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 G 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.

Clonal Lineages, Antimicrobial Resistance, and PVL Carriage of Staphylococcus aureus Associated to Skin and Soft-Tissue Infections from Ambulatory Patients in Portugal

“…These patterns of QRDR mutations were also detected in an earlier study from S. aureus clinical isolates in Lisbon [46]. The GrlA S80F and GyrA S84L mutations are the most commonly described in the literature [47][48][49][50][51] and are characteristic of ST22 and some ST8 lineages [51]. In our study, only one isolate carried both mutations and belonged to ST22.…”

“…The GyrA S84L mutation was also found in one isolate of ST8. The GrlA E84G and S80Y and GyrA E88K mutations are also described in some studies [50,51] but appear to be less frequent. Besides these mutations, the activity of chromosomally-encoded MDR efflux pumps might also be contributing to fluoroquinolone resistance.…”

Expression Levels of Efflux pump mexR and norA Genes in Multi-Drug Resistant in Some Bacteria by Using Quantitative RT-PCR Under Stress of Effect Efflux Pump Inhibitors.

“…Quantitative RT-PCR assay analyzed the mRNA expression of norA and compared its expression with isolates without treatment (control), isolates were exposed to azithromycin antibiotic, and also were exposed to synthetic inhibitors. The result of gene expression was shown in table (4). The fold of gene expression of the norA gene for active efflux in the isolates of S. aureus studied was (4.61), this result was higher than the fold of nonactive efflux norAgene (2.14).…”

“…Staphylococcus aureus pathogen remains one of the more serious bacterial pathogens in the public health field because of its high virulence, and also able to cause multiple diseases varying from complicated skin and skin structure infections (cSSSI) to life-threatening situations, like pneumonia, endocarditis and toxic shock syndrome (4) .Efflux pumps have revealed as specific key drivers for antimicrobial in both Gram-negative and Gram-positive bacteria, EP also is vital in other different physiological processes like stress-adaptations, pathogenicity, virulence factors, and transportation of necessary nutrients (5). The MDR efflux systems are found in whole bacterial species.…”