Vancomycin exposure caused opportunistic pathogens bloom in intestinal microbiome by simulator of the human intestinal microbial ecosystem (SHIME)☆
Graphical abstract
Introduction
Antibiotics are extensively used nowadays and human would exposure to this kind of emerging organic pollutant through inhalation, ingestion of drugs, drinking water, and food, and their risk assessments have attracted increasing attention (Ashbolt et al., 2013; Ben et al., 2019; Le Page et al., 2017; Nnadozie and Odume, 2019). Both human and veterinary antibiotics were detected in the collective gut of the Chinese population through our previous research (Wang et al., 2020). Concerns about the use of antibiotics have traditionally focused on the killing of bacterial pathogens; however overuse and misuse of antibiotics may promote the spread of antibiotic-resistant bacteria (ARB) and the antibiotic resistance genes (ARGs) in human gut, which may limit the treatment efficiency and resulting in chronic and relapsing infections (Bengtsson-Palme et al., 2018; Stecher et al., 2013). And loop forever, host associated fecal with antibiotic residues, ARB, and ARGs would contaminate the environment (Fan et al., 2017; Somnark et al., 2018; Vadde et al., 2019).
Vancomycin, a glycopeptide antibiotic is mainly active against the gram-positive bacteria, many researchers have investigated influences of vancomycin on the human intestinal microbiota in vivo (Isaac et al., 2017; Reijnders et al., 2016; Vrieze et al., 2014). However, in vitro experiments are very important for clarifying direct effects of antibiotics on human intestinal microbiota without the disturbances from neurohumoral regulation, the individual differences, dietary habits, and physiological status. The simulator of the human intestinal microbial ecosystem (SHIME) model is known to be a useful tool for in vitro studies (Marzorati and Van de Wiele, 2016). To the best of our knowledge, there are very few researches that added the antibiotics into this in vitro model (El Hage et al., 2019; Ichim et al., 2018; Marzorati et al., 2017; Van den Abbeele et al., 2012). The effects of antibiotics, including amoxicillin (AMX) and other antibiotics mixture, vancomycin, and clindamycin, were limited to microbiota composition and metabolite.
Fecal microbiota transplantation (FMT) is the introduction of feces from a healthy donor into the intestinal tract of someone with disrupted microbiota, and washed microbiota transplantation is safer, more precise and more quality-controllable than the crude FMT by manual (Zhang et al., 2020). FMT has been confirmed to effectively cure recurrent Clostridioides difficile infection (CDI) in the clinical guideline, consensus and joint-experts (McDonald et al., 2018; Ng et al., 2020). Furthermore, FMT has been confirmed superior to oral vancomycin for the treatment of CDI (Khan et al., 2018; Lubbert et al., 2017), and combination of FMT with vancomycin could be a better choice to treat this kind of infection (Bulow et al., 2018; Hota et al., 2017). However, there is limited information on using fecal microbiota transplantation (FMT) for restoration of intestinal microbiome affected by antibiotics. Therefore, it is exciting for us to investigate whether FMT would restore the human intestinal dysbiosis caused by vancomycin exposure.
Since the reasonable dosage of vancomycin for adult human study is about 500–1500 mg day−1 and only half volume of adult gut can be simulated in the used SHIME model, here the direct effects of 600 mg day−1 (1000 mg L−1) of vancomycin on the composition and human disease-related pathways as well as ARGs of the human gut microbiota were studied (Hota et al., 2017; Reijnders et al., 2016). And 10 mg L−1 and 100 mg L−1 were chosen as lower doses of vancomycin to verify whether these doses of vancomycin would affect human gut microbiota, because the vancomycin agar plate concentration for vancomycin resistant bacteria isolation is between this range according to the CLSI M07-A8 2011 guidelines. In this study, the composition of human intestinal microbiota composition and human disease-related pathways were analyzed by 16S rRNA gene high-throughput sequencing, and the ARGs were quantified by high-throughput quantitative PCR (HT-qPCR). This study achieved a systematical investigation and precisely understanding of the direct effects of vancomycin modulation on the intestinal microbiota and the restoration of FMT using a SHIME model system, which may be valuable for directing future work.
Section snippets
Vancomycin treatment and sample collection
The SHIME model in this study was formed by four double-jacketed reactors designated as the stomach, small intestine, ascending colon, and descending colon, respectively (Fig. S1a). The last two reactors inoculated with a mixture of fecal microbiota collected from a healthy adult volunteer, who did not suffer from gastrointestinal diseases or take any antibiotics in the previous six months according to previous classic studies (Wang et al., 2018; Yu et al., 2016). This would avoid the
Human disease-related pathways and ARGs
The human disease-related pathways and ARGs in samples from the SHIME model were assessed at different time points, which included samples prior to antibiotic treatment (C), sequential doses (10, 100, and 1000 mg L−1) of antibiotic treatments at seven days interval designated as V10, V100, and V1000, followed by 14 days after termination of vancomycin (NR) and 14 days after FMT (FR). The hierarchy cluster heatmap showed that gene numbers of human disease-related pathways, including drug
Vancomycin exposure significantly increased the opportunistic pathogens
In this study, significantly reduction of Bacteroidetes and Firmicutes phyla and enrichment of from Proteobacteria phylum such as Pseudomonas and Klebsiella were shown in human intestinal microbiota following vancomycin administration, which was in agreement with previous in vivo studies (Isaac et al., 2017; Reijnders et al., 2016; Vrieze et al., 2014). Some studies have also reported a drop in richness and alpha diversity and significant effect on beta diversity pursued by vancomycin exposure (
Conclusions
Exposure to vancomycin significantly altered the taxonomic composition and diversity, and it subsequently increased opportunistic pathogens and human disease-related pathways. The changes were “SHIME-compartment” different with a more significant effect on the ascending colon. Moreover, bacteria that increased after vancomycin exposure were positively associated with the human disease-related pathways, while bacterial decreased were positively related to quantified ARGs. The significant shifted
Ethics approval and consent to participate
The study was approved by the Biomedical Ethics Committees of Nankai University. The participant has given written informed consent to understand the study purpose, procedures, risks, benefits, and rights.
CRediT authorship contribution statement
Lei Liu: Methodology, Formal analysis, Writing - original draft, Software. Qing Wang: Funding acquisition, Writing - review & editing. Xinyan Wu: Methodology. Hongmei Qi: Methodology. Ranjit Das: Writing - review & editing. Huai Lin: Methodology. Jingliang Shi: Methodology. Siyi Wang: Methodology. Jing Yang: Methodology. Yingang Xue: Funding acquisition. Daqing Mao: Funding acquisition, Writing - review & editing, Supervision. Yi Luo: Funding acquisition, Writing - review & editing, Supervision.
Declaration of competing interest
The authors declare no potential conflicts of interests.
Acknowledgments
This work was supported by the Key projects of the National Natural Science Foundation of China (41831287), China National Funds for Distinguished Young Scientists (41525013), National Natural Science Foundation of China (31870351, 41703088, 31670509, and 21607016), Key projects of Research and Development of Hebei Province (19273707D), Key projects of Tianjin Natural Science Foundation (19JCZDJC40800). The authors express the sincerest thanks to Professor Bing Wu (School of the Environment in
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This paper has been recommended for acceptance by Jörg Rinklebe.
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L. Liu and Q. Wang contributed equally to the paper.