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Foodborne pathogens are a major public health concern in both developed and developing countries, and account for considerably high cases of human and animal diseases (Tayel and El-Tras 2010). The World Health Organization characterizes this issue as “one of the most prevalent health problems and a major cause of the reduction in economic output” (WHO 2008). Also, the increasing international trade in commodities and food products has raised the risk of spreading pathogenic bacteria from production sites to distant places. Furthermore, contamination with foodborne pathogens is a major problem in livestock production (Sittiwet and Puangpronpitag 2009). This has resulted on extensive use of chemical preservatives to prevent growth of foodborne pathogens in food industry (Natta et al. 2008). However, there has been an increasing consumer demand for foods free from toxic effects of added synthetic preservatives because some chemical preservatives have been associated with gastrointestinal disorders, hypersensitivity, allergic reaction, immunity suppression, and carcinogenic and teratogenic attributes (Pundir et al. 2010). In addition, bacterial resistance to currently used antibiotics has also led to the development of new and safer antimicrobial agents from plant origin to combat against various infectious diseases (Cock 2008).
Moreover, greater consumer awareness regarding the use of synthetic preservatives can lead to negative health consequences, which in turn has encouraged food processors to look for natural, effective and nontoxic food additives for using in food system with a broad spectrum of antimicrobial activity (Burt 2004). Food safety is an essential concern of both consumers and the food industry, especially as the number of reported cases of foodborne infections continues to increase (Alzoreky and Nakahara 2003). Plant-based essential oils are gaining vital importance as potential food preservatives and are considered generally recognized as safe by the United States Food and Drug Administration. Moreover plant-based essential oils showed a wide acceptance from consumers (Burt 2004). The antimicrobial components are commonly found in the essential oil fractions and it is well established that many have a wide spectrum of antimicrobial activity, with the potential for control of foodborne pathogens within food systems (Burt 2004).
Essential oils are natural volatile organic compounds that can be obtained by distillation, enfleurage, expression or solvent extraction, but the method of microwave-assisted distillation is the most commonly used for commercial production (Bousbia et al. 2009). Essential oils have been shown to possess antibacterial, antiviral, antifungal, insecticidal, repellant, anti-inflammatory, spasmolytic and antioxidant properties (Burt 2004; Cakir et al. 2004). Moreover, use of essential oils in consumer goods is expected to increase in the future because of the risk of “green consumerism,” which stimulates the use and development of plant-derived products (De Silva 1996; Burt 2004), as both consumers and regulatory agencies are more comfortable with the use of natural antimicrobials.
Taxus cuspidata Sieb et Zucc. (Japanese yew or spreading yew) is a member of the genus Taxus, native to Japan, Korea, northeast China and Russia, which is a broadly columnar, evergreen shrub with linear, spiny-tipped, dark green leaves. T. cuspidata has been used in traditional medicine in the treatment of inflammation, renal disorders, cancer and diabetes (Wang et al. 2007). Recently, T. cuspidata has been found to possess antidiabetic activity in streptozotocin-induced diabetic mice (Zhang et al. 2012). Previously, an anticancer compound, paclitaxel, isolated from T. cuspidata stem has also confirmed its role on microtubule prevention from disintegration (Schiff et al. 1979; Wang et al. 2007). However, various potentially toxic chemicals containing cardiotoxic taxine alkaloids present in Taxus species are of great concern (Wilson et al. 2001), and more than 150 taxanes and other compounds have been isolated and characterized from T. cuspidata (Wang et al. 2010).
Although antimicrobial efficacy of various essential oils has been reviewed previously, to the best of our knowledge, no systematic research on the chemical composition and mode of antimicrobial action of microwave-assisted extracted T. cuspidata leaf essential oil (TCEO) had been conducted so far against a wide range of foodborne microorganisms. Therefore, this study was undertaken in order to investigate the effectiveness of TCEO on survival and growth of selected foodborne pathogens using in vitro models. Furthermore, antibacterial mechanism of action was investigated by determining the release of extracellular adenosine triphosphate (ATP), potassium ions and cellular materials, and morphological alterations were investigated by scanning electron microscopy (SEM).
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In this study, the results of the antibacterial screening including the results from disc diffusion assay and MIC and MBC values illustrated that TCEO had strong and consistent inhibitory effect against some representative foodborne pathogens as confirmed by its inhibitory effect showing different susceptibility rate against the tested foodborne pathogens. In recent years, several researchers have reported that monoterpene or sesquiterpene hydrocarbons and their oxygenated derivatives, which are the major components of essential oils, exhibit potential antimicrobial activity (Burt 2004; Cakir et al. 2004; Bajpai et al. 2008). These findings strongly support the outcomes of this study as the TCEO was also found to contain oxygenated sesquiterpenes and their respective hydrocarbons, confirming its efficacy as natural antimicrobial agent.
In addition, the results from cell viability assay revealed that exposure of TCEO had a rigorous effect on the cell viability of the investigated foodborne pathogens. The TCEO exerted its maximum bactericidal activity as evident by the significant reduction in microbial counts and complete inhibition of B. cereus ATCC 13061 and E. coli ATCC 43889 cells at the exposure of 160 min for both the tested pathogens. Previously, we have confirmed the inhibitory effects of various plant-based essential oils on the cell viability of various foodborne and food spoilage pathogens (Bajpai et al. 2008, 2009, 2012).
The SEM generated photomicrographs of the investigated pathogens showed changes in cell morphology and topography. The distortion of the cell physical structure may cause the expansion and destabilization of the membrane, hence can increase membrane fluidity, which in turn can increase passive permeability and manifest itself as a leakage of various vital intracellular constituents, such as ions, ATP, nucleic acids and amino acids (Helander et al. 1998; Cox et al. 2001). Changes in membrane fluidity usually occur due to alterations in membrane lipid composition (Sikkema et al. 1995) and are considered to be a compensatory mechanism to counter the lipid disordering effects of the treatment agent. These morphological alterations in bacterial cells could be associated with damage in the cell wall and cell membrane of the investigated foodborne pathogens treated with TCEO, leading to disruption and formation of lysed cell. Previously, such morphological alterations have been reported for foodborne pathogens treated with plant-based essential oils (Bajpai et al. 2009).
The production of ATP in prokaryotes occurs both in the cell wall and in the cytosol by glycolysis (Helander et al. 1998). Hence, it is expected that alterations on intracellular and external ATP balance can be affected due to the action of the essential oils on the cell membrane. Conversely, ATP being a principal energy carrier is used for many cell functions including transport work moving substances across cell membranes, which might be a potential target parameter to understand the mechanism of action of antimicrobial agents. The results of our study showed an increasing rate of extracellular ATP concentrations after B. cereus ATCC 13061 and E. coli ATCC 43889 cells exposed to TCEO at MIC. This might occur due to significant impairment in membrane permeability of the tested bacteria by TCEO, which caused the intracellular ATP leakage through defective cell membrane. Furthermore, reduction in intracellular ATP may also occur due to decreased rate of ATP synthesis and increased rate of ATP hydrolysis inside the cells. Previously, similar findings on this phenomenon have also been reported for various antibacterial agents (Herranz et al. 2001). Burt (2004) also reported that exposure of B. cereus cells to oxygenated monoterpenes resulted in decreased level of intracellular ATP while disproportionately increased the level of extracellular ATP. Similarly, Helander et al. (1998) found that B. subtilis ATCC 6633 cells treated with essential oil components resulted in decreased level of intracellular ATP pool and increased levels of extracellular ATP pool.
Another apparent mechanism of antimicrobial action of TCEO was visualized by the confirmation on the release of 260 nm absorbing materials when the investigated foodborne pathogens were exposed to TCEO at the MIC. The macromolecules of a bacterial cell including DNA and RNA (nucleic acids), which reside throughout the interior of the cell, in the cytosol, are the key structural components. The transfer of cellular information through the processes of translation, transcription and DNA replication occur within the same compartment and can interact with other cytosolic structures. Measurement of specific cell leakage markers such as 260 nm absorbing materials is an indicative of membrane sensitivity to specific antimicrobial agent in relationship to unexposed cells. In this study, exposure of B. cereus ATCC 13061 and E. coli ATCC 43889 to TCEO caused rapid loss of 260 nm absorbing materials from the treated bacterial cells. Previously, similar findings on the leakage of 260 nm absorbing materials have been reported against foodborne pathogens treated with other plant-based essential oil (De Souza et al. 2010).
The plasma membrane is the target of many antimicrobial agents including plant-based essential oils (Bajpai et al. 2012). When bacterial membranes become compromised, small molecules are left out. Potassium ions are the major intracellular cations in bacteria that act as cytosolic-signaling molecules, activating and/or inducing enzymes, and transport systems that allow the cell to adapt to elevated osmolarity. Furthermore, the mechanism of antimicrobial action of TCEO was confirmed on the basis of K+ ions efflux from B. cereus ATCC 13061 and E. coli ATCC 43889 cells when exposed to TCEO at MIC. The bacterial cytosolic membrane provides a permeability barrier to the passage of small ions such as K+ ions and allows cells to control the entry and exit of different compounds. This impermeability to small ions is maintained and even regulated by the structural and chemical composition of the membrane itself. Increases in the efflux of K+ will indicate a disruption of this permeability barrier. Maintaining ion homeostasis is integral to the maintenance of the energy status of the cell in addition to other membrane-coupled energy-transducing processes, such as solute transport, regulation of metabolism, control of turgor pressure, motility and maintains proper enzyme activity (Cox et al. 2001). Therefore, even relatively slight changes to the structural integrity of cell membranes can detrimentally affect cell metabolism and lead to cell death (Cox et al. 2001). This suggests that, in the case of B. cereus ATCC 13061 and E. coli ATCC 43889 cells, monitoring K+ efflux and release of 260 nm absorbing materials may be more sensitive indicators of membrane damage (Cox et al. 2001).
These results suggest that storage of the TCEO components in the plasma membrane causes instant loss of their integrity and become increasingly more permeable to essential cell constituents and ions that might be responsible for setting up an antibacterial activity. Severe leakage of cytosolic materials is used as an indication of gross and irreversible damage to the cell membrane (Cox et al. 1998). From the results that the amount ratio of loss of 260 nm absorbing materials was as extensive as the leakage of K+ ions, this may indicate that the membrane structural damage sustained by B. cereus ATCC 13061 and E. coli ATCC 43889 cells resulted in release of macromolecular cytosolic constituents (Cox et al. 2001). Similarly, the effect of essential oil components such as carvacrol on proton motive force of bacteria has strongly been correlated to leakage of various substances such as ions, ATP, nucleic acids and amino acids (Helander et al. 1998).