Revealing the Genetic Diversity of Chinese Chlamydia trachomatis Strains Directly from Clinical Samples through Selective Whole-Genome Amplification.

BACKGROUND: Chlamydia trachomatis is the causative agent of most prevalent bacterial sexually transmitted infection globally. Whole-genome sequencing is essential for molecular Chlamydia surveillance; however, its application is hampered by the pathogen's low abundance in clinical specimens and the expensive, labor-intensive nature of existing enrichment methodologies for Chlamydia. METHODS: We developed a targeted whole-genome amplification tool termed SWTICH, by integrating phi29 DNA polymerase-mediated amplification with meticulously designed primer sets to enrich Chlamydia trachomatis genome, followed by whole-genome sequencing. This method underwent evaluation through testing synthetic and clinical specimens. RESULTS: SWITCH demonstrated robust ability to achieve up to 98.3% genomic coverage of Chlamydia trachomatis from as few as 26.4 genomic copies present in synthetic specimens and exhibited excellent performance across diverse Chlamydia trachomatis serovars. Utilizing SWITCH, we directly generated 21 Chlamydia genomes from 26 clinical samples, enabling us to gain insights into the genetic relationships and phylogeny of current Chlamydia strains circulating in the country. Remarkably, this study marked the first instance of generating Chinese Chlamydia genomes directly from clinical samples. CONCLUSIONS: SWITCH represents a practical, cost-efficient approach to enrich Chlamydia genome directly from clinical specimens, offering an efficient avenue for molecular surveillance of Chlamydia.

A molecular dynamics study of the complete binding process of meropenem to New Delhi metallo-ß-lactamase 1.

The mechanism of substrate hydrolysis of New Delhi metallo-ß-lactamase 1 (NDM-1) has been reported, but the process in which NDM-1 captures and transports the substrate into its active center remains unknown. In this study, we investigated the process of the substrate entry into the NDM-1 activity center through long unguided molecular dynamics simulations using meropenem as the substrate. A total of 550 individual simulations were performed, each of which for 200 ns, and 110 of them showed enzyme-substrate binding events. The results reveal three categories of relatively persistent and noteworthy enzyme-substrate binding configurations, which we call configurations A, B, and C. We performed binding free energy calculations of the enzyme-substrate complexes of different configurations using the molecular mechanics Poisson-Boltzmann surface area method. The role of each residue of the active site in binding the substrate was investigated using energy decomposition analysis. The simulated trajectories provide a continuous atomic-level view of the entire binding process, revealing potentially valuable regions where the enzyme and the substrate interact persistently and five possible pathways of the substrate entering into the active center, which were validated using well-tempered metadynamics. These findings provide important insights into the binding mechanism of meropenem to NDM-1, which may provide new prospects for the design of novel metallo-ß-lactamase inhibitors and enzyme-resistant antibiotics.