Role of CRISPR-Cas in Modulating Efflux Pump Gene Expression in Acinetobacter baumannii Isolates from Clinical Samples
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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. Methods A 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. Results Out 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. Conclusion This 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.
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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.
Methods
A 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.
Results
Out 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.
Conclusion
This 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.
Competing Interest Statement
The authors have declared no competing interest.
Funding Statement
not receive any funding
Author Declarations
I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.
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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:
Ethical approval for this study was obtained from the ethical committee at Hilla Surgical Teaching Hospital. This study was also approved by a local ethics committee at the College of Medicine, University of Babylon. and the hospital ethics committee under document number [IRB: 399-4/4/2025].
I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.
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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.
Yes
Data Availability
All data produced in the present work are contained in the manuscript
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