Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro

Abstract

In this study the in vitro activities of seven antibiotics (ciprofloxacin, ceftazidime, tetracycline, trimethoprim, sulfamethoxazole, polymyxin B and piperacillin) and six phytochemicals (protocatechuic acid, gallic acid, ellagic acid, rutin, berberine and myricetin) against five P. aeruginosa isolates, alone and in combination are evaluated. All the phytochemicals under investigation demonstrate potential inhibitory activity against P. aeruginosa. The combinations of sulfamethoxazole plus protocatechuic acid, sulfamethoxazole plus ellagic acid, sulfamethoxazole plus gallic acid and tetracycline plus gallic acid show synergistic mode of interaction. However, the combinations of sulfamethoxazole plus myricetin shows synergism for three strains (PA01, DB5218 and DR3062). The synergistic combinations are further evaluated for their bactericidal activity against P. aeruginosa ATCC strain using time-kill method. Sub-inhibitory dose responses of antibiotics and phytochemicals individually and in combination are presented along with their interaction network to suggest on the mechanism of action and potential targets for the phytochemicals under investigation. The identified synergistic combinations can be of potent therapeutic value against P. aeruginosa infections. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals).

Keywords: Synergy; combination therapy; drug resistance.; phytochemicals.

Fig 1

PA01 growth in subminimum inhibitory concentrations of individual antibiotics and phytochemicals and their combination. Dose response and classification of interactions for antibiotics and phytochemicals used in this study. Each panel consists of bars representing measured growth rates (OD_{600 nm}) after 24 hr incubation with sub-inhibitory concentration of the antimicrobials, from left to right: growth control (no antibiotics), antibiotics alone (1/4 x MIC), phytochemicals alone (1/4 x MIC), antibiotics + phytochemicals (1/4 + 1/4 x MIC). The background colour represents the type of interactions, red - additive/indifferent, green - synergistic.

Fig 2

Network interactions between antibiotics and phytochemicals. Antibiotics are classified based on their respective targets and mechanism of action. CE, ceftazidime; PI, piperacillin - cell wall inhibitors; T, trimethoprim; S, sulfamethoxazole - folic acid biosynthesis inhibitors; POL B, polymyxin B - cell membrane inhibitors; TET, tetracycline - 30S ribosome inhibitors; CIP, ciprofloxacin - DNA gyrase and toposiomerase IV inhibitors. Phytochemicals are classified based on their classes. PA, protocatechuic acid; GA, gallic acid; EA, ellagic acid -polyphenol antioxidants (phenolic acids); BER, berberine - alkaloid; RUT, rutin; MYR, myricetin - flavonoids.

Figure 3

Time-kill data for synergistic combinations at ½ x MIC against P. aeruginosa ATCC strain 15692. (A) Combinations of sulfamethoxazole (SMX) and protocatechuic acid (PA); (B) Combinations of sulfamethoxazole (SMX) and gallic acid (GA); (C) Combinations of sulfamethoxazole (SMX) and ellagic acid (EA); (D) Combinations of tetracycline (TET) and gallic acid (GA). Filled diamond, control; Filled square, antibiotics; Filled triangle, phytochemicals; Filled circle, antibiotic and phytochemical combination.

Figure 3

Time-kill data for synergistic combinations at ½ x MIC against P. aeruginosa ATCC strain 15692. (A) Combinations of sulfamethoxazole (SMX) and protocatechuic acid (PA); (B) Combinations of sulfamethoxazole (SMX) and gallic acid (GA); (C) Combinations of sulfamethoxazole (SMX) and ellagic acid (EA); (D) Combinations of tetracycline (TET) and gallic acid (GA). Filled diamond, control; Filled square, antibiotics; Filled triangle, phytochemicals; Filled circle, antibiotic and phytochemical combination.