The CRISPR System and MepA Multidrug Efflux Pump Linked to Antibiotic Resistance in.

Abstract

() is a major zoonotic pathogen. To investigate CRISPR carriage inisolates from cows with mastitis and the role of the CRISPR system and efflux pumps in antibiotic resistance. We analyzed antibiotic resistance genes and CRISPR loci, sequenced spacers, and assessed correlations between CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) presence and antibiotic resistance inisolates. The changes in CRISPR sequences were examined by continuous passage of 360 generations without antibiotic pressure. Subsequently, variations in CRISPR loci and transcript levels were measured under ciprofloxacin (CIP) exposure. In addition, anwas constructed to evaluate changes in antimicrobial sensitivity andtranscript levels in both planktonic and biofilm states. Our results revealed a CRISPR loci detection rate of 7.69% among theisolates, with significantly lower rates of the antibiotic resistance genes,,, and(M) in CRISPR-positive isolates compared to those in CRISPR-negative isolates (< 0.05). CIP-resistant strains exhibited loss of repeat and spacer sequence in CRISPR loci, and the transcript abundance of these loci gradually decreased under CIP pressures, indicating that CRISPR loci deletion or transcript level downregulation under antibiotic stress may be a potential regulatory mechanism of antibiotic resistance. Correlation analysis linked CIP resistance in both planktonic and biofilmtotranscript levels and biofilm integrity. Our study provides insight into the mechanism by whichdevelops antibiotic resistance via the CRISPR system and the MepA efflux pump, offering a theoretical foundation for monitoring the prevalence and resistance of pathogenic bacteria.