Reduction of Gentamicin Standard Dose Combined with Phytochemicals Against Enterobacter Cloacae DSM 3264 BRB Using Response Surface Methodology

Document Type : Original Article


1 Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt.

2 Medical Analysis Central Laboratories for Medical Analysis and Blood Bank of Armed Forces, Al-Khalifa Al-Ma'mun, Cairo, Egypt


Antibiotic-phytochemicals combinations are used for enhancing the antibacterial efficiency against pathogens aiming to prevent microbial resistance development. This study was conducted at the Faculty of Agriculture, Ain Shams University in Cairo, Egypt, to study the in-vitro inhibitory activities of gentamicin antibiotic and phytochemical combinations of Cinnamomum zeylanicum (cinnamon), Syzygium aromaticum (clove) and Mentha piperita (mint) against Enterobacter cloacae DSM 3264 BRB by Minimal inhibitory concentrations (MICs) and Bactericidal inhibitory concentrations (MBCs) of the agents. Inhibitory activity of four recommended antibiotics (ciproflaxacin, cefepime, meropenem and gentamicin) and three oil plant extracts of the selected plants (cinnamon, clove, mint) were tested against E. cloacae DSM 3264 using well diffusion method on Muller-Hinton agar plates by studying MIC and MBC tests. Gentamicin was selected among the four tested antibiotics based on its high potential against Enterobacter cloacae DSM 3264 BRB. For the reduction of gentamicin standard dose, a synergism experiment between gentamicin and the three phytochemicals under investigation (cinnamon, mint and clove) was carried out using a new statistical approach of mixture drug design of response surface methodology against E. cloacae DSM 3264 growth. A mixture design of twenty mixture combination runs using different concentration levels of gentamicin and the three oil plants extracts was performed against Enterobacter cloacae DSM 3264 growth. The software “Design Expert® 12” Stat-Ease was used to analyze the experimental mixture design. Results showed that the standard recommended gentamicin dose 10 µg/100ml may be substituted by oil extract of Cinnamomum zeylanicum 4.75 % (v/v), Syzygium aromaticum 5.0% (v/v), Mentha piperita 5.0%(v/v) and gentamicin 0.25%(w/v) as an antibacterial agents. Analysis of clove, cinnamon and mint oils by GC-MS proved that the major components were 3-allyl-6-methoxyphenol (46.4%) followed by propylene glycol (53.59%), whereas cinnamon contains cinnamic acid and phenethyl ester being 41.79% while Mint contains menthol (32.9%) followed by menthone (27.7 %).