CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases

Abstract

Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction.

Keywords: CRISPR-Cas13a; GES; LAMP; OXA-48; detection.

FIG 1

Workflow of the protocol for diagnosis of infectious diseases based on the CRISPR-Cas13a system.

FIG 2

LoD assay for OXA-48 and GES-producing bacteria inoculated in clinical negative samples that the LAMP-CRISPR-Cas13a technique detects in serial dilutions (1:10) of LB cultures. Thin gray toolings indicate strips spliced for labeling purposes.

FIG 3

(A) HybriDetect lateral flow test strip for GES carbapenemase identification by bla_GES gene detection in inoculated samples and non GES carbapenemase-producing strains (negative controls). (B) HybriDetect lateral flow test strip for OXA-48 carbapenemase identification by bla_OXA-48 gene detection in inoculated samples and non OXA-48 carbapenemase-producing strains (negative controls). (C) Results obtained for bla_GES gene detection. (D) Results obtained for bla_OXA-48 gene detection. (E) Table including the specificity and sensitivity (99% CI) for the LAMP-CRISPR-Cas13a technique calculated by processing data in Fig. 3C and 3D. Thin gray toolings indicate strips spliced for labeling purposes.