Molecular insights into efflux pump genes in ciprofloxacin- and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis clinical isolates from Iraq

Efflux pumps play a critical role in developing antimicrobial resistance (AMR) in S.aureus and S.epidermidis. Therefore, this study aimed to investigate the frequency and distribution of efflux pump resistance genes (norA, mepA, and mdeA), and potentially assess their association with antibiotic susceptibility profiles in ciprofloxacin- and methicillin-resistant S. aureus (CipR-MRSA) and S. epidermidis (CipR-MRSE) using both phenotypic methods and molecular detection. Among 250 clinical samples collected randomly from hospitalized Iraqi patients, 56% were Gram-positive, with 42.1% identified as S.epidermidis and 29.3% as S.aureus. A total of 53 isolates were determined to be ciprofloxacin-resistant (CipR). The majority of CipR-MRSA and CipR-MRSE isolates had mdeA and norA genes. However, the mepA gene was detected only in 13 CipR-MRSA. Additionally, mdeA gene sequence analysis showed that all tested isolates had a novel silent mutation (H388H). However, two isolates,14 and 31, had missense mutations G332V and I338F, located in the transmembrane segment 11 (TMS11) of the MdeA. In silico tools predicted that these mutations might increase the structural stability of the mutated MdeA. Docking analysis revealed that the binding energy of the MdeA wild-type protein with ciprofloxacin was - 5.0 kcal/mol, while for the MdeA mutant (G332V and I338F), it was - 5.8 kcal/mol. Similarly, docking of the MdeA protein with ethidium bromide showed a binding energy of - 5.6 kcal/mol for the wild-type and - 6.7 kcal/mol for the mutant (G332V and I338F). This study found a high frequency of efflux pump genes in CipR-MRSA and CipR-MRSE strains isolated from Iraqi patients. To our knowledge, the mdeA gene was reported, for the first time, in CipR-MRSE. Additionally, identifying sequence-identical MdeA orthologs in both ciprofloxacin-resistant MRSA and MRSE suggests a common evolutionary origin. Furthermore, missense mutations in TMS11 of MdeA may enhance its stability and binding affinity for ciprofloxacin and ethidium bromide, contributing to the understanding of resistance mechanisms in Staphylococcus species.