The spread of antibiotic resistance genes (ARGs), especially those on plasmids, poses a major risk to global health. However, the extent and frequency of ARG transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a new fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. The tool integrates a fluorescent tag immediately downstream ARGs, allowing for gene transfer tracking without affecting host cell antibiotic susceptibility, fitness, conjugation, or transposition. Our experiments show that sfGFP-tagged plasmid-borne mcr-1 can transfer across different bacteria species in feces, including Escherichia, Shigella, Lactobacillus, Staphylococcus, Enterococcus, and Bacillus. CRISPR-AMRtracker offers a powerful tool for monitoring ARG transfer in microbiota communities and can inform strategies to combat the threat of antibiotic resistance.
Aims
The aim of this study was to determine the effects of unsaturated fatty acids on clinical plasmids.
Methods and Results
Two unsaturated fatty acids, linoleic acid (LA) and α‐linolenic acid (ALA) at final concentration 0, 0·03, 0·3 and 3 mmol l−1, respectively, were used to assess the effects on conjugative transfer of a mcr‐1‐harbouring plasmid pCSZ4 (IncX4) in conjugation experiment. The inhibitory mechanisms were analysed by molecular docking and the gene expression of virB11 was quantitated by qRT‐PCR. Target plasmid diversity was carried out by TrwD/VirB11 homology protein sequence prediction analysis. Our results showed that LA and ALA inhibit plasmid pCSZ4 transfer by binding to the amino acid residues (Phe124 and Thr125) of VirB11 with dose‐dependent effects. The expression levels of virB11 gene were also significantly inhibited by LA and ALA treatment. Protein homology analysis revealed a wide distribution of TrwD/VirB11‐like genes among over 37 classes of plasmids originated from both Gram‐negative and Gram‐positive bacteria.
Conclusions
This study demonstrates representing a diversity of plasmids that may be potentially inhibited by unsaturated fatty acids.
Significance and Impact of the Study
Our work reported here provides additional support for application of curbing the spread of multiple plasmids by unsaturated fatty acids.
BackgroundMycophenolate mofetil (MMF) has been widely prescribed for the therapy of neuromyelitis optica spectrum disorders (NMOSD) although not all patients display the optimal clinical response to MMF. Our study investigated a potential role of the gut microbiota in the efficacy of MMF in NMOSD patients.MethodsA total of 34 NMOSD patients treated with MMF were prospectively enrolled and grouped according to the therapeutic efficacy as effect group (EG) versus non-effect group (NEG). The gut microbiota was analyzed using 16S rRNA sequencing with stool samples and purine metabolites were profiled in serum samples from the same patients. To further decipher the role of the gut microbiota in coordinating the MMF efficacy, metabolic profiling was performed in microbiota-depleted mice. ResultsThe gut microbiota compositions could not distinguish EG and NEG patients although Clostridium and Synergistetes had significantly increased for EG and this corresponded to a significant decline in abundance of the Coprococcus genus. Additionally, purine salvage pathway (PSP) metabolites inosine, hypoxanthine, xanthine, guanine and uric acid in the serum of NMOSD patients were elevated 2 – 7-fold in the NEG group compared to EG (p< 0.05). We then used the selected PSP metabolites as indicators of MMF efficacy in a mouse model and found that the MMF response was significantly dampened when the gut microbiota was depleted by vancomycin treatment. The latter microbiomes displayed elevated levels of the PSP metabolites xanthine, inosine and hypoxanthine (p< 0.05).ConclusionsThe activity and metabolic capacities of gut microbiomes are essential for efficient response to MMF in NMOSD patients and implies an active role for the gut microbiota in coordinating host responses to MMF therapy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.