Antibiotic Activity of Rosemary Plant ( Rosmarinus officinalis ) Extract against Antibiotic Resistant Bacteria

Abstract

The aim of the study was to determine the optimum parameters of the rosmarinic acid (RA) extraction of the Rosemary plant ( Rosmarinus officinalis ) and to investigate the antibiotic activity of the extracted RA against antibiotic-resistant Gram-negative bacterial strains. The bacterial strains were Klebsiella pneumoniae , Pseudomonas aeruginosa , and Escherichia coli as compared with the control strains of ATCC which were identified using the VITEK 2 system as parallel compared with the counter-control strains of E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 13883. An extraction protocol using 70% ethanol was optimized for fresh leaves of Rosmarinus officinalis grown in Umm Al Quwain, UAE, after which the bacterial strains were exposed to the prepared extract. The absorption spectrum of the extracted RA was scanned between 200 - 450 nm and the peak of the maximum absorption was determined to be at ( λ max 330 nm) which was used for RA quantification and standardization. The mean value of the moisture contents of rosemary leaves was determined to be 85.27%. The optimum time of RA-equivalent extraction was determined to be 45 min of extraction in 70% ethanol using both fresh and dry leaves. The local isolates of Klebsiella pn eu moniae and Escherichia coli were resistant to ampicillin (10 μg), while both isolates were susceptible to imipenem (10 μg), ceftriaxone (30 μg), and ciprofloxacin (5 μg). The corresponding ATCC reference strains, K. pneumo niae ATCC 13883 and E. coli ATCC 25922, were included as quality-control strains and showed the expected susceptibility profiles. The antibiotic activity of the crude RA treatment on the tested local strains Klebsiella pneumoniae , Pseu domonas aeruginosa, and Escherichia coli showed no inhibition zone and therefore the strains were considered resistant. The control strain K. pneumoniae ATCC 13883 was sensitive to the RA treatment as it showed an inhibition zone of 12.3 mm diameter. The crude rosemary extract also showed weak antibacterial activity against P. aeruginosa ATCC 27853, with inhibition zones increasing from 8.25 ± 0.12 mm at 100 mg/mL to 9.24 ± 0.13 mm at 400 mg/mL. The antibiotic activity of the crude RA treatment on the counter-control strain of E. coli ATCC 25922 showed no inhibition zone and therefore the strain was considered resistant. The inhibition zones for the combined treatment of crude extract with antibiotics against the local strains Klebsiella pneu moniae , Pseudomonas aeruginosa , and Escherichia coli were recorded as 29.34, 27.84, and 27.59 mm, respectively. Furthermore, the inhibition zones for the combined treatment of crude extract with antibiotics against K. pneu moniae ATCC 13883, P. aeruginosa ATCC 27853, and E. coli ATCC 25922 were recorded as 27.95, 22.13, and 30.10 mm, respectively. In the combination assay, neither rosemary crude extract nor equivalent pure RA enhanced the activity of the tested antibiotics, as all combination treatments produced smaller inhibition zones than the antibiotic alone. Overall, UAE-grown rosemary was validated as a quantifiable local source of RA, but its crude extract and equivalent RA exhibited a weak direct antibacterial effect and no antibiotic-enhancing effect against the Gram-negative bacteria under disk diffusion conditions.