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An engineered live biotherapeutic for the prevention of antibiotic-induced dysbiosis

Nature Biomedical Engineering volume 6, pages 910–921 (2022)Cite this article

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Abstract

Antibiotic-induced alterations in the gut microbiota are implicated in many metabolic and inflammatory diseases, increase the risk of secondary infections and contribute to the emergence of antimicrobial resistance. Here we report the design and in vivo performance of an engineered strain of Lactococcus lactis that altruistically degrades the widely used broad-spectrum antibiotics β-lactams (which disrupt commensal bacteria in the gut) through the secretion and extracellular assembly of a heterodimeric β-lactamase. The engineered β-lactamase-expression system does not confer β-lactam resistance to the producer cell, and is encoded via a genetically unlinked two-gene biosynthesis strategy that is not susceptible to dissemination by horizontal gene transfer. In a mouse model of parenteral ampicillin treatment, oral supplementation with the engineered live biotherapeutic minimized gut dysbiosis without affecting the ampicillin concentration in serum, precluded the enrichment of antimicrobial resistance genes in the gut microbiome and prevented the loss of colonization resistance against Clostridioides difficile. Engineered live biotherapeutics that safely degrade antibiotics in the gut may represent a suitable strategy for the prevention of dysbiosis and its associated pathologies.

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Fig. 1: A heterodimeric β-lactamase (spTEM1) system for the extracellular degradation of β-lactam antibiotics.
Fig. 2: Enzymatic activity of the spTEM1 β-lactamase in the mouse intestine does not influence ampicillin concentration in serum.
Fig. 3: L. lactis spTEM1 protects the diversity and composition of the gut microbiota in an ampicillin-induced dysbiosis murine model.
Fig. 4: L. lactis spTEM1 prevents the enrichment of ARGs following the administration of ampicillin in mice.
Fig. 5: L. lactis spTEM1 maintains colonization resistance against C. difficile in a mouse model of ampicillin-induced dysbiosis.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. All DNA sequence data generated in this study are available from the Sequence Read Archive with accession number PRJNA803721. Source data are provided with this paper.

Code availability

All code for the 16S rDNA analysis and for the metagenomics analysis are available on GitHub at https://github.com/maalcantar/eLBP-prevents-dysbiosis-16s-analysis and https://github.com/maalcantar/eLBP-prevents-dysbiosis-metagenomics-analysis.

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Acknowledgements

We are grateful to A. Graveline for help with the animal protocol setup and A. Vernet and M. Sanchez-Ventura for assistance with animal experiments. We thank X. Tan, M. A. English, R. Gayet, D. Morales and J. Cubillos-Ruiz for helpful comments and discussions. We also thank K. Pärnänen for helpful discussion on metagenomic data processing and calculating antibiotic resistance gene abundances from metagenomic data. Additionally, we thank S. Blomquist for help with running analysis pipelines via the Commonwealth Computational Cloud for Data Driven Biology (C3DDB) cluster. This work was supported by funding from the Defense Threat Reduction Agency grant HDTRA1-14-1-0006 (to J.J.C.), Wyss Institute funding (to J.J.C.) and the Paul G. Allen Frontiers Group (to J.J.C.) and Wyss Institute validation project funding (to A.C.-R.). M.A.A. was supported by a National Science Foundation graduate research fellowship (award no. 1122374).

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Authors and Affiliations

  1. Institute for Medical Engineering and Science, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA

    Andrés Cubillos-Ruiz, Miguel A. Alcantar & James J. Collins

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Andrés Cubillos-Ruiz, Nina M. Donghia & James J. Collins

  3. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Andrés Cubillos-Ruiz, Julian Avila-Pacheco & James J. Collins

  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Miguel A. Alcantar, Pablo Cárdenas & James J. Collins

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  1. Andrés Cubillos-Ruiz
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Contributions

A.C.-R. conceptualized the project, designed and performed experiments, analysed and interpreted data, acquired funding and wrote the manuscript. N.M.D. and J.A.-P. performed experiments; M.A.A. analysed data, performed experiments and wrote the manuscript; P.C. and J.A.-P. analysed data; and J.J.C. supervised the work, assisted with manuscript editing and acquired funding.

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Correspondence to James J. Collins.

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J.J.C. is co-founder and SAB chair of Synlogic and EnBiotix. A.C.-R. and J.J.C. have filed a patent application for this work. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Schematic of the antibiotic survival landscape of the bacterial strains in this study.

a. In wildtype cells, the survival to the antibiotic is dictated by the minimum inhibitory concentration (MIC), which is the lowest concentration of an antibiotic that achieves inhibition of growth. In a population of antibiotic-sensitive cells, the landscape partition between survival and death is independent of cell density. b. Antimicrobial resistance factors (that is, native β-lactamase) decrease the susceptibility of the bacterial cell to the antibiotic, increasing the MIC. In a population of antibiotic-resistant cells, the partition between survival and death is also independent of cell density. c. Density-dependent survival effect in spTEM1-expressing strains. Secretion and extracellular assembly of the spTEM1 β-lactamase preclude self-protection in producer cells and makes the partition of the antibiotic survival landscape a function of the cell density. Below the critical cell density threshold, the engineered cells are as susceptible to the antibiotic as wildtype cells. Above the cell density threshold, the engineered cells can survive the antibiotic to the same extent as their neighboring cells, offering population-wide protection.

Extended Data Fig. 2 Determination of L. lactis load in the mouse intestine.

a. Treatment regimen. Mice were orally gavaged with two doses of 1010 CFU of L. lactis. The first dose occurred 2 hours prior to the ampicillin injection and the second simultaneous with the 200 mg/kg ampicillin injection. Intestinal load was evaluated each day for L. lactis spTEM1 and the empty-vector control strain L. lactis EV. b. Enumeration of viable L. lactis cells in feces in each day of treatment demonstrates no difference in the fecal loads of L. lactis spTEM1 (n = 8) and L. lactis EV (n = 8). Box plots display minimum, 25th percentile, median, 75th percentile, and maximum of biological replicate measurements. Unpaired two-sided Wilcoxon test: Day 1, p = 0.7944; Day 2, p = 0.0511; Day 3, p = 0.4093.

Extended Data Fig. 3 Detection of β-lactamase activity in fecal samples of ampicillin-naïve mice.

a. Fecal samples were collected 2 hours after a single dose of L. lactis spTEM1 or delivery vehicle control. b. Nitrocefin hydrolysis assay for the detection of β-lactamase activity. Means and standard deviations of biological replicates are shown. n = 10 mice in the L. lactis spTEM1 and control groups.

Extended Data Fig. 4 Mouse model for parenteral ampicillin-induced dysbiosis and the disruption of colonization resistance against C. difficile.

a. A 3-day intraperitoneal ampicillin administration regimen is evaluated for its effects in abolishing colonization resistance against 5×103 spores of C. difficile at 24 hours after the last ampicillin dose. C. difficile density in feces is evaluated 24 hours after the infection. b. Evaluation of single- or double-dose (8 hours apart) administration regimens for intraperitoneal ampicillin injection indicates that a single dose of ampicillin for 3 days is enough to sensitize the mouse gut to robust C. difficile colonization. n = 5 mice in each treatment group. Means and standard deviations of biological replicates are shown. c. Dose-dependency of single daily intraperitoneal ampicillin injections in the disruption of colonization resistance against C. difficile. n = 5 mice in each treatment group. Means and standard deviations of biological replicates are shown.

Extended Data Fig. 5 Diversity and composition of the gut microbiota during the experiment of preservation of colonization resistance against C. difficile.

a. Determination of the Shannon diversity index for gut microbial communities in mice pre- and post-treatment. The p-values correspond to FDR-corrected Wilcoxon tests between the groups that received L. lactis spTEM1 and L. lactis EV. Adjusted P-values below boxes correspond to FDR-corrected two-sided Wilcoxon tests comparing diversity values against baseline diversity at the pre-treatment timepoint. Box plots display minimum, 25th percentile, median, 75th percentile, and maximum of biological replicate measurements. n = 8 mice in each treatment group. b. Principal coordinates analysis of gut microbial communities in ampicillin-treated mice receiving L. lactis spTEM1 and L. lactis EV control reveals differences in the resulting composition of the community. Close clustering to pre-ampicillin conditions indicates smaller alterations in the community structure. n = 8 mice in each treatment group.

Extended Data Fig. 6 Metagenomic analysis of antimicrobial resistance genes (ARG) during the experiment of preservation of colonization resistance against C. difficile.

a. Analysis of the abundance of ARG reveals significant enrichment in ampicillin-treated mice receiving L. lactis EV but not in mice receiving L. lactis spTEM1. Stacked bar data are presented as reads mapping the different CARD database categories and normalized to the size of the read pool in each sample. Vector-derived ARG in the β-lactam and chloramphenicol classes are presented as a different category to differentiate them from endogenous ARG. Adjusted p-values were calculated with a negative binomial generalized linear model with Tukey’s post hoc test between spTEM1-treated and EV-treated groups. b. Abundance of endogenous and L. lactis spTEM1-derived β-lactamases in the mouse. Elimination of the ampicillin selective pressure by the spTEM1 β-lactamases reduces the enrichment of endogenous β-lactamases in the mouse gut. Rapid elimination from the system of the spTEM1 gene fragments compared to endogenous β-lactamase genes suggests lack of competitive advantage in the spTEM1 strain. n = 8 mice in each L. lactis spTEM1 and L. lactis EV groups. p-value significance corresponds to FDR-corrected two-sided Wilcoxon tests comparing ARG abundance values against baseline values at the pre-treatment timepoint. Means and standard deviations of biological replicates are shown.

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Cubillos-Ruiz, A., Alcantar, M.A., Donghia, N.M. et al. An engineered live biotherapeutic for the prevention of antibiotic-induced dysbiosis. Nat. Biomed. Eng 6, 910–921 (2022). https://doi.org/10.1038/s41551-022-00871-9

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