# Gene Expression Analysis and Whole Genome Sequencing Reveal the Potential Mechanism of Ciprofloxacin Resistance in a Salmonella Dublin Isolate

## TL;DR

This study explores how a Salmonella Dublin isolate becomes resistant to ciprofloxacin, finding that drug efflux activity may play a key role even without major genetic mutations.

## Contribution

The study identifies drug efflux as a potential mechanism of ciprofloxacin resistance in Salmonella Dublin, a serovar where resistance is less commonly reported.

## Key findings

- The ciprofloxacin-resistant Salmonella Dublin isolate showed higher drug efflux activity compared to a susceptible isolate.
- An N868S mutation in the GyrA protein was found in the resistant isolate, though it did not alter protein structure significantly.
- The resistant isolate had elevated expression of efflux-related genes acrA, acrB, ramA, and soxS.

## Abstract

Drug resistance in Salmonella to important antibiotics such as Ciprofloxacin is becoming an increasing public health threat. Ciprofloxacin resistance is, however, well documented in common Salmonella serovars such as Salmonella Typhimurium and Salmonella Enteritidis, while resistance is reported less frequently in isolates like Salmonella Dublin. This study was therefore conducted to investigate potential factors underlying Ciprofloxacin resistance in a Salmonella Dublin isolate. This isolate was identified through antibiotic resistance screening of stored Salmonella bacteria recovered from various samples, using the Kirby-Bauer disk diffusion method, followed by a broth-dilution method. For comparative analysis, the whole genome of this Ciprofloxacin-resistant Salmonella Dublin isolate and another Ciprofloxacin-susceptible isolate from this study cohort were sequenced and screened for resistance genes and plasmids. Also, the two isolates were subjected to gene expression analysis focusing on the efflux genes: acrAB, and the regulator genes marA, ramA, and soxS. Finally, protein modeling and genome comparisons were also done to detect mutations in certain genomic segments of interest and their potential impact. Our results showed that the Ciprofloxacin-resistant Salmonella Dublin isolate had a very efficient drug efflux activity compared to its Ciprofloxacin-susceptible counterpart. A genetic mutation was also identified in this resistant isolate at the amino acid position 868 of the GyrA protein. However, protein modelling analysis did not show any effective change in structure to suggest a change in function. In summary, although this observation was made in a single Ciprofloxacin-resistant Salmonella Dublin isolate, it highlights how an efficient drug efflux activity may contribute to Ciprofloxacin resistance even when no potentially impactful genetic mutations were identified.

There is a growing need to understand ciprofloxacin (CIP) resistance in less prevalent Salmonella serovars like Salmonella Dublin, which causes life-threatening conditions in both humans and animals. This study investigated potential factors contributing to CIP-resistance in a Salmonella Dublin isolate. The isolate was detected from an initial screening of 17 biobanked Salmonella isolates using the Kirby-Bauer disk diffusion (KBDF) method. The minimum inhibitory concentration (MIC) values of the identified CIP-resistant Salmonella Dublin isolate and a CIP-susceptible isolate of the same serovar were also obtained using the broth-dilution (BD) method. The two candidates were then challenged in 1/4 of their respective BD MICs for gene expression analysis, focusing on the acrAB efflux genes and the regulator genes marA, ramA, and soxS. Genomes of the isolates were also sequenced using the Oxford Nanopore sequencing platform, and then analyzed for mutations, antimicrobial resistance genes, and plasmids using ABRicate. The SWISS-MODEL server was used for protein modeling and comparison. For our results, the MIC values (KBDF; BD) for the CIP-resistant and CIP-susceptible Salmonella Dublin isolates were (1.5 μg/mL; 1.95 μg/mL) and (<0.125 μg/mL; 0.03 μg/mL), respectively. Both isolates had genes (mdtK, emrR, emrA, and emrB) notable for fluoroquinolone resistance, with the CIP-susceptible isolate also carrying the IncFII(S) plasmid. Expression of the acrA, acrB, ramA, and soxS genes was markedly higher in the CIP-resistant isolate, which also harbored an Asparagine (N) to Serine (S) mutation at position 868 in the GyrA protein. This mutation, however, caused no significant structural change. Despite reporting on a single CIP-resistant Salmonella Dublin isolate, our result highlights the potentially significant role of an efficient efflux system in contributing to CIP resistance in this isolate, even when no impactful mutations were identified.