Many studies have evaluated the antimicrobial activity of natural products against various microorganisms, but to our knowledge there have been no studies of the possible use of natural products for their antimicrobial activity against Enterobacter hormaechei. In this study, we investigated vanillic acid (VA) for its antimicrobial activities and its modes of action against carbapenem-resistant E. hormaechei (CREH). The MIC of VA against CREH was determined by the agar diffusion method. The antibacterial action of VA against CREH was elucidated by measuring variations in intracellular ATP concentration, intracellular pH, membrane potential, and cell morphology. Moreover, the efficacy of VA against biofilm formation and VA damage to CREH cells embedded in biofilms were further explored. Our results show that VA was effective against CREH with a MIC of 0.8 mg/mL. VA could rupture the cell membrane integrity of CREH, as measured by a decrease of intracellular ATP, pH, and membrane potential, along with distinctive alternations in cell morphology. In addition, VA exerted a remarkable inhibitory effect on the biofilm formation of CREH and also killed CREH cells within biofilms. These findings show that VA has a potent antibacterial and antibiofilm activity against CREH and, hence, has the potential to be used clinically as a novel candidate agent to treat CREH infections and in the food industry as a food preservative and surface disinfectant.
HIGHLIGHTS
Vanillic acid (VA) is a flavoring agent found in edible plants and fruits. Few recent studies exhibited robust antibacterial activity of VA against several pathogen microorganisms. However, little was reported about the effect of VA on carbapenem-resistant Enterobacter cloacae (CREC). The purpose of the current study was to assess in vitro antimicrobial and antibiofilm activities of VA against CREC. Here, minimum inhibitory concentrations (MIC) of VA against CREC was determined via gradient diffusion method. Furthermore, the antibacterial mode of VA against CREC was elucidated by measuring changes in intracellular adenosine triphosphate (ATP) concentration, intracellular pH (pHin), cell membrane potential and membrane integrity. In addition, antibiofilm formation of VA was measured by crystal violet assay and visualized with field emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM). The results showed that MIC of VA against E. cloacae was 600 μg/mL. VA was capable of inhibiting the growth of CREC and destroying the cell membrane integrity of CREC, as confirmed by the decrease of intracellular ATP concentration, pHin and membrane potential as well as distinctive variation in cellular morphology. Moreover, crystal violet staining, FESEM and CLSM results indicated that VA displayed robust inhibitory effects on biofilm formation of CREC and inactivated biofilm-related CREC cells. These findings revealed that VA exhibits potent antibacterial activity against CREC, and thus has potential to be exploited as a natural preservative to control the CREC associated infections.
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