AN INVESTIGATION OF ESSENTIAL OILS AS ANTIMICROBIAL AGENTS AGAINST ANTIBIOTIC-RESISTANT BACTERIA ISOLATED AT SOUTH AFRICAN HOSPICES

Abstract

In developing countries including South Africa information is limited regarding the use of essential oils in treating antibiotic-resistant pathogenic bacteria. Currently, the battle between humans and the multitudes of infection and disease-causing pathogens continues. Emerging at the battlefield as some of the most significant challenges to human health is bacterial resistance to antibiotics and its rapid rise. This has become a major concern in global public health invigorating the need for new antimicrobial compounds. A rational approach to deal with antibiotic resistance problems requires detailed knowledge of the different biological and non-biological factors that affect the rate and extent of resistance development. Besides this approach, there are other approaches to resolving this challenge. For example, modulating the gut microbial community, either through feed additives (e.g. probiotics and prebiotics) or fecal transplantation could be a promising way to prevent certain diseases; non-antibiotic approaches include phage therapy, bacteriocins, and predatory bacteria which are effective against biofilms and can access recalcitrant infections. However, Allen and colleagues (2014) highlighted limitations of these antibiotic alternatives; these limitations include complex regulatory processes of the Food and Drug Administration (FDA) regarding feed additives, the high cost of vaccines as well as limited cross-protection of vaccines against some pathogens. The challenge with bacteriocins is possible sensitivity to proteolysis; phage therapy specificity begets technical limitation of administration against multiple subspecies while possible interactions of predatory with the host and their commensal microbiota are as yet unknown. Therefore, plants and their derivatives, such as essential oils, are currently blooming and represent a potential area for future investigations. This new generation of phytopharmaceuticals may shed light on the development of new pharmacological regimes in fighting antibiotic resistance. This study consolidates and describes the observed antagonistic outcome of essential oils against antibiotic-resistant S. aureus and B. cereus, and highlights the possibilities of essential oils as the potential antimicrobial agent. In the current study, the bacterial cell wall of antibiotic-resistant S. aureus and B. cereus was exposed as one of the targets for thyme essential oil. Thymus vulgaris essential oil showed high antimicrobial activity against the cell wall, cell membrane, and cytoplasm, and in some cases completely changed the morphology of the cells (antibiotic-resistant S. aureus and B. cereus). This indicates that the cell envelope became thinner than normal. In addition, using Gram staining, the current results clearly showed Gram-positive cells to be affected by a cell wall active agent (thyme oil) and stained pink like Gram-negative bacterial cells. This is possibly due to a decrease in peptidoglycan thickness or a disturbance in the cell wall during cell growth in the presence of thyme oil. These observations further indicate the stress placed on bacteria due to exposure to essential oils and this might have resulted in changes in morphology and the formation of Small Colony Variants (SCV) as observed on the agar plate. Small colony variants (SCV) arise within homogeneous microbial populations, largely in response to various environmental stresses, including essential oils. They display unique phenotypic characteristics conferred in part by possible heritable genetic changes. Characteristically slow growing, SCVs comprise a minor proportion of the population from which they arise but persist by virtue of their inherent resilience and host adaptability. To further confirm the influence of thyme oil on the bacterial cell wall of antibiotic-resistant strains of S. aureus and B. cereus, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed. Exposure to thyme oil induced alterations within the bacterial membrane of S. aureus as well as B. cereus, which resulted in a loss of cell wall integrity, as demonstrated by Gram staining, SEM, and TEM. In addition SEM and TEM micrographs showed loss of cellular contents and irregular cytoplasmic membrane. The intracellular leakage and morphological changes of the two treated bacterial cells indicated that Thymus vulgaris essential oil affected the structural organisation of the cytoplasm together with the cell wall of S. aureus and B. cereus cells. It might be proposed that in the primary phase, thyme oil probably binds to the bacterial cell surface and penetrates the cell wall causing cytoplasmic membrane damage and this leads to cell death. This phenomenon indicates that all these observed changes might be stress induced forming SCVs and that the cell wall is the first target of essential oils and this is also an indication that the tested oil indeed affected the structural organisation of both antibiotic-resistant organisms (S. aureus and B. cereus) since this was not observed in the untreated cells. It was evident in this study that the activity of thyme oil is not attributable to a single event but involves a series of events both on the cell surface and within the cytoplasm. The disruption of the cell wall and membrane integrity observed through SEM and TEM electron micrographs was also found to result in reduced saturated and unsaturated fatty acids as observed in the fatty acids profile assessment. Based on the results, it is apparent that thyme essential oil firstly acts on the cell wall and disrupts the outer membrane of antibiotic-resistant S. aureus and B. cereus which could lead to dispersion of the desaturase enzymes and allows them to act on the membrane fatty acids. In addition to direct effects on the fatty acids of the outer membrane, it is believed that thyme oil affected enzymes that are involved in fatty acid synthesis. The tested oil caused a major decrease in unsaturated fatty acids and this could be due to disrupted fatty acyl-CoA desaturase enzyme affected by the essential oil. However, further investigation needs to be done to certainly prove this hypothesis. Again, since lipids are the principal form of stored energy in S. aureus and B. cereus and are major constituents of cellular membranes. It is believed that once fatty acids were depleted due to damaged cell membrane as shown in SEM and TEM electron micrographs, the disrupted cell wall and membrane would inevitably lead to cell death. Therefore, it is speculated from the findings of this study that the membrane disruption effect of the essential oil, and the less amount of fatty acids is the consequence of the inhibited bacterial growth. Except for fatty acids, total proteins were also found to be affected by thyme oil. In the proteomic analysis, a total of three proteins from S. aureus and two proteins from B. cereus bacteria demonstrated reduced expression levels, upon treatment with Thymus vulgaris essential oil. The reduced protein expression of total proteins for both antibiotic-resistant S. aureus and B. cereus could be due to the depletion of fatty acids on the cell membrane. Since lipids serve as anchors for proteins, it is clear from the results that the disrupted fatty acids inevitably results in a damaged bacterial protein with reduced expression levels. Most importantly this study has revealed the importance of using essential oils as possible alternative antimicrobial agents. During microbial analysis, thyme essential oil damaged the cellular membrane of both antibiotic-resistant S. aureus and B. cereus, which led to cell death. Additionally, the depletion in the lipid profile and protein profile shown after the treatments of the resting cells is strictly related to the presence of thyme oil compounds. Therefore, this shows that Thymus vulgaris essential oil has the capability to target numerous bacterial sites (particularly the cell membrane, cytoplasm, lipids, enzymes, and proteins). In addition, from the findings of this study, some of the advantages of using natural antimicrobials such as thyme essential oils were further identified and those factors include: in the health sector the possibility of reducing total dependence on antibiotics and using with essential oils as alternatives can be seen; for the food industry potential use in controlling cross-contaminations by food-borne pathogens as well as improvising food preservation technology can be observed. Thyme essential oil is traditionally believed to be rich in phytochemicals showing rich bioactivity. Their compounds identified in this study could be of interest to the local industry as well as to the general population and could be actively explored for various commercial applications. However, more research will have to be conducted to assess the antimicrobial effects of thyme oil in food matrices since the current study exposed the effects of the tested oil on bacteria without interference from matrices such as food products. Thyme essential oil is therefore considered a potential natural antimicrobial agent. Based on these facts, the present study focused mainly on exploring the diverse mechanisms of action of Thymus vulgaris essential oil and its components against two specific antibiotic-resistant food-borne pathogens namely S. aureus and B. cereus. The scientific details provided in this study on these aspects are expected to be useful for the commercial exploitation of essential oils to develop natural preservative and disinfectant preparations with applicability in the food and pharmaceutical industries.