Abstract
The aim of this work was to test the hypothesis that antimicrobial food additives may alter the composition of human gut microbiota by selectively suppressing the growth of susceptible gut microbes. To explore the influence of antimicrobial food additives on the composition of the human gut microbiota, we examined the susceptibility of both aerobic and anaerobic gut bacteria to sodium benzoate, sodium nitrite, and potassium sorbate, and their combinations, using a broth microdilution method. The tested bacteria exhibited a wide range of susceptibilities to food additives. For example, the most susceptible strain, Bacteroides coprocola, was almost 580 times more susceptible to sodium nitrite than the most resistant strain, Enterococcus faecalis. However, most importantly, we found that gut microbes with known anti-inflammatory properties, such as Clostridium tyrobutyricum or Lactobacillus paracasei, were significantly more susceptible to additives than microbes with known proinflammatory or colitogenic properties, such as Bacteroides thetaiotaomicron or Enterococcus faecalis. Our data show that some human gut microbes are highly susceptible to antimicrobial food additives. We speculate that permanent exposure of human gut microbiota to even low levels of additives may modify the composition and function of gut microbiota and thus influence the host’s immune system. Whether the effect of additive-modified gut microbiota on the human immune system could explain, at least in part, the increasing incidence of allergies and autoimmune diseases remains to be shown.
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Acknowledgments
We would like to thank Radka Stribrna and Jarmila Jarkovska for their invaluable technical support and James Rizzo for editing and proofreading the manuscript.
Funding
This study was supported by the Charles University Grant Agency (No. 906613), the Czech Science Foundation (15-09518S, 15-07268S, 17-07332S, and 17-31248A), and Institutional Research Concept (RVO: 61388971). This work was also supported by Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic, project “PRVOUK” P37/10.
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Table S1
The statistical evaluation of interstrain differences in susceptibility to additives. For each single additive and its combination, the different pairs of IC50 means, averaged from three independent experiments, were compared using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. The table reports p-values adjusted to account for multiple comparisons and asterisks summarizing statistical significance (p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), p ≥ 0.05 (ns)). (XLSX 14 kb)
Table S2
The statistical evaluation of the antibacterial potency of additives. The potency of additives was analyzed by comparing various IC50 means, averaged from all eight bacterial strains for each additive, against the IC50 mean of the most potent additive, sodium nitrite. The statistical significance was evaluated using two-way ANOVA followed by Dunnett’s multiple comparisons test. The table reports p-values adjusted to account for multiple comparisons and asterisks summarizing statistical significance (p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), p ≥ 0.05 (ns)). (XLSX 14 kb)
Fig. S1
The dose-effect linear plots show the growth inhibition effects of each additive alone and its combinations. The growth inhibition effect is shown as a fractional response of bacteria to additives. The fraction affected (Fa) was calculated by the following formula: Fa = 1 - (% growth/100). The Fa ranges from 0 to 1, with the Fa value of 0 meaning no growth inhibition and the value of 1 representing 100% growth inhibition. Symbols represent the mean value ± SD from at least three independent experiments. (PNG 220 kb)
Fig. S2
Polygonograms (at 50% growth inhibition (Fa = 0.5)) for sodium benzoate, sodium nitrite, and potassium sorbate. The solid green line represents synergism (CI < 0.9), the broken thin black line represents additive effects (CI = 0.9–1.1), and the broken red line represents antagonism (CI > 1.1). The specific CI values of all binary AMFA combinations are shown next to the lines. The thickness of the line represents the strength of synergism or antagonism. The CI values were calculated using CompuSyn software and graphs were drawn using Affinity Design software BEN, sodium benzoate; NIT, sodium nitrite; SOR, potassium sorbate; CI, combination index; Fa, fraction affected (PNG 201 kb)
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Hrncirova, L., Hudcovic, T., Sukova, E. et al. Human gut microbes are susceptible to antimicrobial food additives in vitro. Folia Microbiol 64, 497–508 (2019). https://doi.org/10.1007/s12223-018-00674-z
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DOI: https://doi.org/10.1007/s12223-018-00674-z
Keywords
- Gut microbiota
- Autoimmune diseases
- Dysbiosis
- Mucosal immunology
- Food additives
- Chou-Talalay method