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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
AARDA (2011) The cost burden of autoimmune disease: The latest front in the war on healthcare spending. http://www.diabetesed.net/page/_files/autoimmune-diseases.pdf. Accessed 16 Jan 2019
Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y, Taniguchi T, Takeda K, Hori S, Ivanov II, Umesaki Y, Itoh K, Honda K (2011) Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331:337–341. https://doi.org/10.1126/science.1198469
Báez-Saldaña A, Díaz G, Espinoza B, Ortega E (1998) Biotin deficiency induces changes in subpopulations of spleen lymphocytes in mice. Am J Clin Nutr 67:431–437
Balish E, Warner T (2002) Enterococcus faecalis induces inflammatory bowel disease in interleukin-10 knockout mice. Am J Pathol 160:2253–2257. https://doi.org/10.1016/S0002-9440(10)61172-8
Bateman B, Warner JO, Hutchinson E, Dean T, Rowlandson P, Gant C, Grundy J, Fitzgerald C, Stevenson J (2004) The effects of a double blind, placebo controlled, artificial food colourings and benzoate preservative challenge on hyperactivity in a general population sample of preschool children. Arch Dis Child 89:506–511. https://doi.org/10.1136/adc.2003.031435
Bemrah N, Leblanc J-C, Volatier JL (2008) Assessment of dietary exposure in the French population to 13 selected food colours, preservatives, antioxidants, stabilizers, emulsifiers and sweeteners. Food Addit Contam Part B 1:2–14. https://doi.org/10.1080/19393210802236943
Berger-Bächi B (2002) Resistance mechanisms of gram-positive bacteria. Int J Med Microbiol 292:27–35. https://doi.org/10.1078/1438-4221-00185
Bloom SM, Bijanki VN, Nava GM, Sun L, Malvin NP, Donermeyer DL, Dunne WM Jr, Allen PM, Stappenbeck TS (2011) Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease. Cell Host Microbe 9:390–403. https://doi.org/10.1016/j.chom.2011.04.009
Boesten R, Schuren F, Ben Amor K (2011) Bifidobacterium population analysis in the infant gut by direct mapping of genomic hybridization patterns: potential for monitoring temporal development and effects of dietary regimens. Microb Biotechnol 4:417–427. https://doi.org/10.1111/j.1751-7915.2010.00216.x
Chassaing B, Koren O, Goodrich JK, Poole AC, Srinivasan S, Ley RE, Gewirtz AT (2015) Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature 519:92–96. https://doi.org/10.1038/nature14232
Chou T-CC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58:621–681. https://doi.org/10.1124/pr.58.3.10
Chou T-C (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70:440–446. https://doi.org/10.1158/0008-5472.CAN-09-1947
Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzym Regul 22:27–55
Clemente JC, Ursell LK, Parfrey LW, Knight R (2012) The impact of the gut microbiota on human health: an integrative view. Cell 148:1258–1270. https://doi.org/10.1016/j.cell.2012.01.035
Cooper GS, Miller FW, Pandey JP (1999) The role of genetic factors in autoimmune disease: implications for environmental research. Environ Health Perspect 107(Suppl 5):693–700
Cordain L, Miller JB, Eaton SB et al (2000) Plant-animal subsistence ratios and macronutrient energy estimations in worldwide hunter-gatherer diets. Am J Clin Nutr 71:682–692. https://doi.org/10.1038/sj.ejcn.1601353
Davidson PM, Sofos JN, Branen AL (2005) Antimicrobials in food, 3rd edn. CRC Press, New York
Dicksved J, Halfvarson J, Rosenquist M, Järnerot G, Tysk C, Apajalahti J, Engstrand L, Jansson JK (2008) Molecular analysis of the gut microbiota of identical twins with Crohn’s disease. ISME J 2:716–727. https://doi.org/10.1038/ismej.2008.37
Ekmekciu I, Klitzing v E, Fiebiger U et al (2017) The probiotic compound VSL#3 modulates mucosal, peripheral, and systemic immunity following murine broad-spectrum antibiotic treatment. Front Cell Infect Microbiol 7:167. https://doi.org/10.3389/fcimb.2017.00167
Engelkirk PG, Duben-Engelkirk JL (2008) Laboratory diagnosis of infectious diseases. Lippincott Williams and Wilkins, Baltimore
Frank DN, St Amand AL, Feldman RA et al (2007) Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci U S A 104:13780–13785. https://doi.org/10.1073/pnas.0706625104
Furusawa Y, Obata Y, Hase K (2015) Commensal microbiota regulates T cell fate decision in the gut. Semin Immunopathol 37:17–25. https://doi.org/10.1007/s00281-014-0455-3
Gutierrez J, Barry-Ryan C, Bourke P (2009) Antimicrobial activity of plant essential oils using food model media: efficacy, synergistic potential and interactions with food components. Food Microbiol 26:142–150. https://doi.org/10.1016/j.fm.2008.10.008
Hickey CA, Kuhn KA, Donermeyer DL, Porter NT, Jin C, Cameron EA, Jung H, Kaiko GE, Wegorzewska M, Malvin NP, Glowacki RWP, Hansson GC, Allen PM, Martens EC, Stappenbeck TS (2015) Colitogenic Bacteroides thetaiotaomicron antigens access host immune cells in a sulfatase-dependent manner via outer membrane vesicles. Cell Host Microbe 17:672–680. https://doi.org/10.1016/j.chom.2015.04.002
Hrncir T, Stepankova R, Kozakova H, Hudcovic T, Tlaskalova-Hogenova H (2008) Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice. BMC Immunol 9:65. https://doi.org/10.1186/1471-2172-9-65
Hudcovic T, Kolinska J, Klepetar J, Stepankova R, Rezanka T, Srutkova D, Schwarzer M, Erban V, du Z, Wells JM, Hrncir T, Tlaskalova-Hogenova H, Kozakova H (2012) Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice. Clin Exp Immunol 167:356–365. https://doi.org/10.1111/j.1365-2249.2011.04498.x
Husain A, Sawaya W, Al-Omair A et al (2006) Estimates of dietary exposure of children to artificial food colours in Kuwait. Food Addit Contam 23:245–251. https://doi.org/10.1080/02652030500429125
Husain M, Bourret TJ, McCollister BD et al (2008) Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration. J Biol Chem 283:7682–7689. https://doi.org/10.1074/jbc.M708845200
Irwin SV, Fisher P, Graham E, Malek A, Robidoux A (2017) Sulfites inhibit the growth of four species of beneficial gut bacteria at concentrations regarded as safe for food. PLoS One 12:e0186629. https://doi.org/10.1371/journal.pone.0186629
Kim SC, Tonkonogy SL, Albright CA, Tsang J, Balish EJ, Braun J, Huycke MM, Sartor RB (2005) Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria. Gastroenterology 128:891–906. https://doi.org/10.1053/j.gastro.2005.02.009
Lerner A, Jeremias P, Matthias T (2015) The world incidence and prevalence of autoimmune diseases is increasing. Int J Cel Dis 3:151–155. https://doi.org/10.12691/ijcd-3-4-8
Lucera A, Costa C, Conte A, Del Nobile MA (2012) Food applications of natural antimicrobial compounds. Front Microbiol 3:287. https://doi.org/10.3389/fmicb.2012.00287
Malhotra B, Keshwani A, Kharkwal H (2015) Antimicrobial food packaging: potential and pitfalls. Front Microbiol 6. https://doi.org/10.3389/fmicb.2015.00611
McCann D, Barrett A, Cooper A, Crumpler D, Dalen L, Grimshaw K, Kitchin E, Lok K, Porteous L, Prince E, Sonuga-Barke E, Warner JO, Stevenson J (2007) Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: a randomised, double-blinded, placebo-controlled trial. Lancet 370:1560–1567. https://doi.org/10.1016/S0140-6736(07)61306-3
Mischek D, Krapfenbauer-Cermak C (2011) Exposure assessment of food preservatives (sulphites, benzoic and sorbic acid) in Austria. Food Addit Contam Part A 26:1–12. https://doi.org/10.1080/19440049.2011.643415
Miyake S, Kim S, Suda W, Oshima K, Nakamura M, Matsuoka T, Chihara N, Tomita A, Sato W, Kim SW, Morita H, Hattori M, Yamamura T (2015) Dysbiosis in the gut microbiota of patients with multiple sclerosis, with a striking depletion of species belonging to Clostridia XIVA and IV clusters. PLoS One 10:e0137429. https://doi.org/10.1371/journal.pone.0137429
Mudie DM, Murray K, Hoad CL, Pritchard SE, Garnett MC, Amidon GL, Gowland PA, Spiller RC, Amidon GE, Marciani L (2014) Quantification of gastrointestinal liquid volumes and distribution following a 240 mL dose of water in the fasted state. Mol Pharm 11:3039–3047. https://doi.org/10.1021/mp500210c
Nylund L, Nermes M, Isolauri E, Salminen S, de Vos WM, Satokari R (2015) Severity of atopic disease inversely correlates with intestinal microbiota diversity and butyrate-producing bacteria. Allergy 70:241–244. https://doi.org/10.1111/all.12549
Park J-S, Joe I, Rhee PD, Jeong CS, Jeong G (2017) A lactic acid bacterium isolated from kimchi ameliorates intestinal inflammation in DSS-induced colitis. J Microbiol 55:304–310. https://doi.org/10.1007/s12275-017-6447-y
Prinz RJ, Roberts WA, Hantman E (1980) Dietary correlates of hyperactive behavior in children. J Consult Clin Psychol 48:760–769. https://doi.org/10.1037//0022-006x.48.6.760
Pruteanu M, Hyland NP, Clarke DJ, Kiely B, Shanahan F (2011) Degradation of the extracellular matrix components by bacterial-derived metalloproteases. Inflamm Bowel Dis 17:1189–1200. https://doi.org/10.1002/ibd.21475
Reddy D, Lancaster JR, Cornforth DP (1983) Nitrite inhibition of Clostridium botulinum: electron spin resonance detection of iron-nitric oxide complexes. Science 221:769–770. https://doi.org/10.1126/science.6308761
Roca-Saavedra P, Mendez-Vilabrille V, Miranda JM, Nebot C, Cardelle-Cobas A, Franco CM, Cepeda A (2018) Food additives, contaminants and other minor components: effects on human gut microbiota-a review. J Physiol Biochem 74:69–83. https://doi.org/10.1007/s13105-017-0564-2
Rosinger A, Herrick K, Gahche J, Park S (2017) Sugar-sweetened beverage consumption among U.S. youth, 2011–2014 NCHS data brief 1–8 https://www.cdc.gov/nchs/data/databriefs/db271.pdf. Accessed 10 June 2018
Rowe JJ, Yarbrough JM, Rake JB, Eagon RG (1979) Nitrite inhibition of aerobic bacteria. Curr Microbiol 2:51–54. https://doi.org/10.1007/bf02601735
Russell AD (1997) Plasmids and bacterial resistance to biocides. J Appl Microbiol 83:155–165
Scher JU, Ubeda C, Artacho A, Attur M, Isaac S, Reddy SM, Marmon S, Neimann A, Brusca S, Patel T, Manasson J, Pamer EG, Littman DR, Abramson SB (2015) Decreased bacterial diversity characterizes the altered gut microbiota in patients with psoriatic arthritis, resembling dysbiosis in inflammatory. Arthritis Rheum 67:128–139. https://doi.org/10.1002/art.38892
Schillde v M-A, Hörmannsperger G, Weiher M et al (2012) Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines. Cell Host Microbe 11:387–396. https://doi.org/10.1016/j.chom.2012.02.006
Shoenfeld Y, Cervera R, Gershwin ME (2010) Diagnostic criteria in autoimmune diseases. Humana Press, Totowa, NJ
Silbergeld EK, Graham J, Price LB (2008) Industrial food animal production, antimicrobial resistance, and human health. 29:151–169. https://doi.org/10.1146/annurev.publhealth.29.020907.090904
Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, Benyamin FW, Man Lei Y, Jabri B, Alegre ML, Chang EB, Gajewski TF (2015) Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy. Science 350:1084–1089. https://doi.org/10.1126/science.aac4255
Steck N, Hoffmann M, Sava IG, Kim SC, Hahne H, Tonkonogy SL, Mair K, Krueger D, Pruteanu M, Shanahan F, Vogelmann R, Schemann M, Kuster B, Sartor RB, Haller D (2011) Enterococcus faecalis metalloprotease compromises epithelial barrier and contributes to intestinal inflammation. Gastroenterology 141:959–971. https://doi.org/10.1053/j.gastro.2011.05.035
Sugahara H, Odamaki T, Fukuda S, Kato T, Xiao JZ, Abe F, Kikuchi J, Ohno H (2015) Probiotic Bifidobacterium longum alters gut luminal metabolism through modification of the gut microbial community. Sci Rep 5:13548. https://doi.org/10.1038/srep13548
Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G (2001) Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob Agents Chemother 45:1126–1136. https://doi.org/10.1128/AAC.45.4.1126-1136.2001
Technavio (2017) A New Report on the Global Food Additives Market from 2017–2021. https://www.businesswire.com/news/home/20171011005886/en/Food-Additives-Market%2D%2D-Drivers-Forecasts-Technavio. Accessed 10 June 2018
Thapa D, Louis P, Losa R et al (2015) Essential oils have different effects on human pathogenic and commensal bacteria in mixed faecal fermentations compared with pure cultures. Microbiology (Reading, Engl) 161:441–449. https://doi.org/10.1099/mic.0.000009
Tlaskalova-Hogenova H, Stepankova R, Kozakova H et al (2011) The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases. Cell Mol Immunol 8:110–120. https://doi.org/10.1038/cmi.2010.67
Tongnuanchan P, Benjakul S (2014) Essential oils: extraction, bioactivities, and their uses for food preservation. J Food Sci 79:R1231–R1249. https://doi.org/10.1111/1750-3841.12492
Vally H, Thompson PJ (2001) Role of sulfite additives in wine induced asthma: single dose and cumulative dose studies. Thorax 56:763–769. https://doi.org/10.1136/thorax.56.10.763
Versini M, Jeandel P-Y, Bashi T, Bizzaro G, Blank M, Shoenfeld Y (2015) Unraveling the hygiene hypothesis of helminthes and autoimmunity: origins, pathophysiology, and clinical applications. BMC Med 13:2226. https://doi.org/10.1186/s12916-015-0306-7
Vétizou M, Pitt JM, Daillère R et al (2015) Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 350:1079–1084. https://doi.org/10.1126/science.aad1329
Willis AT (2014) Anaerobic bacteriology: clinical and laboratory practice. Butterworths, London
Wu H-J, Ivanov II, Darce J, Hattori K, Shima T, Umesaki Y, Littman DR, Benoist C, Mathis D (2010) Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 32:815–827. https://doi.org/10.1016/j.immuni.2010.06.001
Yuan J, Zhu L, Liu X, Li T, Zhang Y, Ying T, Wang B, Wang J, Dong H, Feng E, Li Q, Wang J, Wang H, Wei K, Zhang X, Huang C, Huang P, Huang L, Zeng M, Wang H (2006) A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705. Mol Cell Proteomics 5:1105–1118. https://doi.org/10.1074/mcp.M500410-MCP200
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
The study was reviewed and approved by the Institutional Review Committee (protocol no. PP77-2014), and all subjects participating in the study signed and dated Informed Consent and Fecal Donor Agreement forms.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12223-018-00674-z