Gabriella Reynolds

All Publications

• Transposable elements are driving rapid adaptation of Enterococcus faecium. Nature Grieshop, M. P., Behr, A. A., Bowden, S., Lin, J. D., Molari, M., Reynolds, G. Z., Brooks, E. F., Doyle, B., Moore, A. A., Rodriguez-Nava, G., Salinas, J. L., Banaei, N., Bhatt, A. S. 2026

  Abstract

  Bacterial pathogens adapt rapidly to clinical and within-host selective pressures1. Insertion sequences (IS) are transposable elements that can contribute to pathogenic adaptation2, but their activity and consequences in contemporary clinical populations are not well characterized. Here, combining large-scale genomic surveys with long-read sequencing of clinical isolates and longitudinal gut metagenomes, we quantify pathogen IS dynamics from global patterns to within-host evolution. Across 19,485 publicly available high-contiguity ESKAPEE pathogen genomes, Enterococcus faecium genomes are the most IS dense, dominated by replicative ISL3 family elements, which have proliferated in clinical lineages over the past 30 years. We find extensive chromosomal structural variation, largely involving ISL3, within a new single-hospital collection of bloodstream isolates. Long-read metagenomic sequencing of 28 longitudinal stool samples from 12 haematopoietic cell transplantation (HCT) recipients demonstrates within-host IS dynamics and their regulatory consequences. In one patient, an ISL3 insertion upstream of a folate transporter formed a strong promoter, increasing transcription and improving relative fitness under folate limitation. Enhanced folate scavenging may enable E. faecium to thrive in the setting of microbiome collapse, which is common in HCT and other critically ill patients3. Together, these results show that a recent ISL3 expansion is driving rapid evolution in healthcare-associated E. faecium, with consequences for its metabolic fitness that may help explain its increasing clinical burden. Several other pathogens also show elevated IS loads in our survey, which suggests that IS expansion-mediated evolution might be more broadly relevant.

  View details for DOI 10.1038/s41586-026-10373-2

  View details for PubMedID 42020750

  View details for PubMedCentralID 7190074

• Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR. Nature protocols Doyle, B., Reynolds, G. Z., Dvorak, M., Maghini, D. G., Natarajan, A., Bhatt, A. S. 2025

  Abstract

  Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.

  View details for DOI 10.1038/s41596-025-01165-5

  View details for PubMedID 40389632

  View details for PubMedCentralID 6750738

• Accurate prediction of absolute prokaryotic abundance from DNA concentration. Cell reports methods Wirbel, J., Andermann, T. M., Brooks, E. F., Evans, L., Groth, A., Dvorak, M., Chakraborty, M., Palushaj, B., Reynolds, G. Z., Porter, I. E., Al Malki, M., Rezvani, A., Gooptu, M., Elmariah, H., Runaas, L., Fei, T., Martens, M. J., Bolaños-Meade, J., Hamadani, M., Holtan, S., Jenq, R., Peled, J. U., Horowitz, M. M., Poston, K. L., Saber, W., Kean, L. S., Perales, M. A., Bhatt, A. S. 2025: 101030

  Abstract

  Quantification of the absolute microbial abundance in a human stool sample is crucial for a comprehensive understanding of the microbial ecosystem, but this information is lost upon metagenomic sequencing. While several methods exist to measure absolute microbial abundance, they are technically challenging and costly, presenting an opportunity for machine learning. Here, we observe a strong correlation between DNA concentration and the absolute number of 16S ribosomal RNA copies as measured by digital droplet PCR in clinical stool samples from individuals undergoing hematopoietic cell transplantation (BMT CTN 1801). Based on this correlation and additional measurements, we trained an accurate yet simple machine learning model for the prediction of absolute prokaryotic load, which showed exceptional prediction accuracy on an external cohort that includes people living with Parkinson's disease and healthy controls. We propose that, with further validation, this model has the potential to enable accurate absolute abundance estimation based on readily available sample measurements.

  View details for DOI 10.1016/j.crmeth.2025.101030

  View details for PubMedID 40300608

• Replicative selfish genetic elements are driving rapid pathogenic adaptation of Enterococcus faecium. bioRxiv : the preprint server for biology Grieshop, M. P., Behr, A. A., Bowden, S., Lin, J. D., Molari, M., Reynolds, G. Z., Brooks, E. F., Doyle, B., Rodriguez-Nava, G., Salinas, J. L., Banaei, N., Bhatt, A. S. 2025

  Abstract

  Understanding how healthcare-associated pathogens adapt in clinical environments can inform strategies to reduce their burden. Here, we investigate the hypothesis that insertion sequences (IS), prokaryotic transposable elements, are a dominant mediator of rapid genomic evolution in healthcare-associated pathogens. Among 28,207 publicly available pathogen genomes, we find high copy numbers of the replicative ISL3 family in healthcare-associated Enterococcus faecium, Streptococcus pneumoniae and Staphylococcus aureus. In E. faecium, the ESKAPE pathogen with the highest IS density, we find that ISL3 proliferation has increased in the last 30 years. To enable better identification of structural variants, we long read-sequenced a new, single hospital collection of 282 Enterococcal infection isolates collected over three years. In these samples, we observed extensive, ongoing structural variation of the E. faecium genome, largely mediated by active replicative ISL3 elements. To determine if ISL3 is actively replicating in clinical timescales in its natural, gut microbiome reservoir, we long read-sequenced a collection of 28 longitudinal stool samples from patients undergoing hematopoietic cell transplantation, whose gut microbiomes were dominated by E. faecium. We found up to six structural variants of a given E. faecium strain within a single stool sample. Examining longitudinal samples from one individual in further detail, we find ISL3 elements can replicate and move to specific positions with profound regulatory effects on neighboring gene expression. In particular, we identify an ISL3 element that upon insertion replaces an imperfect -35 promoter sequence at a folT gene locus with a perfect -35 sequence, which leads to substantial upregulation of expression of folT, driving highly effective folate scavenging. As a known folate auxotroph, E. faecium depends on other members of the microbiota or diet to supply folate. Enhanced folate scavenging may enable E. faecium to thrive in the setting of microbiome collapse that is common in HCT and other critically ill patients. Together, ISL3 expansion has enabled E. faecium to rapidly evolve in healthcare settings, and this likely contributes to its metabolic fitness and may strongly influence its ongoing trajectory of genomic evolution.

  View details for DOI 10.1101/2025.03.16.643550

  View details for PubMedID 40161577

  View details for PubMedCentralID PMC11952509

• Intragenic DNA inversions expand bacterial coding capacity. Nature Chanin, R. B., West, P. T., Wirbel, J., Gill, M. O., Green, G. Z., Park, R. M., Enright, N., Miklos, A. M., Hickey, A. S., Brooks, E. F., Lum, K. K., Cristea, I. M., Bhatt, A. S. 2024

  Abstract

  Bacterial populations that originate from a single bacterium are not strictly clonal and often contain subgroups with distinct phenotypes1. Bacteria can generate heterogeneity through phase variation-a preprogrammed, reversible mechanism that alters gene expression levels across a population1. One well-studied type of phase variation involves enzyme-mediated inversion of specific regions of genomic DNA2. Frequently, these DNA inversions flip the orientation of promoters, turning transcription of adjacent coding regions on or off2. Through this mechanism, inversion can affect fitness, survival or group dynamics3,4. Here, we describe the development of PhaVa, a computational tool that identifies DNA inversions using long-read datasets. We also identify 372 'intragenic invertons', a novel class of DNA inversions found entirely within genes, in genomes of bacterial and archaeal isolates. Intragenic invertons allow a gene to encode two or more versions of a protein by flipping a DNA sequence within the coding region, thereby increasing coding capacity without increasing genome size. We validate ten intragenic invertons in the gut commensal Bacteroides thetaiotaomicron, and experimentally characterize an intragenic inverton in the thiamine biosynthesis gene thiC.

  View details for DOI 10.1038/s41586-024-07970-4

  View details for PubMedID 39322669

  View details for PubMedCentralID 452554

• Nirmatrelvir-Ritonavir and Symptoms in Adults With Postacute Sequelae of SARS-CoV-2 Infection: The STOP-PASC Randomized Clinical Trial. JAMA internal medicine Geng, L. N., Bonilla, H., Hedlin, H., Jacobson, K. B., Tian, L., Jagannathan, P., Yang, P. C., Subramanian, A. K., Liang, J. W., Shen, S., Deng, Y., Shaw, B. J., Botzheim, B., Desai, M., Pathak, D., Jazayeri, Y., Thai, D., O'Donnell, A., Mohaptra, S., Leang, Z., Reynolds, G. Z., Brooks, E. F., Bhatt, A. S., Shafer, R. W., Miglis, M. G., Quach, T., Tiwari, A., Banerjee, A., Lopez, R. N., De Jesus, M., Charnas, L. R., Utz, P. J., Singh, U. 2024

  Abstract

  There is an urgent need to identify treatments for postacute sequelae of SARS-CoV-2 infection (PASC).To assess the efficacy of a 15-day course of nirmatrelvir-ritonavir in reducing the severity of select PASC symptoms.This was a 15-week blinded, placebo-controlled, randomized clinical trial conducted from November 2022 to September 2023 at Stanford University (California). The participants were adults with moderate to severe PASC symptoms of 3 months or longer duration.Participants were randomized 2:1 to treatment with oral nirmatrelvir-ritonavir (NMV/r, 300 mg and 100 mg) or with placebo-ritonavir (PBO/r) twice daily for 15 days.Primary outcome was a pooled severity of 6 PASC symptoms (fatigue, brain fog, shortness of breath, body aches, gastrointestinal symptoms, and cardiovascular symptoms) based on a Likert scale score at 10 weeks. Secondary outcomes included symptom severity at different time points, symptom burden and relief, patient global measures, Patient-Reported Outcomes Measurement Information System (PROMIS) measures, orthostatic vital signs, and sit-to-stand test change from baseline.Of the 155 participants (median [IQR] age, 43 [34-54] years; 92 [59%] females), 102 were randomized to the NMV/r group and 53 to the PBO/r group. Nearly all participants (n = 153) had received the primary series for COVID-19 vaccination. Mean (SD) time between index SARS-CoV-2 infection and randomization was 17.5 (9.1) months. There was no statistically significant difference in the model-derived severity outcome pooled across the 6 core symptoms at 10 weeks between the NMV/r and PBO/r groups. No statistically significant between-group differences were found at 10 weeks in the Patient Global Impression of Severity or Patient Global Impression of Change scores, summative symptom scores, and change from baseline to 10 weeks in PROMIS fatigue, dyspnea, cognitive function, and physical function measures. Adverse event rates were similar in NMV/r and PBO/r groups and mostly of low grade.The results of this randomized clinical trial showed that a 15-day course of NMV/r in a population of patients with PASC was generally safe but did not demonstrate a significant benefit for improving select PASC symptoms in a mostly vaccinated cohort with protracted symptom duration. Further studies are needed to determine the role of antivirals in the treatment of PASC.ClinicalTrials.gov Identifier: NCT05576662.

  View details for DOI 10.1001/jamainternmed.2024.2007

  View details for PubMedID 38848477

• Intragenic DNA inversions expand bacterial coding capacity. bioRxiv : the preprint server for biology Chanin, R. B., West, P. T., Park, R. M., Wirbel, J., Green, G. Z., Miklos, A. M., Gill, M. O., Hickey, A. S., Brooks, E. F., Bhatt, A. S. 2023

  Abstract

  Bacterial populations that originate from a single bacterium are not strictly clonal. Often they contain subgroups that have distinct phenotypes. One way that bacteria generate this heterogeneity is through phase variation: enzyme-mediated, reversible inversion of specific intergenic regions of genomic DNA. These DNA inversions can flip the orientation of promoters within otherwise isogenic populations, impacting fitness and survival. We developed and applied bioinformatic approaches that enabled the discovery of thousands of previously undescribed phase-variable regions in prokaryotes using long-read datasets. We identified 'intragenic invertons', a surprising new class of invertible elements found entirely within genes, across the prokaryotic tree of life. Intragenic invertons allow a single gene to encode two or more versions of a protein by flipping a DNA sequence within the gene, thereby increasing coding capacity without increasing genome size. We experimentally characterize specific intragenic invertons in the gut commensal Bacteroides thetaiotaomicron , presenting a 'roadmap' for investigating this gene-diversifying phenomenon.One-Sentence Summary: Intragenic DNA inversions, identified using long-read sequencing datasets, are found across the prokaryotic tree of life.

  View details for DOI 10.1101/2023.03.11.532203

  View details for PubMedID 36945655