Abstract Non-nutritive sweeteners (NNSs) are widely used in various food products and soft drinks. There is growing evidence that NNSs contribute to metabolic dysfunction and can affect body weight, glucose tolerance, appetite, and taste sensitivity. Several NNSs have also been shown to have major impacts on bacterial growth both in vitro and in vivo. Here we studied the effects of various NNSs on the growth of the intestinal bacterium, E. coli, as well as the gut bacterial phyla Bacteroidetes and Firmicutes, the balance between which is associated with gut health. We found that the synthetic sweeteners acesulfame potassium, saccharin and sucralose all exerted strong bacteriostatic effects. We found that rebaudioside A, the active ingredient in the natural NNS stevia, also had similar bacteriostatic properties, and the bacteriostatic effects of NNSs varied among different Escherichia coli strains. In mice fed a chow diet, sucralose increased Firmicutes, and we observed a synergistic effect on Firmicutes when sucralose was provided in the context of a high-fat diet. In summary, our data show that NNSs have direct bacteriostatic effects and can change the intestinal microbiota in vivo.

Citation: Wang Q-P, Browman D, Herzog H, Neely GG (2018) Non-nutritive sweeteners possess a bacteriostatic effect and alter gut microbiota in mice. PLoS ONE 13(7): e0199080. https://doi.org/10.1371/journal.pone.0199080 Editor: Marie-Joelle Virolle, Universite Paris-Sud, FRANCE Received: February 7, 2018; Accepted: May 31, 2018; Published: July 5, 2018 Copyright: © 2018 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The original sequence data is available at SRA accession: SRP148650 (https://www.ncbi.nlm.nih.gov/sra/SRP148650). The processed data is available in S1 Table. Funding: This work was supported in part through NHMRC project grants APP1026310, APP1029672, APP1028887, APP1046090, APP1042416, APP1086851. G.G.N. is supported by an NHMRC career development fellowship II CDF1111940. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that they have no competing interests.

Introduction Sweet-tasting food is preferred by most people and preloading experiments show that sweet taste, whether delivered by sugar or artificial sweeteners, enhances human appetite [1]. Because of the concern that high sugar intake can increase the risk of developing obesity, pre-diabetes, type 2 diabetes, and cardiovascular disease, non-nutritive sweeteners (NNSs) are increasingly used to replace sugars. NNSs are marketed as dieting tools, and consumers may intuitively choose NNSs over sugar to maintain or lose weight. There is emerging evidence indicating that NNSs have unanticipated effects on human health. NNS consumption has been associated with increased weight gain in rodents [2–6] and humans [6, 7], and glucose intolerance in rodents and humans [6, 8, 9], as well as increased appetite and altered taste sensitivity in Drosophila [10, 11]. Commercially available NNSs include sucralose, acesulfame potassium (Ace K), saccharin, aspartame and stevia. Sucralose, one of the most commonly consumed synthetic NNSs, exists in over 4500 products and accounts for 62% of the $1.146 billion global artificial sweetener market. More recently, naturally occurring NNSs like stevia has been introduced into the market [12]. Steviol glycosides are extracted from the plant stevia Rebaudiana and are perceived as 200–300 times sweeter than sucrose [13, 14]. Commercial steviol glycoside mixtures contain two main active components: stevioside (10–70%) and rebaudioside A (20–70%). Both synthetic and natural NNSs have been shown to exert bacteriostatic effects on a variety of bacteria species. Ace K, cyclamate and saccharin inhibit the anaerobic fermentation of glucose by rat gut flora [15]. Sucralose, saccharin and aspartame inhibit the growth of two common periodontal pathogenic bacterial species [16]. Sucralose also inhibits the growth of the oral bacterium Streptococcus spp. [17, 18] and various environmental microbes [19, 20]. Stevia extract exerts bactericidal effects on E. coli O157:H7, but not Bifidobacterium and Lactobacillus [21], whereas stevia glycosides inhibit the growth of Lactobacillus reuteri in a strain-specific manner [22]. Importantly, many NNSs are not efficiently absorbed through the intestine and may build up in the lumen of the gut. The gut microbiota consists of hundreds of bacterial species and is involved in multiple physiological functions such as carbohydrate and amino acid metabolism [23]. Dysfunction of gut microbiota is associated with obesity and insulin resistance [24]. For example, high-fat diets have been shown to cause an increase in phylum Firmicutes and a decrease in Bacteroidetes, and these changes are sufficient to promote metabolic dysfunction [25, 26]. There is growing evidence indicating that NNSs also have an important impact on gut microbiota in rodents. Saccharin (7.5%, w/v) increases gut aerobic bacteria and decreases anaerobic bacteria in rats [27], and sucralose (1.1–11 mg/kg/d) alters gut microflora in Sprague-Dawley rats [28]. Aspartame (5–7 mg/kg/d) induces significant alterations in intestinal bacteria composition with increased abundance of gut Enterobacteriaceae and Clostridium letptum in Sprague-Dawley rats [29]. Furthermore, the NNSs, saccharin (0.5%, w/v), aspartame (0.4%, w/v), and sucralose (0.5%, w/v) cause dysbiosis of gut bacteria in C57BL/6 mice [8]. Sucralose (15 mg/kg/d) but not Ace K (15 mg/kg/d) affects the amount of gut Clostridium cluster XIVa in C57BL/6 mice [30]. Ace K (37.5 mg/kg/d) alters gut microbiota composition in CD-1 mice [31]. Importantly, all studies on NNS-microbiota interactions so far have been carried out in adult animals, while the effect of NNSs on gut microbiota in young animals remains unexplored. In order to investigate the effects of NNSs on the gut microbiome at the organismal level, we performed phylogenetic analysis using next generation sequencing on the faeces of adolescent mice fed with NNSs. Furthermore, to dissect the effects of NNSs on commensal bacterial species, we use an in vitro E. coli bacterial growth assay. Our study demonstrates that NNSs possess a bacteriostatic effect and alter gut microbiota in mice.

Materials and methods Bacterial strains Escherichia coli strain HB101 and K-12 were purchased from Promega (US) and were maintained in Luria Bertani’s Broth (LB). Non-nutritive sweeteners Sucralose, saccharin, acesulfame potassium and rebaudioside A were purchased from Sigma. LB media and agar were obtained from Invitrogen. Liquid culture assay Sucralose, saccharin, acesulfame potassium and rebaudioside A or an equal molarity of sodium chloride or sucrose were dissolved in Luria’s Broth (LB). E. coli HB101 and E. coli K-12 strains were cultured to mid-log growth phase in LB and inoculated using 30 μL of culture in 3 mL of culture medium (1:100) and incubated at 37°C at 220 rpm in a shaking incubator. After 5 hours, the cultures were removed from the incubator, placed in disposable, plastic cuvettes and the optical density determined by spectrophotometry at 600 nm (OD 600 ). LB without NNSs were used as background references and were generally found to be within 0.006 OD 600 units of the water blank. LB agar plate assay For rebaudioside A experiments, 10-fold serial dilutions of rebaudioside A-treated cultures were performed in LB and a volume of 200 μL of diluted cultures were spread on LB-agar plates and incubated at 37°C. After 24 hours, colonies were counted manually on plates with moderate densities (generally the plates representing 10−6 to 10−7 of the initial culture concentrations) and colony forming units (CFUs) per millilitre of culture was determined. For solid media sucralose experiments, sucralose was dissolved in molten LB-agar and used to make sucralose plates with 1.25% (w/v) sucralose and 2.5% (w/v) sucralose. Colony area is the area in square millimetres that is occupied by each bacterial colony. This was done by calculating the pixel to area ratio in Image J. Bacterial cultures were grown to mid-log growth phase, serially diluted with 10-fold dilutions, plated on treated LB-agar plates using 200 μL of diluted culture and placed in an incubator at 37°C. Standard LB-agar plates were used as treatment controls. After 24 hours, the plates were photographed. Colonies were counted and colony area measurements were determined using Image J software (https://imagej.nih.gov/ij/). Mouse studies Experimental procedures were performed under the ethical standards approved by the Garvan Institute of Medical Research Animal Care and Use Committee. Chow diet (8% calories from fat, 21% calories from protein, 71% calories from carbohydrate, 2.6 kilocalorie (kcal/g)) and high fat diet (HFD, 23% calories from fat, 19.4% calories from protein, 48.2% calories from carbohydrate, 4.7% calories from crude fibre, 4.7% calories from acid detergent fibre, 4.78 kcal/g) were purchased from Gordon’s Specialty Feeds, Glen Forrest, WA, Australia. C57BL/6 mice were housed in pairs in a 12 h light/dark cycle. Animals at 5 weeks of age were divided into four dietary groups based on body weight; chow (12% kcal fat) + water, chow + sucralose solution, high fat diet + water (HFD, 60% kcal fat), HFD + sucralose solution for 8 weeks (n = 8 mice per group). Sucralose (Sigma) solution was made by directly adding sucralose to drinking water (2.5%, w/v). All animals had access to food and fluid ad libitum for an additional 8 weeks prior to sacrifice. The faeces were collected at Zeitgeber time 3 in the daylight cycle (week 5, 8 and 12) for gut microbiota analysis. Bacterial 16s RNA sequencing and analysis Faeces were collected at week 5, 8 and 12 in both chow and HFD fed mice. Total bacterial DNA was extracted and 16s rDNA were sequenced and mapped according to taxonomy. Mice faecal bacterial DNA were extracted by uBiome kit and 16s rDNA were amplified and sequenced by Illumina NextSeq 500. The sequences were mapped according to NCBI taxonomy. The gut microbiota abundance was expressed as percentage of the total amount. For α-diversity, a Shannon index was initially calculated in the vegan package [32] in R. The original sequence data is available at SRA accession: SRP148650. Statistical methods One-way analysis of variance (ANOVA) with Bonferroni’s test for multiple comparisons was used for in vitro sucralose study. Wilcoxon match-pairs signed rank test was used for rebaudioside A in vitro study. Two-way analysis of variance (ANOVA) with Bonferroni’s test for multiple comparisons was used for in vivo mouse study.

Discussion NNSs have been associated with metabolic dysfunction but the underlying mechanisms remain unclear. Recent studies reveal that NNSs affect metabolic traits through gut microbiota [8] or an imbalance in energy intake and taste perception [10]. In this study, we investigated the direct effect of NNSs on E. coli bacteria in vitro and on gut microbiota in vivo. Our results showed that NNSs exert strong bacteriostatic effects on bacteria in vitro, which is consistent with previous studies [15, 17–20, 27]. Moreover, we showed that in vivo, sucralose alters gut microbiota promoting an increase in Firmicutes, and the effects of sucralose synergized with HFD to further augment Firmicutes. Although there are a variety of compounds used as NNSs, all have the ability to mimic the taste of sugar in some regard. So far, all NNSs we have tested show a bacteriostatic effect in some bacterial species. However, the effect on different bacteria varies, even within species [17]. In this study, E. coli K-12 was more sensitive than E. coli HB101 to Ace K and sucralose, while E. coli HB10 was more sensitive to stevia. This difference implies that divergent mechanisms are responsible for the observed bacteriostatic effects. There is some evidence pointing to a mechanism whereby NNSs exert bacteriostatic effects through inhibition of metabolic enzymes or by altering nutrient transportation, or processes that are essential for growth [19, 27]. This difference in susceptibility may in part explain the alterations in bacterial abundance we observed in vivo. Given that there are hundreds of bacteria species living in our intestine[33], NNSs may selectively inhibit the survival of some bacterial populations thus causing a change in the balance of the overall gut microbiota. Of note, our finding that sucralose alters gut microbiota may be a general mechanism for all NNSs, many of which are not efficiently absorbed and may become concentrated in the intestine. Of note, there is some evidence that aspartame can also alter the intestinal microbiota in rats [29], however the mechanism for this effect is unclear, as aspartame is rapidly hydrolysed in the upper GI tract and would not likely directly interact with microbiota in the lower intestine. The abundance and diversity of bacterial species comprising the gut microbiota are in dynamic flux; however, the phyla Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria seem to dominate in intestine. Previous studies showed that Bacteroidetes is reduced and Firmicutes is increased in obese humans [33–35] and that sucralose can cause dysbiosis of gut bacteria in rat [28] and metabolic dysregulation in mice [8]. Our results here highlight that sucralose promotes an increase in Firmicutes, which is partially consistent with the microbiota alterations reported in similar studies with aspartame or Ace K [8, 29, 30]. Of note, in this study, dietary fibre content was different between chow and HFD and may represent a confounding variable, however HFD promotes significant weight gain, and increases Firmicutes while decrease Bacteroidetes abundance in the gut, which is consistent with previous results for HFD [25, 26]. Although HFD caused a significant increase in Clostridium and reduction in Bifidobacterium, sucralose did not have additional effects on these bacteria. The effects of NNSs on weight gain are controversial. Some studies have associated NNS consumption with weight gain in humans, while other studies did not report these effects [36–40]. In our study, sucralose did not promote weight gain either in the context of chow or HFD, and with young mice, we actually observed a decrease in body weight in response to high dose sucralose. This may be due to the high dose of sucralose used in the study, which could be considered super-physiological. Moreover, we found that sucralose promoted increased faecal output, regardless of diet, which could impede nutrient absorption and promote weight loss. Alternatively, it is possible that this study design was not long enough to see increased weight gain, as long-term weight gain and gain in waist circumference are observed in adult humans who consume NNSs daily [6, 41]. Interestingly, maternal consumption of NNSs also appears to impact weight gain in infants and children [41, 42]. Moreover, routine consumption of NNS is associated with increased risk of cardiometabolic diseases [43]. Together our data show that a variety of NNSs can have a direct effect on commensal bacteria. This study further reinforces the notion that NNSs are not biologically inert; rather, consumption of NNSs alters the relative proportion of intestinal microbial phyla through a selective bacteriostatic effect. The mechanisms for this activity and relevance to human disease warrant further investigation.

Supporting information S1 Table. Gut microbiota abundance. The raw sequence reads of gut microbiota. Group: week 0, 2 and 7 indicates weeks of sucralose consumption; Count, read count; Tax-name: taxonomy name; Tax-rank, ranking of the taxonomic classification, Percentage: relative abundance normalized to all counts in each sample. https://doi.org/10.1371/journal.pone.0199080.s001 (XLSX)

Acknowledgments This work was supported in part through NHMRC project grants APP1026310, APP1029672, APP1028887, APP1046090, APP1042416, APP1086851. G.G.N. is supported by an NHMRC career development fellowship II CDF1111940.