Jesse Shapiro

Microbial communities in harsh environments, such as the Canadian High Arctic, remain a relatively unexplored source of antimicrobial natural products. In this study, we developed a pipeline for screening and evaluating bacterial isolates cultured from 23 Canadian High Arctic soil, sediment, and active-layer permafrost samples to discover antibacterial compounds. The pipeline incorporated an undergraduate studentsourcing program and produced a collection of over 350 bacterial isolates with preliminary antibacterial activity. Following dereplication, whole genome sequencing and secondary screening by co-culture assays, five isolates were prioritized to test crude extracts for antibacterial activity. Among bioactive crude extracts from these isolates, the Pseudomonas isolate with the most potent activity against S. aureus was analyzed. Using liquid chromatography bioactivity guided fractionation, we identified a potential bioactive compound known as koreenceine A, a compound previously only isolated from a Pseudomonas from temperate soil and involved in gene expression of other bacteria in their microbial community. Here we expanded the known antibiotic activity of koreenceine A and confirmed that a Δ kecBCD genetic knockout of the putative biosynthetic gene cluster significantly abrogated the antibacterial activity. Importance: Environmental bacteria naturally produce antimicrobial compounds to compete and suppress other microbes around them. Polar bacteria are relatively understudied, especially from the Canadian High Arctic (CHA) region. In this study we report on the development of a pipeline which integrated a studentsourcing program to screen environmental samples from the CHA, followed by characterization of a collection of CHA bacterial isolates with antibacterial activity against human pathogens. This work contributes to a better understanding of the antibacterial-producing capabilities of Arctic bacteria.

Antibiotics are known to trigger prophage induction in controlled laboratory settings, but it remains unclear whether this also occurs within microbiomes in nature. Current methods investigating the link between antibiotics and prophage induction within the human gut rely on in vitro culturing of human gut bacterial isolates. Using a metagenomic approach, we aimed to measure prophage induction, and whether it is associated with antibiotic exposure. In two independent human cohorts, we compared prophage to bacterial host read depth ratios (P:H) across known or measured antibiotic exposures. We found that induction is not broadly associated with antibiotic exposures at the level of the overall microbiome, but that ciprofloxacin increases P:H ratios in certain bacterial species. We documented heterogeneous trajectories of P:H ratios over the course of antibiotic exposure, sometimes increasing and remaining high, or returning to baseline. This study complements experimental models by providing in vivo evidence of induction in the human gut. IMPORTANCE: Bacteriophages are viruses that infect a bacterial host. The lytic and lysogenic cycles are the two classic outcomes of phage infection. In the lytic cycle, the phage immediately replicates and lyses its host cell to release new viral particles. In the lysogenic cycle, the phage, now called a prophage, integrates its genome into that of its host without killing it. Prophages can switch to the lytic cycle in a process called induction, in which the viral genome is replicated, the host cell is lysed, and viral particles are released. The most immediate consequence of induction is host cell death, which can impact bacterial populations and communities. Since prophages are mobile genetic elements that can move between bacteria, they are also an important vehicle for horizontal gene transfer. While induction has been well studied in vitro , whether and how induction occurs within the complex microbial ecosystem in humans is less well characterized. Understanding prophage induction in vivo is therefore critical in corroborating in vitro observations.

Virulent bacteriophages (phages) can kill bacterial prey, potentially reducing burden of infection. In cholera, a high phage to Vibrio cholerae ratio is associated with mild dehydration, yet the relative importance of this ratio in disease severity remains unclear. We used machine learning to rank select host, microbial, and environmental factors as determinants of dehydration severity in over 600 cholera patients from across Bangladesh. We found the phage:pathogen ratio ranked among the top classifiers for mild dehydration, behind age and location. We advocate that phage predation be included as a key factor in cholera characterization for scientific, clinical and epidemiological applications.

SUMMARY: Early postnatal life is a highly dynamic period in which the intestine is inundated with billions of microbes that not only battle for colonization, but also strongly influence immune system function and long-term health. This brief period of development provides a window of opportunity to imprint resilience against disease, yet the intercellular dialogue that dictates tissue protection are only beginning to be discovered. By performing a kinetic analysis of immune cell activation in the colon of mice from early life to adulthood, we identified transient activation of a microbiota-dependent type 3 immune response during the weaning period. This response was characterized by a selective increase in IL-17 production by fetal-derived γδ T cells that occurred in an IL-1 receptor-dependent, cell-intrinsic manner. The subsequent differentiation of IL-10 producing Rorγt+ T regulatory cells extinguished IL-17 production by γδ T cells and prevented immunopathology. Microbial gain-of-function approaches determined that colonic γδ T cell activation occurred in response to passive acquisition of C. difficile at birth. In turn, IL-17 limited C. difficile growth and intestinal barrier breach by the commensal microbiota. Collectively, our results reveal how an agile immune cell network meets the demands of a maturing microbiota to support gut health during early life. Highlights: - Colonic γδ17 T cells are activated during early life in a microbiota-specific manner - Fetal-derived γδ17 T cells require IL-1 signals for activation - Passive acquisition of C.difficile induces γδ17 T cell activation - IL-17 limits C. difficile growth and systemic dissemination of commensals

Oxford Nanopore Technologies (ONT) rapid library preparation kits use transposase-mediated tagmentation to attach click chemistry functionalized oligonucleotide duplexes to fragmented DNA, followed by click chemistry to conjugate Rapid Adapter (RA) sequencing adapters. A similar protocol is used in 16S rRNA gene amplicon and PCR-amplified rapid whole-genome sequencing workflows. Here, we describe custom oligonucleotides with dibenzocyclooctyne (DBCO) added onto PCR primer 5′ termini. After standard PCR amplification, DBCO-modified amplicons react spontaneously with RA sequencing adapters, producing sequencing-ready libraries in minutes without enzymatic processing. All configurations employ an asymmetric design in which the DBCO modification is restricted to a single primer, leaving the opposite primer available for barcoding at low cost. We validate three primer architectures: (i) direct attachment of DBCO to a target-specific primer, (ii) a universal DBCO-modified oligonucleotide used in a two-step PCR workflow, and (iii) a three-primer single-pot reaction combining the universal DBCO oligonucleotide with unmodified target-specific primers. These configurations are validated using full-length 16S rRNA gene amplicons sequenced on a PromethION flow cell. DBCO-modified primers are synthesized either commercially or in-house via DBCO-TFP ester conjugation to 5′-amino oligonucleotides and remain fully active through standard PCR thermocycling. The best-performing configuration used a two-step PCR with a universal oligonucleotide and achieved higher pore occupation and reads than comparable commercial solutions. This approach reduces library preparation reagent costs compared to available kits, as the initial synthesis cost is lower than existing amplicon sequencing kits, while providing enough material for hundreds or thousands of PCR reactions. This is further applicable to an unlimited number of gene targets beyond 16S sequencing.

Antibiotics are known to trigger prophage induction in controlled laboratory settings, but it remains unclear whether this also occurs within microbiomes in nature. Current methods investigating the link between antibiotics and prophage induction within the human gut rely on in vitro culturing of human gut bacterial isolates. Using a metagenomic approach, we aimed to measure prophage induction and whether it is associated with antibiotic exposure. Across two independent human cohorts, we compared prophage to bacterial host read depth ratios (P:H) across known or measured antibiotic exposures. We found that induction is not broadly associated with antibiotic exposures at the level of the overall microbiome, but that ciprofloxacin increases P:H ratios in specific bacterial species. We documented heterogeneous trajectories of P:H ratios over the course of antibiotic exposure, sometimes increasing and remaining high, or returning to baseline. This study complements experimental models by providing in vivo evidence of induction in the human gut. Importance: Bacteriophages are viruses that infect a bacterial host. The lytic and lysogenic cycles are the two classic outcomes of phage infection. In the lytic cycle, the phage immediately replicates and lyses its host to release new viral particles. In the lysogenic cycle, the phage, now called a prophage, integrates its genome into that of its host without killing it. Prophages can switch to the lytic cycle in a process called induction, in which the viral genome is replicated, the host cell is lysed, and viral particles are released. The most immediate consequence of induction is host cell death which can impact bacterial populations and communities. Since prophages are mobile genetic elements that can move between bacteria, they are also an important vehicle for horizontal gene transfer. While induction has been well studied in vitro , whether and how induction occurs within the complex microbial ecosystem in humans is less well characterized. Understanding prophage induction in vivo is therefore critical in corroborating in vitro observations.

Global climate change and nutrient pollution from agricultural systems increase cyanobacterial bloom and toxin release events, and will pose a significant concern for public health and safety over the coming decades. Many in situ studies have focused on environmental, chemical, and microbial community changes and their impact on cyanobacterial bloom frequency and toxicity. However, fine-scale genetic differences in the genomes of bloom-forming cyanobacteria may also impact the quantity and types of toxins produced. Metagenomic approaches allow resolution of strain- and nucleotide-level changes within microbial communities and can improve our understanding of the factors that affect cyanobacterial bloom dynamics and toxicity. Here, we conduct a metagenomic analysis of the bloom-forming cyanobacterial genus Microcystis across a 10-month lake time series from the Valle de Bravo Reservoir, to assess how within-genus genotype-level changes are linked to intracellular toxin production and extracellular toxin release, as well as how single nucleotide variation may affect the types of microcystin toxins produced. Our results demonstrate that the abundances of both toxigenic and non-toxigenic Microcystis genotypes are significantly related to microcystin toxin concentrations. In addition to these genome-wide (”strain”-level) associations, specific single nucleotide variants show strong associations with chemical variants of microcystin in the environment. Our work highlights the importance of fine-scale analysis of microbial community composition for understanding cyanobacterial bloom dynamics and toxin production.

Among dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen by dnd and ssp gene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possessed dnd or ssp genes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis revealed several previously undescribed PT synthesis systems, including type 4 Bacteriophage Exclusion (BREX) type 4 brx genes, which we genetically validated in Bacteroides salyersiae. Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessing dnd, ssp, and brx genes revealed 8 PT dinucleotide settings confirmed in 10 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.

Bacterial populations evolve rapidly in the lab when faced with experimentally applied selective pressures. Yet how bacteria evolve in nature, in more complex multi-species communities, is both challenging to study and essential to our understanding of ecosystem responses to rapid anthropogenic change. To track bacterial evolution in a semi-natural context, we applied Roundup, a glyphosate-based herbicide (GBH) that interferes with aromatic amino acid synthesis, as a selective pressure to 1000 L ponds containing bacterioplankton communities from a pristine lake. We show that both ecological and evolutionary changes can occur on short timescales after a strong selective pressure. Using metagenome-assembled genomes as a proxy for species, we found that GBH treatment substantially affected community diversity but did not purge within-species genetic diversity over the 4 weeks of the experiment. We identified several functional categories of genes consistently targeted by GBH selection across seven different species of bacteria. Genes involved in amino acid transport and metabolism were more likely to experience GBH-driven changes in allele frequency, including the gene aroA targeted by glyphosate, along with other potentially novel targets of selection. Together, these results show how environmental change can rapidly affect bacterial community structure and select for specific genetic targets without purging genetic diversity genome-wide.