Artificial Sweeteners Induce Bacterial Drug Resistance and Modulate Gene Expression

Xin Jin,1 Juan Xu,2 Yu Cao,1 Zhen Dai,1 Ruirong Wang,1 Xiaoming Ge,1 Rui Cai,3 Ziyan Hu,1 Xiaojie Sun,1 Jing Chen,4 Ling Zhang1 1Nanjing Institute for Food and Drug Control, Jiangsu, Nanjing, People’s Republic of China; 2Nanjing Institute of Technology, Jiangsu, Nanjing, People’s Republic of China; 3Jiangsu Province Hospital of Chinese Medicine, Jiangsu, Nanjing, People’s Republic of China; 4China Pharmaceutical University, Jiangsu, Nanjing, People’s Republic of ChinaCorrespondence: Xin Jin; Ling Zhang, Nanjing Institute for Food and Drug Control, No. 199, Wenfang Road, Jianye District, Nanjing, Jiangsu, People’s Republic of China, Email xjin14@fudan.edu.cn; 2459437556@qq.comObjective: To explore the effects of artificial sweeteners on reduced antibiotic susceptibility and expression of antibiotic resistance and virulence-related genes in bacteria exposed to permitted daily intake levels.Methods: We first evaluated the antibacterial effects of five commonly used artificial sweeteners (saccharin, cyclamate, aspartame, acesulfame potassium, and sucralose) on Escherichia coli and Bacillus subtilis, and detected changes in reactive oxygen species (ROS) production. We investigated the expression of genes associated with resistance, oxidative stress, and virulence using transcriptome sequencing after 2 h of exposure.Results: The minimum inhibitory concentration (MIC) of artificial sweeteners for E. coli and B. subtilis were higher than the Codex Alimentarius Commission (CAC) defined daily intake limits. Except for saccharin, these sweeteners did not significantly affect the bacterial growth within 48 h. However, at these limiting concentrations, artificial sweeteners are associated with reduced antibiotic susceptibility and upregulation of resistance-related genes and enhance ROS production. Transcriptome analysis at 2 h revealed that artificial sweeteners upregulated genes associated with resistance, oxidative stress, and virulence compared to the glucose control. Additionally, we observed the effects of artificial sweeteners on iron uptake-related genes in E. coli, suggesting potential implications for bacterial ferroptosis that require further validation.Conclusions: Exposure to artificial sweeteners at CAC-permitted doses is associated with reduced antibiotic susceptibility and may affect bacterial function. Therefore, the safety of artificial sweeteners as substitutes for natural sugars requires careful consideration and further in vivo validation. This study assesses artificial sweetener safety by investigating their effects on antibiotic susceptibility and gene expression in E. coli and B. subtilis. Phenotypic assays (MIC, growth curves, ROS) demonstrated that sweetener exposure reduces antibiotic sensitivity. Transcriptome sequencing and heatmap analysis revealed significant gene expression alterations, indicating that artificial sweeteners modulate bacterial drug susceptibility and transcriptional programs.Artificial sweeteners modulate antibiotic susceptibility and gene expression in E. coli and B. subtilis.