose, high-fat diet, mice that received the human NAFL microbiota (NAFLR) gained more weight and had a higher liver triglycerides level and higher plasma LDL cholesterol than mice that received the human healthy microbiota (HR). Metabolomic analyses revealed that it was associated with lower and higher plasma levels of glycine and 3-Indolepropionic acid in NAFLR mice, respectively. Moreover, several bacterial genera and OTUs were identified as differently represented in the NAFLR and HR microbiota and therefore potentially responsible for the different phenotypes observed. Altogether, our results confirm that the gut bacteria play a role in obesity and steatosis development and that targeting the gut microbiota may be a preventive or therapeutic strategy in NAFLD management.

A unified catalog of 204,938 reference genomes from the human gut microbiome.

Comprehensive, high-quality reference genomes are required for functional characterization and taxonomic assignment of the human gut microbiota. We present the Unified Human Gastrointestinal Genome (UHGG) collection, comprising 204,938 nonredundant genomes from 4,644 gut prokaryotes. These genomes encode >170 million protein sequences, which we collated in the Unified Human Gastrointestinal Protein (UHGP) catalog. The UHGP more than doubles the number of gut proteins in comparison to those present in the Integrated Gene Catalog. More than 70% of the UHGG species lack cultured representatives, and 40% of the UHGP lack functional annotations. Intraspecies genomic variation analyses revealed a large reservoir of accessory genes and single-nucleotide variants, many of which are specific to individual human populations. The UHGG and UHGP collections will enable studies linking genotypes to phenotypes in the human gut microbiome.

Antimicrobial Resistance Profiles of Adherent Invasive Escherichia coli Show Increased Resistance to ß-Lactams.

The adherent invasive Escherichia coli (AIEC) pathotype has been associated with the aetiology of Crohn's disease (CD). Scarce reports have shown the antimicrobial resistance (AMR) profiles of AIEC. Despite antibiotics not being recommended to treat CD, antimicrobial therapy could be useful in stratified patients, such as AIEC carriers. We examined the antimicrobial resistance profiles of AIEC strains to identify which therapies could be effective or confer a risk for such patients. Phenotypic resistance to 30 antimicrobials was tested according to CLSI standards. AIEC (n = 22) and non-pathogenic E. coli (non-AIEC) strains (n = 37) isolated from the gut mucosa of 31 CD patients and 18 controls were studied. De novo genome sequencing was carried out for 39 of the 59 strains, and AMR genes were searched using the DeepARG database in these genomes and 33 additional AIEC publicly available genomes. The strains isolated from CD and controls showed similar phenotypic AMR profiles. The genomic analysis did not reveal an increased prevalence of AMR genes. However, AIEC strains were more frequently resistant to ß-lactams than non-AIEC strains (11 AIEC (50%) and 5 non-AIEC (22%) strains were resistant to at least one ß-lactam; p < 0.042). Two AIEC strains were resistant to expanded-spectrum cephalosporins. One strain carried a plasmid-mediated AmpC ß-lactamase (CMY-69), and the other presented mutations in the promotor of the intrinsic chromosomal AmpC related to the hyperproduction of this enzyme. The rest of the strains were resistant to ß-lactams not including expanded-spectrum cephalosporins. The majority carried TEM-related ß-lactamases. Genomic analysis including external AIEC revealed that the gene sul1 encoding for sulphonamide resistance was more frequent in AIEC strains than non-AIEC strains (34.6% vs. 9.5%, p = 0.030). AMR in AIEC is a matter of concern regarding the putative implication of the pathotype in CD. The high proportion of AIEC resistant to ß-lactams warrants caution about the risk there may be in the use of these antimicrobials in AIEC-colonized CD patients.

A heritable subset of the core rumen microbiome dictates dairy cow productivity and emissions.

A 1000-cow study across four European countries was undertaken to understand to what extent ruminant microbiomes can be controlled by the host animal and to identify characteristics of the host rumen microbiome axis that determine productivity and methane emissions. A core rumen microbiome, phylogenetically linked and with a preserved hierarchical structure, was identified. A 39-member subset of the core formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (methane emissions, rumen and blood metabolites, and milk production efficiency). These phenotypes can be predicted from the core microbiome using machine learning algorithms. The heritable core microbes, therefore, present primary targets for rumen manipulation toward sustainable and environmentally friendly agriculture.

Scalable Workflows and Reproducible Data Analysis for Genomics.

Biological, clinical, and pharmacological research now often involves analyses of genomes, transcriptomes, proteomes, and interactomes, within and between individuals and across species. Due to large volumes, the analysis and integration of data generated by such high-throughput technologies have become computationally intensive, and analysis can no longer happen on a typical desktop computer.In this chapter we show how to describe and execute the same analysis using a number of workflow systems and how these follow different approaches to tackle execution and reproducibility issues. We show how any researcher can create a reusable and reproducible bioinformatics pipeline that can be deployed and run anywhere. We show how to create a scalable, reusable, and shareable workflow using four different workflow engines: the Common Workflow Language (CWL), Guix Workflow Language (GWL), Snakemake, and Nextflow. Each of which can be run in parallel.We show how to bundle a number of tools used in evolutionary biology by using Debian, GNU Guix, and Bioconda software distributions, along with the use of container systems, such as Docker, GNU Guix, and Singularity. Together these distributions represent the overall majority of software packages relevant for biology, including PAML, Muscle, MAFFT, MrBayes, and BLAST. By bundling software in lightweight containers, they can be deployed on a desktop, in the cloud, and, increasingly, on compute clusters.By bundling software through these public software distributions, and by creating reproducible and shareable pipelines using these workflow engines, not only do bioinformaticians have to spend less time reinventing the wheel but also do we get closer to the ideal of making science reproducible. The examples in this chapter allow a quick comparison of different solutions.

Exome sequences and multi-environment field trials elucidate the genetic basis of adaptation in barley.

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi-environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype-by-environment (G×E) modelling. Sub-populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock-related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large-effect alleles. Our analysis supports a gene-level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50).

Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2 n = 50) and the swamp (2 n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1.

Johne's disease in cattle: an in vitro model to study early response to infection of Mycobacterium avium subsp. paratuberculosis using RNA-seq.

Johne's disease is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which affects ruminants worldwide and has a significant economic impact. MAP has also been associated with human Crohn's disease, although this connection is not well established. MAP is highly adapted for survival within host macrophages and prevents macrophage activation, blocks phagosome acidification and maturation, and attenuates presentation of antigens to the immune system. The consequence is a very long silent infection before clinical signs are observed. The present work examined the transcriptome of bovine monocyte-derived macrophages (MDM) infected with the L1 strain of MAP at 2h, 6h and 24h post infection using RNA-seq. Pathway over-representation analysis of genes differentially expressed between infected vs. control MDM identified that immune related pathways were affected. Genes belonging to the cytokine-cytokine receptor interaction pathway and members of the JAK-STAT pathway, which is involved in the regulation of immune response, were up-regulated. However, in parallel inhibitors of immune functions were activated, including suppressor of cytokine signaling (SOCS) and cytokine-inducible SH2-containing protein (CISH), which most likely suppresses IFNγ and the JAK/STAT signaling cascade in infected MDM, which may favour MAP survival. After exposure, macrophages phagocytise pathogens, activate the complement cascade and the adaptive immune system through the antigen presentation process. However, data presented here suggest that genes related to phagocytosis and lysosome function are down regulated in MAP infected MDM. Genes of MHC class II and complement pathway were also down-regulated. This study therefore shows that MAP infection is associated with changes in expression of genes related to the host immune response that may affect its ability to survive and multiply inside the host cell.

The ruminal microbiome associated with methane emissions from ruminant livestock.

Methane emissions from ruminant livestock contribute significantly to the large environmental footprint of agriculture. The rumen is the principal source of methane, and certain features of the microbiome are associated with low/high methane phenotypes. Despite their primary role in methanogenesis, the abundance of archaea has only a weak correlation with methane emissions from individual animals. The composition of the archaeal community appears to have a stronger effect, with animals harbouring the Methanobrevibacter gottschalkii clade tending to be associated with greater methane emissions. Ciliate protozoa produce abundant H2, the main substrate for methanogenesis in the rumen, and their removal (defaunation) results in an average 11% lower methane emissions in vivo, but the results are not consistent. Different protozoal genera seem to result in greater methane emissions, though community types (A, AB, B and O) did not differ. Within the bacteria, three different 'ruminotypes' have been identified, two of which predispose animals to have lower methane emissions. The two low-methane ruminotypes are generally characterized by less abundant H2-producing bacteria. A lower abundance of Proteobacteria and differences in certain Bacteroidetes and anaerobic fungi seem to be associated with high methane emissions. Rumen anaerobic fungi produce abundant H2 and formate, and their abundance generally corresponds to the level of methane emissions. Thus, microbiome analysis is consistent with known pathways for H2 production and methanogenesis, but not yet in a predictive manner. The production and utilisation of formate by the ruminal microbiota is poorly understood and may be a source of variability between animals.

QTL mapping and candidate genes for resistance to Fusarium ear rot and fumonisin contamination in maize.

BACKGROUND: Fusarium verticillioides is a common maize pathogen causing ear rot (FER) and contamination of the grains with the fumonisin B1 (FB1) mycotoxin. Resistance to FER and FB1 contamination are quantitative traits, affected by environmental conditions, and completely resistant maize genotypes to the pathogen are so far unknown. In order to uncover genomic regions associated to reduced FER and FB1 contamination and identify molecular markers for assisted selection, an F2:3 population of 188 progenies was developed crossing CO441 (resistant) and CO354 (susceptible) genotypes. FER severity and FB1 contamination content were evaluated over 2 years and sowing dates (early and late) in ears artificially inoculated with F. verticillioides by the use of either side-needle or toothpick inoculation techniques. RESULTS: Weather conditions significantly changed in the two phenotyping seasons and FER and FB1 content distribution significantly differed in the F3 progenies according to the year and the sowing time. Significant positive correlations (P < 0.01) were detected between FER and FB1 contamination, ranging from 0.72 to 0.81. A low positive correlation was determined between FB1 contamination and silking time (DTS). A genetic map was generated for the cross, based on 41 microsatellite markers and 342 single nucleotide polymorphisms (SNPs) derived from Genotyping-by-Sequencing (GBS). QTL analyses revealed 15 QTLs for FER, 17 QTLs for FB1 contamination and nine QTLs for DTS. Eight QTLs located on linkage group (LG) 1, 2, 3, 6, 7 and 9 were in common between FER and FB1, making possible the selection of genotypes with both low disease severity and low fumonisin contamination. Moreover, five QTLs on LGs 1, 2, 4, 5 and 9 located close to previously reported QTLs for resistance to other mycotoxigenic fungi. Finally, 24 candidate genes for resistance to F. verticillioides are proposed combining previous transcriptomic data with QTL mapping. CONCLUSIONS: This study identified a set of QTLs and candidate genes that could accelerate breeding for resistance of maize lines showing reduced disease severity and low mycotoxin contamination determined by F. verticillioides.

Application of meta-omics techniques to understand greenhouse gas emissions originating from ruminal metabolism.

Methane emissions from ruminal fermentation contribute significantly to total anthropological greenhouse gas (GHG) emissions. New meta-omics technologies are beginning to revolutionise our understanding of the rumen microbial community structure, metabolic potential and metabolic activity. Here we explore these developments in relation to GHG emissions. Microbial rumen community analyses based on small subunit ribosomal RNA sequence analysis are not yet predictive of methane emissions from individual animals or treatments. Few metagenomics studies have been directly related to GHG emissions. In these studies, the main genes that differed in abundance between high and low methane emitters included archaeal genes involved in methanogenesis, with others that were not apparently related to methane metabolism. Unlike the taxonomic analysis up to now, the gene sets from metagenomes may have predictive value. Furthermore, metagenomic analysis predicts metabolic function better than only a taxonomic description, because different taxa share genes with the same function. Metatranscriptomics, the study of mRNA transcript abundance, should help to understand the dynamic of microbial activity rather than the gene abundance; to date, only one study has related the expression levels of methanogenic genes to methane emissions, where gene abundance failed to do so. Metaproteomics describes the proteins present in the ecosystem, and is therefore arguably a better indication of microbial metabolism. Both two-dimensional polyacrylamide gel electrophoresis and shotgun peptide sequencing methods have been used for ruminal analysis. In our unpublished studies, both methods showed an abundance of archaeal methanogenic enzymes, but neither was able to discriminate high and low emitters. Metabolomics can take several forms that appear to have predictive value for methane emissions; ruminal metabolites, milk fatty acid profiles, faecal long-chain alcohols and urinary metabolites have all shown promising results. Rumen microbial amino acid metabolism lies at the root of excessive nitrogen emissions from ruminants, yet only indirect inferences for nitrogen emissions can be drawn from meta-omics studies published so far. Annotation of meta-omics data depends on databases that are generally weak in rumen microbial entries. The Hungate 1000 project and Global Rumen Census initiatives are therefore essential to improve the interpretation of sequence/metabolic information.

Elucidating the genetic basis of an oligogenic birth defect using whole genome sequence data in a non-model organism, Bubalus bubalis.

Recent strong selection for dairy traits in water buffalo has been associated with higher levels of inbreeding, leading to an increase in the prevalence of genetic diseases such as transverse hemimelia (TH), a congenital developmental abnormality characterized by absence of a variable distal portion of the hindlimbs. Limited genomic resources available for water buffalo required an original approach to identify genetic variants associated with the disease. The genomes of 4 bilateral and 7 unilateral affected cases and 14 controls were sequenced. A concordance analysis of SNPs and INDELs requiring homozygosity unique to all unilateral and bilateral cases revealed two genes, WNT7A and SMARCA4, known to play a role in embryonic hindlimb development. Additionally, SNP alleles in NOTCH1 and RARB were homozygous exclusively in the bilateral cases, suggesting an oligogenic mode of inheritance. Homozygosity mapping by whole genome de novo assembly also supported oligogenic inheritance; implicating 13 genes involved in hindlimb development in bilateral cases and 11 in unilateral cases. A genome-wide association study (GWAS) predicted additional modifier genes. Although our data show a complex inheritance of TH, we predict that homozygous variants in WNT7A and SMARCA4 are necessary for expression of TH and selection against these variants should eradicate TH.

Transcriptome analysis of Pseudomonas mediterranea and P. corrugata plant pathogens during accumulation of medium-chain-length PHAs by glycerol bioconversion.

Pseudomonas corrugata and P. mediterranea are soil inhabitant bacteria, generally living as endophytes on symptomless plants and bare soil, but also capable of causing plant diseases. They share a similar genome size and a high proteome similarity. P. corrugata produces many biomolecules which play an important role in bacterial cell survival and fitness. Both species produce different medium-chain-length PHAs (mcl-PHAs) from the bioconversion of glycerol to a transparent film in P. mediterranea and a sticky elastomer in P. corrugata. In this work, using RNA-seq we investigated the transcriptional profiles of both bacteria at the early stationary growth phase with glycerol as the carbon source. Quantitative analysis of P. mediterranea transcripts versus P. corrugata revealed that 1756 genes were differentially expressed. A total of 175 genes were significantly upregulated in P. mediterranea, while 217 were downregulated. The largest group of upregulated genes was related to transport systems and stress response, energy and central metabolism, and carbon metabolism. Expression levels of most genes coding for enzymes related to PHA biosynthesis and central metabolic pathways showed no differences or only slight variations in pyruvate metabolism. The most relevant result was the significantly increased expression in P. mediterranea of genes involved in alginate production, an important exopolysaccharide, which in other Pseudomonas spp. plays a key role as a virulence factor or in stress tolerance and shows many industrial applications. In conclusion, the results provide useful information on the co-production of mcl-PHAs and alginate from glycerol as carbon source by P. mediterranea in the design of new strategies of genetic regulation to improve the yield of bioproducts or bacterial fitness.

The Possible Innovative Use of Bifidobacterium longum W11 in Association With Rifaximin: A New Horizon for Combined Approach?

GOALS: The aim of the study was to unequivocally demonstrate the nontransmissibility of the genes mediating the resistance of the strain Bifidobacterium longum W11 (LMG P-21586) to rifaximin. BACKGROUND: Most antibiotic treatments can induce unfavorable side effects such as antibiotic-associated diarrhea, which is largely attributable to the disruption of the intestinal microbiota. The parallel intake of probiotic bacteria might reduce these events, even if with generally very poor results. In this regard, the use of antibiotic-resistant beneficial bacteria could represent a worthy strategy. STUDY: Rifaximin was tested in parallel with rifampicin, rifapentine, and rifabutin, all rifamycin derivates, using 5 different concentrations. Susceptibility tests were performed by the disc diffusion method of Kirby-Bauer, and inhibition zones were measured after incubation at 37°C. B. longum BL03 was used as comparison. The B. longum W11 genome was sequenced on Illumina MiSeq with a 250 PE reads module. After mapping the reads with the reference bacterial genome, the alignment data were processed using FreeBayes software. RESULTS: B. longum BL03 was inhibited by all antibiotics even at the lowest concentration. In contrast, the W11 strain was inhibited by rifampicin, rifabutin, and rifaximin only at the highest concentration (512 µg/mL). The genomic analysis showed a mutation into the chromosomal DNA. No transposable elements were found, and the genetic locus was not flanked by close mobile genetic elements. CONCLUSIONS: B. longum W11 could be used in combined therapy with rifaximin, thus opening new focused frontiers in the probiotic era while preserving the necessary safety of use for consumers.