The Expression of Efflux Pump Genes in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Blood Cultures in Iran

Abstract

Background: Efflux pump is a significant resistance mechanism in Staphylococcus aureus. A total of 100 patients with bacteremia from Shahid Beheshti University Hospitals of Tehran in Iran were tested for the expression of efflux pump genes, contributing to S. aureus antimicrobial resistance. Objectives: this study was conducted to identify resistance pattern, and to evaluate the inhibitory effect of efflux pump, MIC of ciprofloxacin, and expression levels of norA, norB, and norC efflux pump genes in the presence of an efflux pump inhibitor against MDR S. aureus. Methods: A total of 100 MRSA isolates were investigated in different hospitals of Shahid Beheshti University of Medical Sciences from April 2017-2018. Owing to new consensus guidelines from the Clinical and Laboratory Standards Institute (CLSI), both the Kirby-Bauer disk diffusion test and micro-dilution method were used to evaluate antimicrobial susceptibility. Efflux pump activity using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was identified as a chemical efflux pump inhibitor. E-test was used to determine Vancomycin-resistant antibiotic. Broth micro-dilution method for S. aureus isolates resistant to ciprofloxacin has been developed for minimum inhibitory concentration (MIC) of ciprofloxacin and CCCP and their composition. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to investigate the expression level of norA, norB, and norC efflux pump genes. Results: A total of 38 of 45 MRSA isolates (84.4%) showed resistance to ciprofloxacin. Moreover, 100% of isolates had the norA and norB genes. Further, 95% of S. aureus isolates had the norC gene. According to this study, ciprofloxacin MIC has decreased by CCCP compared to ciprofloxacin. There was an increase in the expression level of norA, norB, and norC efflux pump genes in methicillin-resistant and ciprofloxacin-resistant S. aureus strains based on RT- PCR. In this study, four different spA types were obtained as the most prevalent type of spA by t037and t790 (23.3%) and t030 (14.1%) and t044 (12.2%). Conclusions: This study indicates that the prevalence of ciprofloxacin-resistant S. aureus strains has a rising trend among MRSA clinical isolates. The ability of S. aureus isolates to be converted into drug-resistant strains using efflux pump mechanism has become a widespread concern.

Expression of norA, norB and norC efflux pump genes mediating fluoroquinolones resistance in MRSA isolates

“…In our study, the resistance rate to ciprofloxacin was 60% when performing MICs by broth micro-dilution method which was similar to that observed in Egypt (58%), Ethiopia (61.5%) and Brasil (64.5%) [20,22,23]. However, our finding was lower than other studies conducted in Egypt (96%) [24], Brasil (79%) [25] and Iran (84.4%, 70%) [15,21].…”

“…Moreover, Tsige et al found that the isolated MRSA showed high resistance to erythromycin (61.5%), ciprofloxacin (61.5%) and gentamicin (53.8%) [20]. However, compared to our results, higher resistance rates of MRSA isolates against erythromycin (91.1%), ciprofloxacin (84.4%) and clindamycin (80%), were detected by Bostanmaneshrad et al [15] These variations in the antimicrobial drug resistance rates among different countries might be attributed to the availability and indiscriminate use of these antibiotics in certain areas which indicates that the antibiotic resistance patterns vary according to regional and geographical factors.…”

“…Rehman et al [18] and Garoy et al [19] reported a higher prevalence of MRSA at 78.3% and 72.0%, respectively. Lower frequencies of MRSA detected by Tsige et al [20], Bostanmaneshrad et al [15] and Hassanzadeh et al [21] were 28.3%, 45% and 50%, respectively by using cefoxitin disc diffusion method and PCR of the mecA gene. MRSA prevalence varies widely between countries and is attributed to different infection control policies that are followed by these countries.…”

“…Amplification was accomplished on Biometra thermal cyclers (Biometra, Göttingen, Germany) using the following cycling conditions: primary denaturation for 5 min at 95 °C, 30 cycles of amplification composed of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s for all genes, and extension at 72 °C for 30 s, followed by a step of final extension at 72 °C for 5 minutes. Amplified products were electrophoresed using 2.5% agarose gel and visualized with ultraviolet irradiation [15].…”

“…Negative controls were utilized to exclude presence of contamination. The expression of norA, norB, and norC genes were normalized to the expression of gmK housekeeping gene (internal control) [15] and evaluated based on 2 (-∆∆CT) relative expression quantification method [16,17].…”