In vitro combinational efficacy of ceftriaxone and leaf extract of Combretum albidum G. Don against multidrug-resistant Pseudomonas aeruginosa and host-toxicity testing with lymphocytes from human cord blood

Abstract

To study the in vitro antibacterial activity of methanolic leaf extract of Combretum albidum in combination with the antibiotic ceftriaxone (CE) against multidrug-resistant (MDR) Pseudomonas aeruginosa, and to assess the host toxicity of the leaf extract with human cord blood-derived lymphocytes in vitro. The synergistic/antagonistic action of the leaf extract with CE was evaluated with the checkerboard procedure. The leaf extract and the antibiotic were added into the wells of a microtiter plate in serially proportionate combinations along with nutrient broth, bacterial inocula, and 2,3,5-triphenyltetrazolium chloride to achieve visible growth of the bacterium. Lymphocytes from human cord blood were cultured and the toxicity of the leaf extract was assessed by both acridine orange/ethidium bromide (AO/EB) staining method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The test results were analyzed by the probit method. Minimum inhibitory concentration (MIC) values of the methanolic leaf extract and CE separately were 0.866 and 0.0384 mg/mL, respectively. The MIC value of the leaf extract with the antibiotic was in the range of 0.102–0.866 mg/mL. Synergistic results in the in vitro control were obtained in all combinations of the antibiotic and the extract (i.e., from 9:1 to 5:5); ratios 1:9, 2:8, and 3:7 caused antagonism, whereas an indifference was seen at the ratio 4:6. Individual minimum bactericidal concentration (MBC) values of the leaf extract and CE were 4.39 and 87.7 μg/mL, respectively, whereas those of all combinations, 9:1 to 1:9, ranged from 0.33 to 3.07 mg/mL. The MBC value of the leaf extract alone was 4.39 mg/mL, whereas in various combinations with CE (at ratios from 9:1 to 1:9), the value varied from 0.70 to 3.07 mg/mL. The MBC value of CE was only 0.877 mg/mL in all combinations with the leaf extract. The plant extract level of 58.88 mg/mL was considered the lethal concentration 25 (LC25) because of its lethal effects on lymphocytes, whereas the LC25 value for CE was 380.19 μg/mL according to the AO/EB staining method. In the MTT assay, however, the LC25 value of leaf extract was 53.70 mg/mL. Therefore, we used a leaf extract concentration of 50 mg/mL, which was a little below the LC25 value of leaf extract obtained in the MTT assay (i.e., 53.70 mg/mL). This study provided the result of synergistic activity of the combination of crude leaf extract of C. albidum with CE against MDR P. aeruginosa. The leaf extract was nontoxic to human lymphocytes.