«An accurate amplification-free CRISPR/Cas12a-based assay for GES ß-lactamase detection».

Fecha de publicación: 03/04/2025 Fecha Ahead of Print: 03/04/2025

Autores de IDIVAL

Daniel Pablo Marcos

Autor
Maria Consuelo Fernandez Diego

Autor
Jorge Rodríguez Grande

Autor
Nuria Valcárcel Fraile

Autor
Samuel García García

Autor
Sergio García Fernández

Autor
Alain Antonio Ocampo Sosa

Autor
Jorge Calvo Montes

Autor

Autores ajenos al IDIVAL

Ortiz-Cartagena C
Pacios O
Blasco L
Tomás M

Unidades

ENFERMEDADES INFECCIOSAS Y MICROBIOLOGIA CLÍNICA

Abstract

Guiana-Extended-Spectrum (GES) ß-lactamases belong to the minor class A ß-lactamases and are probably underdiagnosed due to a lack of specific diagnostic tests. There is therefore an urgent need to develop new molecular diagnostic tools that will be able to fill the gap in the detection of rare ß-lactamases. Here, we propose an optimized, amplification-free CRISPR/Cas12a-based assay for the accurate detection of GES ß-lactamases and we validate its application with clinical isolates (Graphic Abstract). Based on the results of examination of 79 standard collection, the proposed assay exhibited 100% sensitivity and specificity, as well as 100% positive and negative predictive values in less than 1.5 hours. We optimized the CRISPR/Cas12a method by harnessing a multiplex crRNA strategy, a highly efficient DNA reporter (TTATT-5C) and the Murine RNase Inhibitor to prevent crRNA degradation. This yielded limits of detection of 1ng/µL and 3ng/µL in Enterobacterales and Pseudomonas aeruginosa, respectively. The observed difference is due to the location of the bla(GES) gene. The gene occurs in a chromosomal integron present only in one to three copies in P. aeruginosa, whereas it occurs in plasmids present in multiple copies in Enterobacterales. The proposed method could be established as a routine diagnostic tool in clinical microbiology laboratories to fill the gap in availability of commercial diagnostic tests for GES ß-lactamases.

Datos de la publicación

ISSN/ISSNe:: 0924-8579, 1872-7913
Tipo:: Article
Páginas:: 107506-107506
DOI:: 10.1016/j.ijantimicag.2025.107506
PubMed:: 40187665

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Métricas

Filiaciones

Pablo-Marcos D:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain

Multidisciplinary and Translational Microbiology group (MicroTM), Biomedical Research Institute of A Coruña (INIBIC), Microbiology Service, University Hospital of A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
Fernández-Diego L:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain
Rodríguez-Grande J:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain
Fraile-Valcárcel N:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain
Ortiz-Cartagena C:: Multidisciplinary and Translational Microbiology group (MicroTM), Biomedical Research Institute of A Coruña (INIBIC), Microbiology Service, University Hospital of A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain

Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Madrid, Spain
Pacios O:: Multidisciplinary and Translational Microbiology group (MicroTM), Biomedical Research Institute of A Coruña (INIBIC), Microbiology Service, University Hospital of A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain

Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Madrid, Spain
García-García S:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain
García-Fernández S:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain

CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
Blasco L:: Multidisciplinary and Translational Microbiology group (MicroTM), Biomedical Research Institute of A Coruña (INIBIC), Microbiology Service, University Hospital of A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain

Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Madrid, Spain
Ocampo-Sosa A:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain

CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
Calvo-Montes J:: Instituto de Investigación. Microbiology Service, University Hospital Marqués de Valdecilla - IDIVAL, Santander, Spain

CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
Tomás M:: Multidisciplinary and Translational Microbiology group (MicroTM), Biomedical Research Institute of A Coruña (INIBIC), Microbiology Service, University Hospital of A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain

Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Madrid, Spain

Keywords

CRISPR; Cas12a; GES ß-lactamase; amplification-free

Campos de Estudio

Proyectos asociados

CB21/13/00068 Fariñas Álvarez, María Del Carmen

Investigador Principal: María del Carmen Fariñas Álvarez

CB21/13/00068 . INSTITUTO DE SALUD CARLOS III. . 2022