Role of CRISPR-Cas in Modulating Efflux Pump Gene Expression in Acinetobacter baumannii Isolates from Clinical Samples

Abstract

The CRISPR-Cas system serves as an adaptive immune defence in bacteria, protecting against foreign genetic elements. In Acinetobacter baumannii, efflux pumps are major contributors to multidrug resistance (MDR). This study investigates the potential regulatory role of the CRISPR-Cas system on efflux pump genes, specifically adeB, and its association with antibiotic resistance. MethodsA total of 100 clinical specimens were collected from patients admitted to the Wound Unit at Al-Hilla Teaching Hospital between March and May 2025. Standard bacteriological methods were used for isolation and identification. Antimicrobial susceptibility testing (AST) was conducted using the disk diffusion technique and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2025 guidelines. PCR assays were used to detect the presence of CRISPR-Cas system components and the blaOXA-51 gene. Quantitative real-time PCR (qRT-PCR) was employed to assess the expression levels of the adeB efflux pump gene. ResultsOut of the 100 clinical samples (44 females and 55 males, aged 10-55 years), 15 (15%) isolates were confirmed as A. baumannii. AST results indicated high resistance rates to oxacillin (100%), benzylpenicillin (93.3%), erythromycin (73.3%), and tetracycline (66.7%). The isolates exhibited the highest sensitivity to tigecycline (93.3%), trimethoprim/sulfamethoxazole (93.3%), and rifampicin (86.7%). All isolates were positive for the blaOXA-51 gene.Molecular analysis revealed that the I-Fb subtype of the cas1 gene was present in 86.7% of the isolates. Expression profiling showed that adeB was overexpressed in 66.6% of the isolates. Notably, isolates harboring complete CRISPR-Cas components exhibited downregulation of adeB, suggesting a possible repressive regulatory effect of CRISPR-Cas on efflux pump expression. ConclusionThis study demonstrates variability in the distribution of CRISPR-Cas elements among clinical A. baumannii isolates and suggests a potential inverse correlation between CRISPR-Cas system presence--particularly the I-Fb-cas1 subtype--and adeB efflux pump gene expression. These findings highlight the potential of CRISPR-Cas systems to modulate resistance mechanisms in A. baumannii, warranting further investigation into their therapeutic implications.