Molecular Characterization of Fluoroquinolone Resistance of Methicillin –Resistant Clinical Staphylococcus aureus Isolates from Rawalpindi, Pakistan

Abstract

We have characterized 10 multidrug-resistant Staphylococcus aureus clinical isolates from Pakistan, with respect to their fluoroquinolone resistance, mutations in gyrA/B, and grlA/B genes, and the presence and expression of the efflux pump genes norA, norB, norC, and mdeA. All the isolates were highly resistant to ciprofloxacin, enrofloxacin, levofloxacin, and norfloxacin and possessed one or more efflux pump genes. The efflux pumps, along with the single and double point mutations S84L, S84L and G106D observed in gyrA; and S80Y, S81P, E84G, and S80F and E84G in grlA genes; enhanced fluoroquinolone resistance in these isolates.  A single deletion of nucleotide “A” upstream of the putative Fur-binding box and the presence of two novel mutations, G291D in isolate 30, 32, 35, and 41 and V371I in isolate 10 within the norA coding region, were also observed. An in silico structural analysis revealed an increase in NorA_G291D stability with a predicted DDG of 0.48 kcal/mol, but a decrease in NorA_V371I stability by -1.12 kcal/mol. Moreover, an increased transcriptional expression of the norA, norB, norC, and mdeA genes and relatively higher fluoroquinolone resistance in isolates with a G291D mutation, compared to that of the isolate with a V371I mutation, suggests a possible role of these mutations in fluoroquinolone resistance. The data provide new insights into the mechanism of fluoroquinolone resistance and may lead to an enhanced understanding of multidrug resistance observed in MRSA isolates and the development of effective mitigation strategies.