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      Development of omics‐based protocols for the microbiological characterization of multi‐strain formulations marketed as probiotics: the case of VSL#3

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          Summary

          The growing commercial interest in multi‐strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi‐strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single‐cell analysis was used to evaluate the cell viability, and β‐galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi‐strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.

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          FLASH: fast length adjustment of short reads to improve genome assemblies.

          Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.
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            The comprehensive antibiotic resistance database.

            The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.
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              Complete genome sequence of Lactobacillus plantarum WCFS1.

              The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.
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                Author and article information

                Contributors
                diego.mora@unimi.it
                Journal
                Microb Biotechnol
                Microb Biotechnol
                10.1111/(ISSN)1751-7915
                MBT2
                Microbial Biotechnology
                John Wiley and Sons Inc. (Hoboken )
                1751-7915
                12 August 2019
                November 2019
                : 12
                : 6 , Thematic Issue on Fungal Biotechnology ( doiID: 10.1111/mbt2.v12.6 )
                : 1371-1386
                Affiliations
                [ 1 ] Department of Food Environmental and Nutritional Sciences (DeFENS) University of Milan Milan Italy
                [ 2 ] Laboratory of Biochemistry Wageningen University Wageningen The Netherlands
                [ 3 ] Laboratory of Microbiology Wageningen University Wageningen The Netherlands
                [ 4 ] Human Microbiome Research Program Unit, Faculty of Medicine University of Helsinki Helsinki Finland
                Author notes
                [*] [* ] For correspondence. E‐mail diego.mora@ 123456unimi.it ; Tel. +39 0250319133; Fax +39 0250319238.

                Author information
                https://orcid.org/0000-0002-6692-9301
                Article
                MBT213476
                10.1111/1751-7915.13476
                6801179
                31402586
                8ba559bb-33a2-4ac1-b987-907a55c44021
                © 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 30 March 2019
                : 30 July 2019
                : 31 July 2019
                Page count
                Figures: 5, Tables: 4, Pages: 16, Words: 12154
                Funding
                Funded by: Netherlands Organization for Scientific Research
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                mbt213476
                November 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.7.0 mode:remove_FC converted:20.10.2019

                Biotechnology
                Biotechnology

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