Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii

Abstract

Polymerase chain reaction (PCR), especially the multiplex PCR assay, enables simultaneous detection of multiple genes and is highly effective for diagnostic applications. The CRISPR-associated (Cas) system consists of several genes, and complete gene clusters are essential for its activity; multiplex PCR is an excellent method for detecting these multiple genes. This study focuses on the development and validation of a multiplex PCR protocol for the specific detection of CRISPR-Cas subtypes I-F1 and I-F2 found in A. baumannii, which is classified as a critical ESKAPE pathogen. The multiplex PCR method achieved a 100 % detection rate for isolates containing Cas subtypes I-F1 and I-F2 in clinical A. baumannii isolates. Testing across various genera and Acinetobacter species confirmed the high specificity of the assay, with no false positives, establishing it as a reliable tool for large-scale clinical applications. Of the 96 clinical A. baumannii isolates analysed, 29.167 % (n = 28) were multiplex PCR positive for a CRISPR-Cas system. Among these, 71.43 % (n = 20) had subtype I-F1, while 28.57 % (n = 8) had subtype I-F2. No clear association was found between Cas subtypes and resistance to the tested antibiotics or carbapenem genes. This study provides a valuable tool for monitoring CRISPR-Cas systems and can aid in various experimental and novel strategies to manage multidrug-resistant A. baumannii.