Cantillon, D. | bioRxiv Science

bioRxiv2026

Comparison of multiple cefiderocol susceptibility testing methods against genomic determinants of resistance in blaNDM carbapenemase producing Enterobacterales

BackgroundCefiderocol is a siderophore-conjugated cephalosporin antibiotic used to treat multi drug resistant Gram negative infections, including metallo-beta-lactamase producing Enterobacterales. Antimicrobial useage is guided by antimicrobial susceptibility testing (AST) which is hampered by differences between EUCAST and CLSI breakpoints, methodological challenges of AST, and lack of information on clinical outcome related to AST. ObjectivesThis study assessed the agreement between AST methods under EUCAST and CLSI breakpoints in a collection of 57 blaNDM producing Enterobacterales isolated from a UK hospital network. MethodsAll isolates, including Klebsiella pneumoniae, Enterobacter hormaechei, Escherichia coli and Citrobacter freundii, were whole-genome sequenced and tested with disk diffusion and MIC gradient test strip, and broth microdilution MICs were determined for a subset. Categorical agreement between methods was calculated using both EUCAST and CLSI breakpoints. Mutations and acquired resistance genes associated with cefiderocol resistance were identified and compared with AST results. ResultsThe disk diffusion method, based on EUCAST interpretation, classified 94.7% of isolates as cefiderocol resistant and 5.3% as susceptible, with 22.8% within the Area of Technical Uncertainty. The CLSI breakpoint classified one isolate as resistant (1.8%) and 5.26% intermediate. Category agreement of broth microdilution and disk diffusion for E. coli using EUCAST guidelines was 38.5%. Mutations associated with cefiderocol resistance were highly prevalent and varied between species. ConclusionsThe discordant EUCAST and CLSI breakpoint values provided have large impacts on the classification of isolates susceptibility to cefiderocol, which will impact global cefiderocol usage and surveillance of resistance, further complicated by poor agreement between AST methods.

microbiology↗

bioRxiv2024

A mycobacterial DNA extraction protocol designed for resource limited settings generates high quality whole genome sequencing

Mycobacteria are major global human pathogens and include Mycobacterium tuberculosis, the causative agent of tuberculosis, and M. abscessus, an emerging multidrug resistant pathogen. M. abscessus affects people with structural lung disease and those who are immunocompromised, most commonly causing pulmonary disease but also disseminated infections in the central nervous system and skin. High quality whole genome sequencing is essential to research mycobacterial epidemiology, pathogenesis and antimicrobial resistance. However, current DNA extraction protocols are time consuming, use toxic chemicals, require cold chain storage for certain reagents and can often result in poor quality, degraded DNA that directly impacts whole genome sequencing outputs. This is a particular challenge in low-income settings. Here, we report a novel optimised DNA extraction workflow for M. tuberculosis and M. abscessus that invariably generates high quality Illumina short read sequencing data. We evaluated input culture CFU and physical cell disruption times. DNA quantity was determined using a Qubit fluorometer system with DNA integrity assessed using the Agilent TapeStation platform. We showed that this protocol facilitated complete genome assemblies of M. abscessus and M. tuberculosis reference strains. There is no requirement for cold chain transport or storage of reagents, solvent extractions, or boiling to heat inactivate cultures, and the method does not require surfactant chemicals such as cetyltrimethylammonium bromide (CTAB).

microbiology↗