Division of Microbiology , National Center for Toxicological Research, U.S. Food and Drug Administration , 3900 NCTR Rd , Jefferson , Arkansas 72079 , United States.
ACS Appl Mater Interfaces. 2019 Jul 24;11(29):25708-25719. doi: 10.1021/acsami.9b07635. Epub 2019 Jul 12.
The increased use of graphene in consumer products such as food contact materials requires a thorough understanding of its effects on the gastrointestinal commensal bacterial population. During the first phase of study, three representative commensal bacterial species (, , and ) were exposed to different concentrations (1, 10, and 100 μg/mL) of pristine graphene for 3, 6, and 24 h in the Bioreactor Rotary Cell Culture System (BRCCS) which allowed a continuous interaction of intestinal microbiota with the pristine graphene without precipitation of test material. The results showed that pristine graphene had dose-dependent effects on the growth of selective bacteria. To study the interaction of graphene with more diverse consortia of intestinal microbiota, fresh fecal samples from laboratory rats were used. Rat fecal slurry (3%) was maintained in an anaerobic environment and treated with different concentrations (1, 10, and 100 μg/mL) of pristine graphene for 3, 6, and 24 h. Counts of viable aerobic and anaerobic bacteria were assessed and fecal slurries were also collected for microbial population shift analysis using quantitative real-time PCR, as well as 16s rRNA sequencing. The results showed a significant two-fold increase in both aerobic and anaerobic bacterial counts (expressed as colony forming unit; CFU) during the first 3 h of exposure to all pristine graphene concentrations. However, 24 h of continuous exposure resulted in a 120% decrease in the CFU of aerobic bacteria at the highest concentration and the anaerobic bacteria CFU remained unchanged. Multivariate analysis of the q-PCR data showed that the exposure time, as well as the graphene concentrations, impacted the bacterial population abundance. Community analysis of graphene-treated fecal samples by 16S sequencing revealed significant alteration of 15 taxonomic groups, including 9 species. The increased abundance of butyrate-producing bacteria (, , and ) was correlated with an increase of the short-chain fatty acid, butyric acid after exposure to graphene. These results clearly indicate that graphene may cause adverse effects on the intestinal microbiome at the doses equal to 100 μg/mL. Further experiments using intestinal explants (nonanimal model) could reveal the mechanisms by which graphene could perturb the microbe-host intestinal mucosa homeostasis.
在食品接触材料等消费产品中越来越多地使用石墨烯，这就需要深入了解其对胃肠道共生细菌种群的影响。在研究的第一阶段，三种代表性的共生细菌（、和）分别在生物反应器旋转细胞培养系统（BRCCS）中暴露于不同浓度（1、10 和 100μg/ml）的原始石墨烯中 3、6 和 24 小时，这使得肠道微生物群与原始石墨烯连续相互作用，而不会使测试材料沉淀。结果表明，原始石墨烯对选择性细菌的生长具有剂量依赖性影响。为了研究石墨烯与更多样化的肠道微生物群落的相互作用，使用了来自实验室大鼠的新鲜粪便样本。大鼠粪便浆（3%）在厌氧环境中维持，并分别用不同浓度（1、10 和 100μg/ml）的原始石墨烯处理 3、6 和 24 小时。评估了需氧和厌氧活菌的计数，并收集粪便浆进行微生物种群转移分析，使用定量实时 PCR 以及 16s rRNA 测序。结果表明，在所有原始石墨烯浓度暴露的前 3 小时内，需氧和厌氧细菌的数量（表示为菌落形成单位；CFU）均显著增加了两倍。然而，24 小时的连续暴露导致最高浓度下需氧细菌的 CFU 减少了 120%，而厌氧细菌的 CFU 保持不变。q-PCR 数据分析的多元分析表明，暴露时间以及石墨烯浓度均影响细菌种群丰度。通过 16S 测序对石墨烯处理粪便样本的群落分析显示，15 个分类群发生了显著变化，包括 9 个种。丁酸盐产生菌（、和）的丰度增加与暴露于石墨烯后短链脂肪酸丁酸的增加有关。这些结果清楚地表明，在 100μg/ml 剂量下，石墨烯可能对肠道微生物组产生不良影响。使用 肠道外植体（非动物模型）进行的进一步实验可以揭示石墨烯干扰微生物-宿主肠道黏膜稳态的机制。
