Antimicrobial Susceptibility Profiles and In Vitro Antibacterial and Anti-Virulence Effects of Aqueous Pomegranate Peel Extract on Clinical Vibrio Cholerae Isolates from Erbil, Iraq

Abstract

This study investigated the antimicrobial susceptibility patterns of clinical Vibrio cholerae isolates from Erbil, Iraq and evaluated the in vitro antibacterial and anti-virulence activity of aqueous pomegranate (Punica granatum) peel extract. Fifteen confirmed non-duplicate clinical V. cholerae isolates obtained from 257 diarrheal stool specimens (2024-2025) were identified by conventional microbiological methods, O1/O139 serotyping, VITEK 2 and confirmed by 16S rRNA gene amplification. Antimicrobial susceptibility testing was performed across ten antimicrobial agents according to CLSI guidelines. Antibacterial efficacy of the aqueous peel extract was evaluated by minimum inhibitory concentration (MIC), agar well diffusion and growth kinetic assays. Transcriptional responses of virulence-associated genes (flaA, vpsT and luxO) following exposure to 10 and 20 mg/mL of extract were quantified by RT-qPCR using the 2^(-ΔΔC_T ) method. All isolates (100%) were resistant to amoxicillin-clavulanic acid, whereas high susceptibility was retained for chloramphenicol (93.3%), imipenem (86.7%), amikacin (80.0%), tetracycline (80.0%) and doxycycline (73.3%), with intermediate responses in erythromycin (60.0%) and ciprofloxacin (53.3%). The aqueous peel extract demonstrated dose-dependent antibacterial activity, with MIC values ranging from 3 to 50 mg/mL MIC50 = 25 mg/mL (; MIC90 = 50 mg/mL) and inhibition zones of 19-24 mm at 50 mg/mL. Significant downregulation of flaA and vpsT was observed following 10 and 20 mg/mL treatment (p<0.001), whereas luxO expression showed variable, moderate reduction. These findings provide updated regional baseline data on antimicrobial resistance in clinical V. cholerae and demonstrate that aqueous pomegranate peel extract exhibits direct in vitro growth-inhibitory and transcriptional modulatory activity. These findings remain strictly limited to in vitro laboratory observations and do not establish therapeutic efficacy; further chemical profiling of the aqueous phase, phenotypic motility/biofilm assays and in vivo functional validation are required.