Acquisition of a Stable and Transferable blaNDM-5-Positive Plasmid With Low Fitness Cost Leading to Ceftazidime/Avibactam Resistance in KPC-2-Producing Klebsiella pneumoniae During Treatment.

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in blaKPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in blaKPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored blaKPC-2, blaSHV-182 and blaTEM-1B. Compared with KP135194, KP137060 harbored an additional blaNDM-5 positive plasmid. blaNDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that blaKPC-2- and blaNDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a blaNDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.

A network pharmacology-based investigation on the bioactive ingredients and molecular mechanisms of Gelsemium elegans Benth against colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. Gelsemium elegans Benth (GEB) is a traditional Chinese medicine commonly used for treatment for gastrointestinal cancer, including CRC. However, the underlying active ingredients and mechanism remain unknown. This study aims to explore the active components and the functional mechanisms of GEB in treating CRC by network pharmacology-based approaches. METHODS: Candidate compounds of GEB were collected from the Traditional Chinese Medicine@Taiwan, Traditional Chinese Medicines Integrated Database, Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine, and published literature. Potentially active targets of compounds in GEB were retrieved from SwissTargetPrediction databases. Keywords "colorectal cancer", "rectal cancer" and "colon cancer" were used as keywords to search for related targets of CRC from the GeneCards database, then the overlapped targets of compounds and CRC were further intersected with CRC related genes from the TCGA database. The Cytoscape was applied to construct a graph of visualized compound-target and pathway networks. Protein-protein interaction networks were constructed by using STRING database. The DAVID tool was applied to carry out Gene Ontology and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis of final targets. Molecular docking was employed to validate the interaction between compounds and targets. AutoDockTools was used to construct docking grid box for each target. Docking and molecular dynamics simulation were performed by Autodock Vina and Gromacs software, respectively. RESULTS: Fifty-three bioactive compounds were successfully identified, corresponding to 136 targets that were screened out for the treatment of CRC. Functional enrichment analysis suggested that GEB exerted its pharmacological effects against CRC via modulating multiple pathways, such as pathways in cancer, cell cycle, and colorectal cancer. Molecular docking analysis showed that the representative compounds had good affinity with the key targets. Molecular dynamics simulation indicated that the best hit molecules formed a stable protein-ligand complex. CONCLUSION: This network pharmacology study revealed the multiple ingredients, targets, and pathways synergistically involved in the anti-CRC effect of GEB, which will enhance our understanding of the potential molecular mechanism of GEB in treatment for CRC and lay a foundation for further experimental research.

Molecular Mechanism of Gelsemium elegans (Gardner and Champ.) Benth. Against Neuropathic Pain Based on Network Pharmacology and Experimental Evidence.

Gelsemium elegans (Gardner and Champ.) Benth. (Gelsemiaceae) (GEB) is a toxic plant indigenous to Southeast Asia especially China, and has long been used as Chinese folk medicine for the treatment of various types of pain, including neuropathic pain (NPP). Nevertheless, limited data are available on the understanding of the interactions between ingredients-targets-pathways. The present study integrated network pharmacology and experimental evidence to decipher molecular mechanisms of GEB against NPP. The candidate ingredients of GEB were collected from the published literature and online databases. Potentially active targets of GEB were predicted using the SwissTargetPrediction database. NPP-associated targets were retrieved from GeneCards, Therapeutic Target database, and DrugBank. Then the protein-protein interaction network was constructed. The DAVID database was applied to Gene Ontology and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis. Molecular docking was employed to validate the interaction between ingredients and targets. Subsequently, a 50 ns molecular dynamics simulation was performed to analyze the conformational stability of the protein-ligand complex. Furthermore, the potential anti-NPP mechanisms of GEB were evaluated in the rat chronic constriction injury model. A total of 47 alkaloids and 52 core targets were successfully identified for GEB in the treatment of NPP. Functional enrichment analysis showed that GEB was mainly involved in phosphorylation reactions and nitric oxide synthesis processes. It also participated in 73 pathways in the pathogenesis of NPP, including the neuroactive ligand-receptor interaction signaling pathway, calcium signaling pathway, and MAPK signaling pathway. Interestingly, 11-Hydroxyrankinidin well matched the active pockets of crucial targets, such as EGFR, JAK1, and AKT1. The 11-hydroxyrankinidin-EGFR complex was stable throughout the entire molecular dynamics simulation. Besides, the expression of EGFR and JAK1 could be regulated by koumine to achieve the anti-NPP action. These findings revealed the complex network relationship of GEB in the "multi-ingredient, multi-target, multi-pathway" mode, and explained the synergistic regulatory effect of each complex ingredient of GEB based on the holistic view of traditional Chinese medicine. The present study would provide a scientific approach and strategy for further studies of GEB in the treatment of NPP in the future.