Effect of antibiotic ceftriaxone on elimination of ABI and GV3101 strains of Agrobacterium tumefaciens

I. R. Gorbatyuk, I. S. Gnatyuk, M. A. Bannikova © 2015 I. R. Gorbatyuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.181.5:602.64


Introduction
Various Agrobacterium tumefaciens strains mediate the genetic transformation of plants.The antibiotics of β-lactam group are used for the bacterial cell elimination during Agrobacterium-mediated transformation [1,2].They kill bacteria by specific interfering with biosynthesis of the peptidoglycan component of the bacterial cell wall via binding to Penicillin-Binding Proteins (PBPs) whereas there is only little or no detrimental effect on the eukaryotic plant cells [3][4][5].The antibiotics cefotaxime and carbenicillin are commonly used for the Agrobacterium cell elimination.Nowadays the usage of carbenicillin becomes undesirable because of its toxicity.Cefotaxime is mostly used in concentrations of 500 mg/l and higher [6].However, the numerous studies prove the negative effect of its high concentrations on the organogenesis, embryogenesis and shoot regeneration of most plant species [4].Now, timentin and ceftriaxone (β-lactam group) are frequently used instead of cefotaxime [7,8].These antibiotics eliminate A. tumefaciens efficiently during the genetic transformation in vitro and do not affect the regeneration frequency in most cases.Thus, ceftriaxone may be considered as an alternative to cefotaxime.
Ceftriaxone, a third-generation cephalosporin, characterized by a prolonged half-value period, has the broad-spectrum activity.The successful elimination of various Agrobacterium tumefaciens strains (EHA105, LBA4404, AGL_1) by means of ceftriaxone has been earlier reported [9][10][11][12][13].However, it has not been yet used for the elimination of ABI Agrobacterium strain.
The aim of our study was to find out the concentration of ceftriaxone, the antibiotic of β-lactam group, causing the eliminating effect on the ABI and GV3101 strains of Agrobacterium tumefaciens.

Materials and Methods
The ceftriaxone effect on the ABI and GV3101 Agrobacterium tumefaciens strains was evaluated by ISSN 1993-6842 (on-line) disk diffusion method [13].Cefotaxime (500 mg/l) was used as a control.We also compared the eliminating capability of ceftriaxone and timentin.

Results and Discussion
The inhibiting effects of ceftriaxone, cefotaxime and timentin on the cells of ABI and GV3101 Agro bacterium tumefaciens strains were compared in our study.
The suppression zone of 12.5 mm diameter was shown for ABI strain in case of using the solutions: 400 mg/l ceftriaxone, 500 mg/l cefotaxime and 350 mg/l timentin (Fig. 1).The inhibiting zone was 10.5 mm in case of using the mentioned concentrations of antibiotics for GV3101 strain.This way, ceftriaxone was proved to inhibit the Agrobacterium growth more effectively than cefotaxime and less effectively than timentin.We consider that ceftriaxone should be used in the concentration of 400 mg/l for elimination of the GV3101 Agrobacterium strain.

Conclusions
Ceftriaxone has been shown to inhibit the Agro bacterium growth more effectively comparing to the cefotaxime effect: the zones of inhibition were proved to be of the same size in cases of using the 400 mg/l ceftriaxone and 500 mg/l cefotaxime solutions for both studied Agrobacterium strains (ABI and GV3101).