isolateR: an R package for generating microbial libraries from Sanger sequencing data.

MOTIVATION: Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. RESULTS: isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. AVAILABILITY AND IMPLEMENTATION: isolateR is available at: https://github.com/bdaisley/isolateR.

Assessing phage-host population dynamics by reintroducing virulent viruses to synthetic microbiomes.

Microbiomes feature complex interactions between diverse bacteria and bacteriophages. Synthetic microbiomes offer a powerful way to study these interactions; however, a major challenge is obtaining a representative bacteriophage population during the bacterial isolation process. We demonstrate that colony isolation reliably excludes virulent viruses from sample sources with low virion-to-bacteria ratios such as feces, creating "virulent virus-free" controls. When the virulent dsDNA virome is reintroduced to a 73-strain synthetic gut microbiome in a bioreactor model of the human colon, virulent viruses target susceptible strains without significantly altering community structure or metabolism. In addition, we detected signals of prophage induction that associate with virulent predation. Overall, our findings indicate that dilution-based isolation methods generate synthetic gut microbiomes that are heavily depleted, if not devoid, of virulent viruses and that such viruses, if reintroduced, have a targeted effect on community assembly, metabolism, and prophage replication.

Metabolism of azo food dyes by bacterial members of the human gut microbiome.

OBJECTIVES: We set out to survey the capacities of bacterial isolates from the human gut microbiome to reduce common azo food dyes in vitro. METHODS: A total of 206 strains representative of 124 bacterial species and 6 phyla were screened in vitro using a simple azo dye decolorization assay. Strains which showed azoreductive activity were characterized by studies of azoreduction kinetics and bacterial growth. RESULTS: Several groups of gut bacteria, including ones not previously associated with azoreduction, reduced one or more of the four azo food dyes commonly used in Canada: Allura Red, Amaranth, Sunset Yellow, and Tartrazine. Strains within some species differed in their azoreductive capabilities. Some strains displayed evidence of effects on growth related to the presence of azo dyes and/or the products of their azoreduction. CONCLUSION: The continued widespread use of food azo dyes requires re-evaluation in light of the potential for disturbance of the gut microbial ecosystem resulting from azoreduction and the possibility of consequences for human health.

Exploring functional interplay amongst Escherichia coli efflux pumps.

Bacterial efflux pumps exhibit functional interplay that can translate to additive or multiplicative effects on resistance to antimicrobial compounds. In diderm bacteria, two different efflux pump structural types - single-component inner membrane efflux pumps and cell envelope-spanning multicomponent systems - cooperatively export antimicrobials with cytoplasmic targets from the cell. Harnessing our recently developed efflux platform, which is built upon an extensively efflux-deficient strain of Escherichia coli, here we explore interplay amongst a panel of diverse E. coli efflux pumps. Specifically, we assessed the effect of simultaneously expressing two efflux pump-encoding genes on drug resistance, including single-component inner membrane efflux pumps (MdfA, MdtK and EmrE), tripartite complexes (AcrAB, AcrAD, MdtEF and AcrEF), and the acquired TetA(C) tetracycline resistance pump. Overall, the expression of two efflux pump-encoding genes from the same structural type did not enhance resistance levels regardless of the antimicrobial compound or efflux pump under investigation. In contrast, a combination of the tripartite efflux systems with single-component pumps sharing common substrates provided multiplicative increases to antimicrobial resistance levels. In some instances, resistance was increased beyond the product of resistance provided by the two pumps individually. In summary, the developed efflux platform enables the isolation of efflux pump function, facilitating the identification of interactions between efflux pumps.