- ItemWhole-exome sequencing identifies novel pathogenic variants across the ATP7B gene and some modifiers of Wilson's disease phenotype(John Wiley & Sons, Inc., 2018-09-19) Kluska, Anna; Kulecka, Maria; Litwin, Tomasz; Dziezyc, Karolina; Balabas, Aneta; Piatkowska, Magdalena; Paziewska, Agnieszka; Dabrowska, Michalina; Mikula, Michal; Kaminska, Diana; Wiernicka, Anna; Socha, Piotr; Czlonkowska, Anna; Ostrowski, Jerzy; National Science CentreBackground & Aims: Wilson's disease (WD) is an autosomal recessive disorder associated with disease-causing alterations across the ATP7B gene, with highly variable symptoms and age of onset. We aimed to assess whether the clinical variability of WD relates to modifier genes. Methods: A total of 248 WD patients were included, of whom 148 were diagnosed after age of 17. Human exome libraries were constructed using AmpliSeq technology and sequenced using the IonProton platform. Results: ATP7B p.His1069Gln mutation was present in 215 patients, with 112 homozygotes and 103 heterozygotes. Three other mutations: p.Gln1351Ter, p.Trp779Ter and c.3402delC were identified in >10 patients. Among patients, 117 had a homozygous mutation, 101 were compound heterozygotes, 27 had one heterozygous mutation, and 3 other patients had no identifiable pathogenic variant of ATP7B. Sixteen mutations were novel, found as part of a compound mutation or as a sole, homozygous mutation. For disease phenotype prediction, age at diagnosis was a deciding factor, while frameshift allelic variants of ATP7B and being male increased the odds of developing a neurological phenotype. Rare allelic variants in ESD and INO80 increased and decreased chances for the neurological phenotype, respectively, while rare variants in APOE and MBD6 decreased the chances of WD early manifestation. Compound mutations contributed to earlier age of onset. Conclusions: In a Polish population, genetic screening for WD may help genotype for four variants (p.His1069Gln, p.Gln1351Ter, p.Trp779Ter and c.3402delC), with direct sequencing of all ATP7B amplicons as a second diagnostic step. We also identified some allelic variants that may modify a WD phenotype.
- ItemConjugated linoleic acid production and probiotic assessment of Lactobacillus plantarum isolated from Pico cheese(Elsevier Ltd., 2018-01-02) Ribeiro, Susana C.; Stanton, Catherine; Yang, Bo; Ross, R. Paul; Silva, Célia C. G.; Fundação para a Ciência e a Tecnologia; Science Foundation Ireland; Fundo Regional para a Ciência e TecnologiaLactic acid bacteria isolated from a traditional Azorean cheese were screened for their ability to convert free linoleic acid to conjugated linoleic acid (CLA). Two strains of Lactobacillus plantarum were recognized as potential CLA producers. GC analysis identified cis-9, trans-11 C18:2 as the predominant isomer (10–14 μg/mL), followed by trans-9, trans-11 C18:2 (4–6 μg/mL). The CLA producing strains demonstrated strong biofilm capacity, high cell surface hydrophobicity and good auto-aggregation ability. These strains were capable of surviving in the presence of bile salts (0.3%) and pancreatin (0.1%), but only the highest CLA producer (L3C1E8) was able to resist low pH (2.5). Moreover, the CLA-producers showed good adhesion capacity to intestinal human cells (Caco-2 and HT-29) and were able to prevent colonization of Escherichia coli. Of the two strains, Lactobacillus plantarum L3C1E8 revealed superior probiotic properties and great potential for producing food products enriched in the two CLA isomers, cis-9, trans-11 C18:2 (60%) and trans-9, trans-11 C18:2 (25%).
- ItemThe impact of primer design on amplicon-based metagenomic profiling accuracy: detailed insights into bifidobacterial community structure(MDPI, 2020-01) Mancabelli, Leonardo; Milani, Christian; Lugli, Gabriele A.; Fontana, Federico; Turroni, Francesca; van Sinderen, Douwe; Ventura, Marco; Horizon 2020; Science Foundation IrelandNext Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.
- ItemMobilome and resistome reconstruction from genomes belonging to members of the Bifidobacterium genus(MDPI, 2019-12) Mancino, Walter; Lugli, Gabriele A.; van Sinderen, Douwe; Ventura, Marco; Turroni, Francesca; Horizon 2020 Framework Programme; Science Foundation IrelandSpecific members of the genus Bifidobacterium are among the first colonizers of the human/animal gut, where they act as important intestinal commensals associated with host health. As part of the gut microbiota, bifidobacteria may be exposed to antibiotics, used in particular for intrapartum prophylaxis, especially to prevent Streptococcus infections, or in the very early stages of life after the birth. In the current study, we reconstructed the in silico resistome of the Bifidobacterium genus, analyzing a database composed of 625 bifidobacterial genomes, including partial assembled strains with less than 100 genomic sequences. Furthermore, we screened bifidobacterial genomes for mobile genetic elements, such as transposases and prophage-like elements, in order to investigate the correlation between the bifido-mobilome and the bifido-resistome, also identifying genetic insertion hotspots that appear to be prone to horizontal gene transfer (HGT) events. These insertion hotspots were shown to be widely distributed among analyzed bifidobacterial genomes, and suggest the acquisition of antibiotic resistance genes through HGT events. These data were further corroborated by growth experiments directed to evaluate bacitracin A resistance in Bifidobacterium spp., a property that was predicted by in silico analyses to be part of the HGT-acquired resistome.
- ItemBifidobacterium breve UCC2003 exopolysaccharide modulates the early life Microbiota by acting as a potential dietary substrate(MDPI, 2020) Püngel, Deborah; Treveil, Agatha; Dalby, Matthew J.; Caim, Shabhonam; Colquhoun, Ian J.; Booth, Catherine; Ketskemety, Jennifer; Korcsmaros, Tamas; van Sinderen, Douwe; Lawson, Melissa A. E.; Hall, Lindsay J.; Wellcome Trust; H2020 Marie Skłodowska-Curie Actions; Science Foundation IrelandBACKGROUND: Bifidobacterium represents an important early life microbiota member. Specific bifidobacterial components, exopolysaccharides (EPS), positively modulate host responses, with purified EPS also suggested to impact microbe-microbe interactions by acting as a nutrient substrate. Thus, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system. METHODS: Differential gene expression and growth characteristics were determined for each strain; Bifidobacterium breve UCC2003 and corresponding isogenic EPS-deletion mutant (B. breve UCC2003del). Model colon vessels were inoculated with B. breve and microbiome dynamics monitored using 16S rRNA sequencing and metabolomics (NMR). RESULTS: Transcriptomics of EPS mutant vs. B. breve UCC2003 highlighted discrete differential gene expression (e.g., eps biosynthetic cluster), though overall growth dynamics between strains were unaffected. The EPS-positive vessel had significant shifts in microbiome and metabolite profiles until study end (405 h); with increases of Tyzzerella and Faecalibacterium, and short-chain fatty acids, with further correlations between taxa and metabolites which were not observed within the EPS-negative vessel. CONCLUSIONS: These data indicate that B. breve UCC2003 EPS is potentially metabolized by infant microbiota members, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow development of EPS-specific strategies to promote infant health.