Overexpression of the major facilitator superfamily efflux pump gene nfa56470 mediates ciprofloxacin resistance in Nocardia farcinica.

OBJECTIVES

To explore the molecular basis of ciprofloxacin resistance in N. farcinica, providing a scientific basis for clinical antibiotic use and controlling the spread of resistant strains.

METHODS

We analyzed 20 N. farcinica strains. The quinolone resistance-determining regions (QRDRs) of gyrA and gyrB were sequenced and compared to the reference strain IFM 10152. The impact of efflux pumps was assessed by measuring changes in ciprofloxacin minimum inhibitory concentration (MIC) in the presence of three efflux pump inhibitors (EPIs). RT-qPCR was used to quantify the expression of 2 porin genes and 26 putative major facilitator superfamily (MFS) efflux pump genes, with and without ciprofloxacin induction.

RESULTS

Sequencing revealed no resistance-associated mutations in gyrA or gyrB. The ciprofloxacin MICs were significantly reduced (4- to 64-fold) upon exposure to EPIs, confirming efflux pump activity. Porin gene expression was modestly downregulation but did not correlate with resistance. Notably, three MFS efflux pump genes (nfa56470, nfa29840, and nfa34160) were significantly upregulated under ciprofloxacin pressure. Among these, nfa56470 emerged as the most consistently and highly overexpressed gene in resistant strains.

CONCLUSION

This study identifies the overexpression of specific MFS efflux pump genes, particularly nfa56470, as a primary mechanism of ciprofloxacin resistance in N. farcinica, supported by both gene expression data and functional EPI assays. This finding provides a clear target for future research into combating nocardial resistance.