Monitoring of Pseudomonas aeruginosa mutational resistome dynamics using an enrichment panel for direct sequencing of clinical samples.

Autor:	Cortes-Lara S; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Medina-Reatiga P; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Barrio-Tofiño ED; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Gomis-Font MA; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Cabot G; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Gómez-Romano F; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Ayestarán I; Servicio de Medicina Intensiva, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Colomar A; Servicio de Medicina Intensiva, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain., Palou-Rotger A; Servicio de Neumología, Hospital Universitario Son Espases, IdISBa, Palma de Mallorca, Spain., Oteo-Iglesias J; Laboratorio de Referencia en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, CIBERINFEC, Instituto de Salud Carlos III, Madrid, Spain., Campo RD; Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERINFEC, Madrid, Spain., Cantón R; Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERINFEC, Madrid, Spain., Horcajada JP; Servicio de Enfermedades Infecciosas, Hospital del Mar, Hospital del Mar Research Institute, Universitat Pompeu Fabra (UPF) Barcelona, Spain. CIBERINFEC, Instituto de Salud Carlos III, Madrid, Spain., López-Causapé C; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain. Electronic address: carla.lopez@ssib.es., Oliver A; Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain. Electronic address: antonio.oliver@ssib.es.
Jazyk:	angličtina
Zdroj:	EBioMedicine [EBioMedicine] 2024 Oct; Vol. 108, pp. 105367. Date of Electronic Publication: 2024 Sep 26.
DOI:	10.1016/j.ebiom.2024.105367
Abstrakt:	Background: Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples. Methods: A panel for enrichment before sequencing of close to 200 genes related to P. aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated in vitro using a multidrug-resistant ST175 isolate and representative isolates from major P. aeruginosa clades. In vivo testing included ventilator associated pneumonia by MDR P. aeruginosa in ICU (3-10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P. aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample. Findings: In vitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. In vivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel β-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and in vitro studies, was evidenced in vivo. Interpretation: This proof-of-concept study demonstrates that resistance genomics of P. aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of in vivo population dynamics in bacterial infections. Funding: Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU. Competing Interests: Declaration of interests AO and RC have participated in educational programs organised by MSD, Pfizer and Shionogi and conducted research studies financed by MSD and Shionogi. JPH has participated in educational programs organised by MSD, Pfizer, Menarini and Angelini and in advisory boards organised by Advanz Pharma, Tillots, and GILEAD. (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)
Databáze:	MEDLINE
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