Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are thought to reduce intestinal colonization by pathogens, and thus to reduce susceptibility to infection. However, the mechanisms that underlie these effects remain poorly understood. Here we report that the consumption of probiotic Bacillus bacteria comprehensively abolished colonization by the dangerous pathogen Staphylococcus aureus in a rural Thai population. We show that a widespread class of Bacillus lipopeptides, the fengycins, eliminates S. aureus by inhibiting S. aureus quorum sensing—a process through which bacteria respond to their population density by altering gene regulation. Our study presents a detailed molecular mechanism that underlines the importance of probiotic nutrition in reducing infectious disease. We also provide evidence that supports the biological significance of probiotic bacterial interference in humans, and show that such interference can be achieved by blocking a pathogen’s signalling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.
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We thank R. Kolter, Harvard Medical School, for providing the B. subtilis srfA mutant; D. Dubnau, Rutgers University, for the SPP1 phage; S. Holtfreter, University of Greifswald, and W. P. Zeng, Texas Tech University Health Sciences Center, for providing strain JSNZ/ST88; B. Krismer, University of Tübingen, for plasmid pKX15; F. DeLeo, National Institute of Allergy and Infectious Diseases (NIAID), for anti-Panton–Valentine-leucocidin; and N. A. Amissah for technical assistance. This work was supported by the Intramural Research Program of the NIAID, US National Institutes of Health (NIH) (project ZIA AI000904-16, to M.O.); and the Thailand Research Fund through the Royal Golden Jubilee PhD Program (grant number PHD/0072/2557, to P.P. and P.K.). P.K. was also supported by the Faculty of Medicine, Siriraj Hospital, Mahidol University, grant number (IO) R015833012; P.P. by the Graduate Partnership Program of the NIH; and S.W.D. by the Postdoctoral Research Associate Program of the National Institute of General Medical Sciences (1FI2GM11999101).
Nature thanks A. Baumler, M. Parsek and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
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Extended data figures and tables
The microbiota of n = 20 randomly selected S. aureus carriers (red) and n = 20 non-carriers (blue) were analysed in faecal samples. a–c, Rarefaction (species-richness) curves based on 16S rRNA gene sequences. Data are mean ± s.d. a, Shannon index. b, Observed species. c, Chao1 index. d, Comparison of relative taxa abundance between S. aureus carriers (red) and non-carriers (blue). e, f, Beta diversity, represented by a principal coordinate analysis plot based on unweighted UniFrac (e) and weighted UniFrac (f) metrics for samples from S. aureus carriers (red) and non-carriers (blue).
Data from strains USA300 LAC and ST88 JSNZ. The experimental set-up is the same as in Fig. 2: mice received by oral gavage either 100 μl containing 108 CFU ml−1 of wild-type S. aureus strain USA300 LAC or ST88 JSNZ plus another 100 μl of 108 CFU ml−1 of the corresponding isogenic agr mutant (n = 5 per group; competitive experiment shown in a, b); or 200 μl containing 108 CFU ml−1 wild-type, isogenic agr mutant or Agr (RNAIII)-complemented agr mutant (n = 5 per group, non-competitive experiment shown in c). CFU in the faeces were determined two, four and six days after infection. At the end of the experiment (day seven), CFU in the small and large intestines were determined. a, b, Competitive experiment. Total obtained CFU are shown as dot plots; also shown are mean ± s.d. Bars show the percentage of wild-type among total determined CFU, of which 100 were analysed for tetracycline resistance that is present only in the agr mutant. No agr mutants were detected in any experiment; thus, all bars show 100%. Given that 100 isolates were tested, the competitive index wild-type/agr mutant in all cases is ≥100. c, Non-competitive experiment with genetically complemented strains. Wild-type and isogenic agr mutant strains all harboured the pKXΔ16 control plasmid; Agr-complemented strains harboured pKXΔRNAIII and thus constitutively expressed RNAIII, which is the intracellular effector of Agr. During the experiment, mice received 200 μg ml−1 kanamycin in their drinking water to maintain plasmids. Statistical analysis was performed using Poisson regression versus values obtained with the agr mutant strains. *P < 0.0001. Data are mean ± s.d. Note that no bacteria were found in the faeces or intestines of any mouse receiving S. aureus Δagr with vector control. The corresponding zero values are plotted on the x axis of the logarithmic scale.
a, Influence of heat and proteases on Agr inhibition. B. subtilis culture filtrate was subjected to heat (95 °C for 20 min) or digestion with proteinase K (50 μg ml−1, 37 °C, 1 h) and the effect on inhibition of Agr activity was measured using the luminescence assay with the USA300 P3–luxABCDE reporter strain (see Fig. 3a). RLU, relative light units. The experiment was performed with n = 2 independent biological samples. Lines connect the means. (The observed additional suppression of Agr activity in the proteinase-K-treated sample at 6 h, compared with the B. subtilis culture filtrate sample, is expected owing to proteolytic inactivation of intrinsic AIP.) b, Preparative RP chromatography of B. subtilis culture filtrate to determine the Agr-inhibiting substance. The peaks labelled 2 and 3 showed substantial Agr-inhibiting activities in the Agr-activity assay and were identified as fengycins using subsequent RP-HPLC/ESI-MS and MS/MS analysis (see c, d). The peaks labelled 1 and 4–6 also contained fengycin species (see e). AU, arbitrary units. The applied gradient (% buffer B) is shown in green. c, Fractions corresponding to Agr-inhibitory peaks 2 and 3 from the preparative RP run (b) were subjected to RP-HPLC/ESI-MS. Top, total ion chromatograms (TICs) of the RP-HPLC/ESI-MS runs; bottom, ESI mass spectrogram of the major peaks. d, MS/MS analysis of the peak 2 and 3 fractions. Peaks that are characteristic of a given fengycin subtype (A or B in this case) are marked in colour. ‘Parent’ refers to the relevant numbered peak in the spectrograms above. e, Analysis of further fengycin-containing fractions. Peaks 1, 4, 5 and 6 from the preparative RP run (b) were also found to contain fengycin species as determined by subsequent RP-HPLC/ESI-MS analysis. Shown are the mass spectrograms of the major peaks of those runs and the tentative characterization for fengycin type. The preparative and analytical chromatography and RP-HPLC/ESI-MS analyses (as shown in b, d) were repeated multiple (more than ten) times for fengycin purification, with similar results. MS/MS analyses were not repeated. f, Analysis of fengycin and surfactin lipopeptide expression by the B. subtilis wild-type strain and its isogenic ΔfenA mutant.
a, RP-HPLC run. b, Agr inhibition at different concentrations in the luminescence assay. RLU, relative light units. Statistical analysis was by two-way ANOVA with Tukey’s post-test. Comparisons shown are those versus DMSO control. c, Agr inhibition as measured by inhibition of expression of RNAIII by qRT–PCR. *P < 0.0001 (one-way ANOVA with Tukey’s post-test; comparisons shown are those versus 0 μM value). The experiments in b, c were performed with n = 3 independent biological samples. Data are mean ± s.d.
Extended Data Fig. 5 Inhibition of S. aureus colonization by dietary fengycin-producing Bacillus spores in a mouse model.
a, Concentration of AIP-I during S. aureus growth. Strain LAC (USA300) was grown in TSB, and AIP-I concentrations were measured by RP-HPLC/ESI-MS. Calibration was performed using synthetic AIP-I. The detection limit of this assay is around 0.3 μM. The experiment was performed with n = 3 independent biological samples. Data are mean ± s.d. b, B. subtilis colonization kinetics in the mouse intestinal colonization experiment. Mice (n = 5) received 200 μl of a 108 CFU ml−1 suspension of wild-type B. subtilis or ΔfenA mutant spores by oral gavage; CFU in the faeces were analysed up to five days afterwards. Data are mean ± s.d. c–f, Inhibition mouse model with strains USA300 LAC and ST88 JSNZ. The experimental set-up was as shown in Fig. 5a. In brief, n = 4 or 5 mice per group received 200 μl of 108 CFU ml−1 S. aureus strains USA300 LAC or ST88 JSNZ by oral gavage. On the next day and every following second day, the mice received 200 μl of 108 CFU ml−1 spores of wild-type B. subtilis or its isogenic fenA mutant, also by oral gavage. CFU in the faeces were determined two, four and six days after infection. At the end of the experiment (day seven), CFU in the small and large intestines were determined. The experiment was performed with (c, d) or without (e, f) antibiotic pretreatment. Statistical analysis was performed using Poisson regression versus values obtained with wild-type B. subtilis spore samples. *P < 0.0001. Data are mean ± s.d. Note that no S. aureus were found in the faeces or intestines of any mouse challenged with any S. aureus strain that also received Bacillus wild-type spores. The corresponding zero values are plotted on the x axis of the logarithmic scale.
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Piewngam, P., Zheng, Y., Nguyen, T.H. et al. Pathogen elimination by probiotic Bacillus via signalling interference. Nature 562, 532–537 (2018). https://doi.org/10.1038/s41586-018-0616-y
- Probiotic Bacillus
- National Institute Of Allergy And Infectious Diseases (NIAID)
- Methicillin-resistant S. Aureus (MRSA)
- Fengycin Production
- Quorum Quenching
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