Multi drug-resistant bacterial infections are responsible for greater portion of infectious disease burden worldwide. For the last twenty years, the emergence of antimicrobial resistance along with the adverse effects of antibiotics has directed the researchers for the exploration of new antimicrobial substances specifically from herbal extracts, which can be able to solve the above problems . Moreover, the therapeutic potential of herbal plants has been studied and identified to use them as a source of new drugs. According to the guidelines of WHO in 1997, beneficial plants can be used as an alternative of drugs .
During the course of the study, different clinical samples were collected from patients suffering from a variety of infections. These samples were processed for isolation of MDR human pathogens, which were screened against different antibiotics in order to determine their antibiotic susceptibility pattern. Methyl gallate exhibited remarkable antimicrobial activity against both MDR gram-positive (MIC = 250 µg/ml) and gram-negative bacteria (MICs = 125–250µg/ml). According to one study, phytochemicals with MIC between 100–1000µg/ml are considered as potential antimicrobial agents . Earlier reports stated the antimicrobial activity of Methyl gallate against MRSA with (MIC = 1.25mg/ml) [4, 5]. MRSA is one of the most important pathogens that accounts for respiratory tract, skin, and surgical site infections with high mortality and morbidity . Previous studies indicated the antimicrobial activity of Methyl gallate against non-MDR E. coli, S. typhi and Vibrio Cholerae [7, 8]. Time kill kinetics study of Methyl gallate showed dose-dependent bactericidal activity in both gram-negative and gram-positive bacteria. Profound effects of Methyl gallate on cell morphology of MRSA and MDR E. coli were seen by Scanning Electron Microscopy. Disruption in cell wall, incomplete cell division and distorted structure suggested that the site of action of Methyl gallate was cell wall.
Gallic acid showed antimicrobial activity (MIC = 500–1000µg/ml) in the current study, whereas, previous study endorsed our results . Antimicrobial activity of Gallic acid was observed in one study with (MIC = 100µg/ml, 1100µg/ml, 1250µg/ml) against non- MDR E. coli, P. aeruginosa and S. aureus, respectively . Moreover, Ethyl gallate showed antibacterial activity against MDR E. coli and E. coli ATCC 25922 (MIC = 500µg/ml). Another study supports the findings of our research work .
It was reported that the antimicrobial activity of alkyl esters of GA depends on specific lipophilic characteristics, assuming that cytoplasmic membrane could be a possible site of action. There are various modes of actions regarding the antimicrobial activities of phenolic compounds including interaction with including sulfhydryl group, change in permeability of cytoplasmic membrane, inactivation of enzymes, insoluble complexes with amino acids and proteins. One more study explained the antibacterial activity of Ethyl gallate against P. aeruginosa ATCC 27853 . However, Dodecyl gallate and Propyl gallate did not show antimicrobial activity against MDR bacteria in the present study but another study reported good antimicrobial activity of both compounds against MRSA . Propyl gallate performed antibacterial activity against non-MDR E. coli, P. aeruginosa and S. typhi . Gram-negative bacteria have specialized cell wall structure, therefore, they are difficult to kill as compared to gram-positive bacteria. The diffusion of antibiotic and active compounds is obstructed due to the presence of periplasmic space and murein layer. Mutation in active efflux pumps out anti-bacterial agent through the efflux pumps, which further promotes the development of intrinsic resistance for gram-negative bacteria . Chemical structure including saturated chain length position and number in the benzene ring of phenolic compounds plays a key role in determining their anti-microbial activity. Phenolic acids had lower antimicrobial activity compared with their butyl and methyl ester . The antimicrobial effect increased with increasing length of the alkyl chain .
Hydroxybenzoic and hydroxycinnamic acids occurring in plants exhibit diversity with respect to the number of hydroxyl or methoxy groups. Alkyl gallates disrupt the respiratory mechanism of bacteria by acting as pro-oxidants, which produce free radicles. These ROS cause oxidation of unsaturated fatty acids in the cell membrane, leading to disruption of its structure and function . However, current knowledge on structure–function relationships of the antimicrobial activity of phenolic acids does not account for this diversity of compounds. Para hydroxy benzoic acid was found to be a potent antimicrobial agent against MRSA but the present study exhibited no antimicrobial activity of Parahydroxy benzoic acid and its ethyl and methyl esters against MDR gram-positive and gram-negative bacteria . Methyl 4 hydroxy benzoic acid showed weak antimicrobial activity against non-MDR P. aeruginosa, E. coli and S. aureus . Similarly, Vanillic acid exhibited no antimicrobial activity in the present study. Earlier reports displayed antimicrobial activity against MRSA, MDR E. coli and P. mirabilus. Antimicrobial activity of Trimethyl gallic acid against non-MDR gram-positive and gram-negative bacteria was reported (350). The previous study explained antimicrobial activity of Syringic acid against MRSA, MDR E. coli and P. mirabilus, which supports the results of the present study .
The absence of antibacterial activity does not mean that there is no potential of inhibiting microbes. It may be possible that the active compound was not in sufficient quantity or diluted with other non-antimicrobial compounds. Sometimes seasonal variations, geographical area, age of plant and method of extraction also influence the activity of the compound . Extraction techniques are usually based on the chemical nature of compounds and their solubility in polar and non-polar solvents .
The approach of using antimicrobial substances that suppress the mechanism of antimicrobial resistance, hence increasing the efficiency of antimicrobial product, is known as synergism . Synergistic combinations of different drugs or with herbal formulations play an important role in treating antibiotic resistance. Alkyl gallates are documented to possess resistance modifying action on various antibiotics against MRSA . Therefore, we used different combinations of antibiotics with Methyl gallate. Our study exhibited strong synergistic combinations of Ciprofloxacin and Amoxycillin with Methyl gallate against MDR E. coli and MRSA, respectively. An earlier study explained synergistic combination of Ciprofloxacin and Methyl gallate against non-MDR S. typhi . There are different mechanisms involved in the augmentation of the antibacterial activity of antibiotics by natural compounds such as interaction on multiple target sites in bacteria; bioavailability and solubility of antibiotics or aimed for a specific bacterial resistance mechanism . These interactions prevent the activity of drugs from enzyme degradation and alterations in transport proteins, thus bypassing the mechanism of antimicrobial resistance . Amoxycillin is a β-lactam antibiotic that inhibits bacterial cell wall synthesis by binding one or more of the penicillin-binding proteins (PBPs). Stimulation of mecA gene and gene variants resulted in the formation of PBP2a, which attaches to lower affinity with antibiotics and leads to drug resistance . We assume that methyl gallate interferes with the synthesis of PBP2a in cell wall of bacteria. This could be the explanation of synergistic combinations in the present study.