Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus

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Abstract

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

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Fig. 1: BPSCSK expresses a lantibiotic in vivo that inhibits VRE.
Fig. 2: BPSCSK colonizes the gastrointestinal tract and broadly inhibits Gram-positive pathogens while preserving some commensal species.
Fig. 3: Lantibiotic genes are present in human microbiomes of healthy individuals and gut resident, lantibiotic-producing species inhibit VRE.
Fig. 4: Enrichment of lantibiotic genes correlates with reduced E. faecium in patient faecal samples.

Data availability

Microbiome sequencing data are available from Bioproject with the accession number 394877.

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Acknowledgements

This work was supported by grants RO1 AI42135, RO1 AI95706, UO1 AI124275, and P30 CA008748 from the US National Institutes of Health (NIH) and the Tow Foundation and Lucille Castori Center for Microbes, Inflammation and Cancer to E.G.P. S.G.K. is supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences, NIH (award T32GM07739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program). S.B. was supported by an Early Postdoc Mobility Fellowship from the Swiss National Science Foundation and an Irvington Fellowship from the Cancer Research Institute. We thank members of the Pamer laboratory for discussions and comments on the manuscript.

Author information

S.G.K. and E.G.P. designed the experiments and wrote the manuscript. S.G.K. performed and analysed most experiments. S.B. helped to design experiments, performed and analysed fluorescence in situ hybridization and RNA-sequencing analysis on caecal content. T.U.M., S.C. and V.E. cultured bacterial isolates from faecal samples and analysed whole-genome sequences of isolates. P.V.S. and R.C.H. performed peptide purifications and subsequent characterization by mass spectrometry. E.R.L. performed bioinformatic analyses and metagenomic sequence data. R.S. assisted in bacterial culturing and animal experiments. I.M.L. and R.S. maintained and screened mouse strains. M.G. generated and characterized bacterial isolates from faecal samples. W.Q., R.J.J.F.R. and J.R.C. contributed to the development of methods to purify bacterial lantibiotics for biochemical analyses. E.F., L.A. and R.W. performed DNA extractions, 16S MiSeq Illumina sequencing and analysed microbiome sequence data. Z.-M.X.W. assisted in ileal homogenization, western blot, and RT–qPCR analyses. H.-J.J. contributed to the cloning and expression of the lantibiotic gene. S.M.M. and Y.T. enrolled patients undergoing allogeneic haematopoietic cell transplantation in the prospective faecal collection protocol and contributed to the analysis of sequence data. S.N. and K.H. contributed human-derived commensal bacterial strains that were included in this study. J.U.P. and M.R.M.v.d.B. contributed to the analyses of patient-derived faecal samples.

Correspondence to Eric G. Pamer.

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Competing interests

K.H. is co-founder and scientific advisor to Vedanta Biosciences. M.R.M.v.d.B. is on the advisory board of and has financial holdings in Seres Therapeutics Inc., serves on the DKMS medical council, has received speaker honoraria from Merck and Acute Leukemia Forum, holds patents that receive royalties from Seres Therapeutics Inc., has received honorarium and research support (1 January 2017 to present) from Seres Therapeutic Inc., and IP licensing with Seres Therapeutics Inc. and Juno. J.U.P. reports research funding, intellectual property fees, and travel reimbursement from Seres Therapeutics. E.G.P. has received speaker honoraria from Bristol-Myer Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis, and Ferring Pharmaceuticals; is an inventor on patent application no. WPO2015179437A1, entitled ‘Methods and compositions for reducing Clostridium difficile infection’ and no. WPO2017091753A1, entitled ‘Methods and compositions for reducing vancomycin-resistant Enterococci infection or colonization’; and holds patents that receive royalties from Seres Therapeutics Inc.

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Peer review information Nature thanks Eran Elinav, Michael Gilmore, Barbara Murray and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 BPSCSK directly inhibits VRE through a contact-independent mechanism.

ad, VRE was co-cultured with each CBBPSCSK isolate (n = 15 biologically independent samples from three independent experiments) and growth was monitored. e, VRE was inoculated in conditioned media from each CBBPSCSK isolate culture (n = 15 biologically independent samples from three independent experiments). fi, VRE was inoculated in conditioned media from each CBBPSCSK isolate culture (−VRE), or each CBBPSCSK isolate co-cultured with VRE (+VRE) (n = 5 biologically independent samples from five independent experiments) and growth was monitored. j, VRE was inoculated in conditioned media from Blautia species cultures (n = 6 strains, 15 biologically independent samples from three independent experiments). VRE (ATCC 700221) was used in all experiments shown. Data are median ± range (ae, j) or mean ± s.d. (fi). ****P < 0.0001, two-tailed Mann–Whitney U-test for comparisons with a negative control.

Extended Data Fig. 2 BPSCSK, but not BPcontrol, reduces VRE colonization in vivo.

a, b, Faecal samples collected from antibiotic- treated, VRE-dominated mice (n = 4 mice from one independent experiment) orally gavaged with CBBPSCSK (a) or CBBPcontrol (b) were shotgun sequenced and the relative abundance of each species was determined by 16S rRNA. c, d, Antibiotic-treated (c) or germ-free (d) mice (n = 8 mice from two independent experiments) were orally gavaged with VRE. Three days later, VRE-dominated mice received an oral gavage of CBBPSCSK or CBBPcontrol and VRE colonization was monitored by quantifying VRE in faecal samples. eg, Antibiotic-treated mice (n = 4 mice from one independent experiment) were orally gavaged with different strains of clinical VRE isolates. Three days later, VRE-dominated mice received an oral gavage of CBBPSCSK or CBBPcontrol and VRE colonization was monitored by quantifying VRE in faecal samples. The following VRE strains were used: strain 0151F is an E. faecium MLST type ST80 (e); strain 1107 is an E. faceium MLST type ST412 (f); strain V583 is an E. faecalis strain (g). VRE strains used were VRE (ATCC 700221) (ad), VRE (0151F) (e), VRE (1107) (f), and VRE (V583) (g). Data are mean ± s.d. *P < 0.05, ***P < 0.001, two-tailed Mann–Whitney U-test.

Extended Data Fig. 3 BPSCSK colonizes the large intestine.

Antibiotic-treated mice (n = 5 mice from one independent experiment) were orally administered CBBPSCSK. Two weeks later, BPSCSK localization around the mucosal epithelium (top) and lumen (bottom) of the caecum were visualized by fluorescence in situ hybridization. Entire caecum cross-sections were hybridized with a probe specific for BPSCSK. Sections were counterstained with Hoechst dye to visualize the nuclei. Representative images are shown. Scale bars, 25 μm.

Extended Data Fig. 4 CBBPSCSK mediates VRE colonization resistance by producing an inhibitor.

a, Antibiotic-treated mice (n = 8 mice from two independent experiments) received treatment by oral gavage containing CBBPSCSK, CBBPcontrol, PBS or VRE. One week later, VRE was inoculated into the caecal content and growth was monitored 6 h after inoculation. bi, Antibiotic-treated mice received an oral gavage containing CBBPSCSK (n = 4 mice from one independent experiment) or PBS (n = 3 mice from one independent experiment). Wild-type mice (n = 4 mice from one independent experiment) were untreated and received no antibiotics. Four days later, RNA and proteins were extracted from the distal ileum, and RegIIIγ was measured by RT–qPCR (b) and western blot (c). Other genes involved in host-derived antimicrobial peptide production, including angiogenin-4 (ang4) (d), defensin-1 (def1) (e), amphiregulin (areg) (f), and deleted in malignant brain tumours 1 (dmbt1) (g); or inflammatory mediators including cytochrome b beta (cybb) (h) and calgranulin A (s100a8) (i) were measured by RT–qPCR. j, Rag2/Il2rg−/− mice were treated with antibiotics, and orally gavaged with VRE. Three days later, VRE-dominated mice received CBBPSCSK or CBBPcontrol by oral gavage and VRE colonization was monitored by quantifying VRE in faecal samples. VRE (ATCC 700221) was used in experiments in a and j. *P < 0.05 (0.0286), ***P < 0.001, ****P < 0.0001, two-tailed Mann–Whitney U-test. Data are median ± range (a) or mean ± s.d. (b, di).

Extended Data Fig. 5 BPSCSK encodes a lantibiotic.

a, VRE was inoculated in media conditioned with BPSCSK or BPcontrol culture protein precipitate fractions (n = 8 biologically independent samples from two independent experiments), and monitored for growth. b, c, BPSCSK was whole-genome sequenced, assembled and annotated. b, Schematic comparing the lantibiotic operon discovered in the genome of BPSCSK to the nisA operon from L. lactis. Gene functions are based on the characterization of homologous genes in the nis operon. c, Amino acid sequence alignment comparing the BPSCSK lantibiotic precursor (LanA1–LanA4) and the nisin-A precursor (NisA). Sequence features are based on the characterization of nisin. d, The molecular formula for the mature, post-translationally modified BPSCSK LanA1–LanA4 lantibiotic with a predicted mass of 3152.45 Da. Abu, alpha-aminobutyric acid; Dha, dehydroalanine;Dhb, dehydrobutyrine. e, Media conditioned with BPSCSK or BPcontrol culture protein precipitates, or commercial nisin-A, were incubated with proteinase K for 3 h at 37 °C, boiled at 100 °C, or left untreated. The treated protein precipitate (n = 8 biologically independent samples from four independent experiments) was serially diluted and VRE was inoculated and cultured for 24 h. The MIC value was the highest mean dilution in which VRE inhibition was observed. f, Proteins were precipitated from BPSCSK or BPcontrol, or nisin-A spiked cultures and applied to a SP sepharose column. Each fraction was serially diluted and VRE was inoculated and cultured for 24 h to determine the MIC (n = 4 biologically independent samples from four independent experiments). VRE (ATCC 700221) was used in experiments in a, e and f. ***P < 0.001, ****P < 0.0001, two-tailed Mann–Whitney U-test for comparisons with a negative control. Data are mean ± s.d. (a, f) or mean values (e).

Extended Data Fig. 6 Heterologous expression of BPSCSK LanA1–LanA4 lantibiotic.

a, Genes involved in biosynthesis of the BPSCSK lantibiotic (His-tagged-LanA, LanB and LanC) were cloned into expression vectors (pRSFDuet-1/LanA+LanB, pCDFDuet-1/LanC) and heterologously expressed in E. coli. a, Schematic map indicating where each lantibiotic gene was inserted into the respective expression vectors. b, c, The His-tagged LanA1–LanA4 lantibiotic was purified from E. coli lysates by HiTrap HP nickel affinity chromatography and subsequently purified to homogeneity by reversed-phase high-performance liquid chromatography. The leader sequence and His tag were removed by trypsin digestion to yield the mature lantibiotic. The purified His-tag product (b) and the purified mature lantibiotic (c) were analysed by electrospray ionization–mass spectrometry (ESI–MS) and the spectrum was deisotoped and deconvoluted using the Xtract algorithm in Xcalibur. The signals with labels correspond to the predicted mass of the His-tagged lantibiotic (M) and its incomplete forms that did not dehydrate all nine residues (for example, M + 1·H2O and M + 2·H2O).

Extended Data Fig. 7 Oral administrations of BPSCSK protein precipitate reduce VRE colonization in vivo.

Antibiotic-treated mice (n = 9 mice from three independent experiments) were administered BPSCSK or BPcontrol protein precipitate. Three hours later, VRE was orally gavaged, followed by oral administrations of BPSCSK or BPcontrol protein precipitate every 3 h for 12 h and VRE colonization was monitored by quantifying VRE in faecal samples. VRE (ATCC 700221) was used. *P = 0.0232, two-tailed Mann–Whitney U-test. Data are mean ± s.d.

Extended Data Fig. 8 The BPSCSK lantibiotic has a narrower spectrum of activity against Gram-positive commensal strains.

a, VRE was inoculated in media conditioned with BPSCSK, L. lactis or BPcontrol culture protein precipitate (n = 4 biologically independent samples from four independent experiments) and growth was monitored 24 h after inoculation. b, c, Culture broth was conditioned with proteins precipitated from BPSCSK, BPcontrol or commercial nisin-A and serially diluted. The MIC value was determined for common nosocomial pathogens (b) or 158 strains from a commensal biobank (n = 2 biologically independent samples from two independent experiments) (c) by calculating the highest dilution factor that inhibited growth. The resistance index is a ratio between MIC of BPcontrol-conditioned media over the MIC of BPSCSK or nisin-A-conditioned media (b). The lantibiotic sensitivity ratio was calculated as the MIC of nisin-A to the MIC of the BPSCSK lantibiotic for each strain (c). *P < 0.05, ****P < 0.0001, two-tailed Mann–Whitney U-test for comparisons with a negative control (a) or between two experimental conditions (b, c). Box plots are as defined in Fig. 2.

Extended Data Fig. 9 Identification of lantibiotic sequences from metagenomic sequencing of healthy human faecal samples.

a, The profile hidden Markov model used to identify the gallidermin superfamily domain, illustrated as a logo. b, Multiple sequence alignment of lantibiotic precursor sequences identified from shotgun sequencing of healthy-donor faecal samples. Detected lantibiotic sequences are the assembly of lantibiotic reads from shotgun metagenomic faecal samples. c, A total of 421 species were individually isolated from healthy human faecal samples, whole-genome sequenced, assembled, annotated and mined for lantibiotic precursor sequences to identify a strain of R. faecis encoding a homologous lantibiotic. The precursor lantibiotic sequence is compared to the sequences of BPSCSK LanA1–LanA4 lantibiotic and nisin-A by multiple alignment.

Extended Data Fig. 10 Lantibiotic sequences identified from metagenomic sequencing of hospitalized patient faecal samples.

a, Stacked heat map matrices represent a single patient. The top row illustrates abundance of the lantibiotic gene (RPKM). The bottom row illustrates relative abundance of E. faecium (percentage of 16S). Columns represent the sample collection day relative to transplant.

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