Plasmid-encoded gene duplications of extended-spectrum ß-lactamases in clinical bacterial isolates.

Introduction: The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.

Characterization of blaOXA-48-carrying plasmids and small non-AMR-coding plasmids collected from Ukrainian patients.

PURPOSE: Carbapenemase-producing Enterobacterales (CPE) pose a serious threat for healthcare facilities worldwide, yet the mode of transmission is often unclear. Recently, we recorded an increase of blaOXA-48-harboring isolates at our hospital associated with patients with previous medical treatment in the Ukraine. We used long-read whole genome sequencing (lrWGS) to characterize these isolates including their plasmids. METHODS: Samples were collected as part of clinical routine diagnostic or screening of multi-drug resistance bacteria (MDRB). Antimicrobial susceptibility testing was performed and all MDRB (n = 10) were sequenced by lrWGS for genotyping, identification of antimicrobial resistance (AMR) genes, and characterization of plasmids. RESULTS: While routine analysis of core genome multilocus sequence typing (cgMLST) did not show any genetic similarities between isolates, we found an unexpected high similarity in the plasmid diversity of different Enterobacterales in patients with previous medical treatment in the Ukraine. This included an IncL/M plasmid carrying blaOXA-48 and additional small non-AMR-coding plasmids. CONCLUSION: Our results show that lrWGS can be used in the routine setting to uncover similarities in plasmids and may give further information about potential epidemiologic associations. In the future, analysis of both AMR and non-AMR plasmids may provide an additional layer of information for molecular surveillance of CPE.