Antibacterial effects and action modes of asiatic acid

In this study, the antibacterial effects and action modes of asiatic acid against the foodborne bacterial pathogens Escherichia coli O157:H7, Salmonella Typhimurium DT104, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus were examined. Minimal inhibitory concentrations (MICs) of asiatic acid against these bacteria were in the range of 20-40 μg/mL. Minimum bactericidal concentrations of asiatic acid were in the range of 32-52 μg/mL. Asiatic acid at 2X MIC effectively reduced bacterial numbers from 6 log10 to < 2 log10 in all test bacteria within 6 h (P < 0.05). The antibacterial activity of asiatic acid was not affected by heat treatments from 25 to 100°C. Asiatic acid at 1 or 2X MICs caused 40-56% and 71-89% membrane damage in test bacteria within 2 h, respectively In addition, asiatic acid at 1 or 2X MICs led to 1.5-2.4 ppm and 2.9-4.1 ppm K+ release within 2 hr, respectively. Asiatic acid treatments dose-dependently increased bacterial nucleotide leakage (P < 0.05). After 3 days of storage at 25°C, the addition of asiatic acid dose-dependently inhibited the growth of test bacteria in ground beef (P < 0.05), in which 8 mg asiatic acid treatments led to bacterial levels (log CFU/g) in said ground beef lower than 2. These findings suggest that asiatic acid might be a potent antibacterial agent to prevent food contamination.

Bacterial cell membrane integrity and permeability play crucial roles for bacterial survival and growth. Thus, any agent with the ability to destroy bacterial cell membrane integrity and/or disrupt membrane permeability may cause bacterial cell damage, and even death. In addition, the rupture of bacterial cytoplasmic membrane promotes the release of intracellular components such as potassium ions and nucleotides, which in turn diminishes bacterial ability to repair and replicate [16,17]. So far, the assays regarding the variation in bacterial membrane integrity, K + efflux, and nucleotides release have been widely used to examine the antibacterial actions of some select agents [18][19][20]. Thus, if asiatic acid could cause bacterial membrane damage and/or enhance the release of potassium ions and nucleotides, its antibacterial action could be explained.
The major purpose of this study was to investigate the inhibitory effects of asiatic acid against seven foodborne bacterial pathogens. The influence of this compound upon the membrane damage, potassium ions and nucleotides loss in these bacteria was also evaluated. Ground beef was used as a food model to examine the antibacterial effects of asiatic acid at various doses.

Materials
Asiatic acid (98%) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Asiatic acid, based on its hydrophobic characteristic, was first dissolved in dimethyl sulphoxide (DMSO, 20 mg/ml), and then used for other preparations. The final concentration of DMSO in the culture medium was maintained at 0.5% (v/v). The impact of DMSO upon the growth of test bacteria was not significant (data not shown).

Test organisms
Three Gram-negative bacteria, E. coli O157:H7, S. Typhimurium DT104, P. aeruginosa, and 4 Gram-positive bacteria, L. monocytogenes, S. aureus, E. faecalis and B. cereus were recovered from contaminated chicken, duck, and dairy products, as well as seafood from May, 2012 to August, 2013 by using a surface swab technique. Swab samples were directly streaked onto a Chromocult Coliform agar plate, a Brilliant Green agar plate, a Cetrimide agar plate, a Baird Parker agar plate, a Listeria selective agar plate, a brain heart infusion agar plate or B. cereus selective agar plate for E. coli O157:H7, S. Typhimurium DT104, P. aeruginosa, S. aureus, L. monocytogenes, E. faecalis or B. cereus enumeration, respectively. These selective agars were purchased from Oxoid Ltd. (Basingstoke, UK). After sample streaking, agar plates were incubated for 24 h at 37°C. One isolated colony from a contaminated food was defined as 1 isolate. In this study, 16 isolates from 16 different contaminated foods for each test bacterial strain were used for experiments.

MIC and minimum bactericidal concentration (MBC) determination
MICs of asiatic acid against test bacteria were determined according to the Clinical and Laboratory Standards Institute guideline [21]. Each bacterial strain culture at 0.1 ml, containing 10 6 CFU/ ml as determined by plates count, was inoculated into a 9.9 ml Mueller Hinton (MH) broth (Difco, MI, USA) supplemented with asiatic acid at concentrations ranging from 2 to 512 μg/ml in tubes. All tubes were then incubated at 35°C for 24 h in an incubator (Model LE-30D, Yih Der Co., Taipei, Taiwan). MIC was recorded as the lowest concentration of asiatic acid to inhibit visible growth of test bacteria, which was reflected by no variation in turbidity. Turbidity was assayed by an optical density (OD) measurement at 600 nm with a UV spectrophotometer (Model UV-1800, Shimadzu Co., Tokyo, Japan). By sub-culturing from the MIC assay tubes onto MH agar plates and incubating at 35°C for another 24 h, MBC was the lowest concentration of asiatic acid to inhibit visible growth on agar plates. All experiments were performed in triplicate.

Time-kill study assay
In vitro time-kill of asiatic acid at 0.5, 1 and 2X MICs (Table 1) against test bacterial strains was monitored in a 10 ml MH broth at 35°C, after inoculation with culture at 10 6 CFU/ml. At 0, 3, 6, 9 and 12 h, bacterial suspensions at 100 µl were cultured on MH plates for determination of CFU/ml. The plates were incubated at 35°C for 24 h, and the colonies were counted. The detection limit was 20 CFU/ml.

Heat treatment
A 10 ml beaker with a solution of asiatic acid (20 mg/ml) was sealed with parafilm and placed in a water bath incubator (Model BH-230D, Yih Der Co., Taipei, Taiwan). The temperature of the beakers was maintained at 25, 50, 75 or 100°C for 60 min in this incubator. After cooling down to room temperature, the inhibitory zone of these solutions against the test bacteria was determined.

Inhibitory zone measurement
The inhibitory zone was determined and compared by disc diffusion method. A sterile blank disc (6 mm diameter, Difco, MI, USA) was soaked in asiatic acid solution for 30 min, and then placed on the surface of a MH agar plate previously seeded with a 100 µl bacterial suspension containing 10 6 CFU/ml test bacteria. The inhibitory zone was measured after 24 h incubation at 35°C. Data are expressed as mean ± SD (n = 16).

Bacterial membrane damage
A LIVE/DEAD BacLight kit containing SYTO-9 and propidium iodide dyes purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA) was used to measure bacterial membrane damage. Briefly, bacteria were grown in MH broth to an OD 600 of 0.3, which was equal to 10 7 CFU/ml determined by plates count, and followed by asiatic acid treatments at 0.5, 1 or 2X MIC for 2 h at 37°C. After centrifugation at 10, 000 × g for 15 min, the pellet was collected and resuspended in a buffer containing 5 µM SYTO-9 and 30 µM propidium iodide in the dark for 15 min at room temperature. Green fluorescence, which reflected intact cell membranes, was read at 530 nm; and red fluorescence, which reflected damaged membranes, was read at 645 nm with an excitation wavelength at 485 nm. The ratio of green to red fluorescence intensities determined by a fluorescence microplate reader (Model MTP-601Lab, Hitachi High Technologies, Tokyo, Japan) was normalized to the bacterial sample without asiatic acid treatment.

Intracellular K + concentration
The K + concentration (ppm) released from the test bacteria was measured by a flame atomic absorption spectrometry (Model 5000, Perkin Elmer Inc., Norwark, CT, USA), according to the method of Li et al. [22]. Briefly, bacteria were grown in an MH broth to an OD 600 of 0.3 (10 7 CFU/ml). One mL of bacterial suspension was treated with asiatic acid at 0, 0.5, 1 or 2X MIC. After 2 h incubation at 37°C, and centrifugation at 10, 000 × g for 15 min, supernatants were collected, and the K + concentration was measured.

Nucleotide leakage
The release of nucleotides was determined by a spectrophotometer (Model U-2000, Hitachi High Technologies, Tokyo, Japan) according to the method of Lou et al. [23]. Briefly, bacteria were grown in an MH broth to an OD 600 of 0.3 (10 7 CFU/ml). Asiatic acid at 0.5, 1 or 2X MIC was added into a 1 ml bacterial suspension, and followed by incubating for 2 h at 37°C. After centrifugation at 10,000 × g for 10 min, supernatants were collected, and the absorbance at 260 nm was measured.

Ground beef preparation
Beef semimembranosus muscle (top round) purchased locally was trimmed of all visible extramuscular fat. The beef muscle was then ground via a 4.5 m/m head on a grinder (Model TS-285, Ta-sin Ltd., Taichung City, Taiwan), and divided into several portions for the following experiments.

Antibacterial assay in ground beef
Asiatic acid at 0, 2, 4 or 8 mg was mixed with 100 g ground beef.
One ml of each test bacterial culture at 10 4 CFU/ml was added into the 100 g ground beef previously treated with asiatic acid. The inoculated ground beef was then mixed at low speed in a meat mixer (Model TS-383, Ta-sin Ltd., Taichung City, Taiwan) to assure uniform distribution of inoculum. Uninoculated beef samples were also used as negative controls. After 3 days storage at 25°C, 20 g of ground beef was homogenized with 100 ml of deionized water in a Waring blender (Model 31BL91, Sunwei Ltd., Taichung City, Taiwan) at high speed. Then, 1 ml beef ho-mogenate was serially diluted with 9 ml of 0.5% peptone water, and a 0.1 ml portion of each dilution was spread on selective agar plates for enumeration. Plates were incubated at 35°C for 24 h, and colonies were counted and reported as a log of CFU/g ground beef.

Statistical analysis
All data were expressed as mean ± SD (n = 16). Differences among means were determined by the Least Significance Difference Test with significance defined at P < 0.05.

Results
The MICs and MBCs of asiatic acid against the test bacteria are presented in Table 1. MICs were in the range of 20-40 µg/ml; MBCs were in the range of 32-52 µg/ml. The time-kill curves of asiatic acid at 0.5, 1 or 2X MICs against test bacteria strains are presented in Figure 2. After 6 h incubation, the bactericidal effects of asiatic acid upon test strains increased with increasing asiatic acid concentrations from 0.5 to 2 X MIC (P < 0.05). Asiatic acid at 2X MIC effectively reduced bacteria numbers from 6 log 10 to < 2 log 10 in all test bacteria within 6 h (P < 0.05).
The influence of heat treatments upon the antibacterial effect of asiatic acid, determined by the inhibitory zone, is shown in Table  2. Compared with the 25°C asiatic acid treatment, the 50, 75, or 100°C heat treatments did not significantly affect the antibacterial activity of asiatic acid (P > 0.05). As shown in Figure 3, asiatic acid dose-dependently impaired membrane integrity (P < 0.05). Asiatic acid at 1 or 2X MICs led to 40-56% and 71-89% membrane damage, respectively, in test bacteria. Asiatic acid treatments also dose-dependently increased bacterial intracellular K + concentrations in test bacteria ( Figure  4, P < 0.05). Asiatic acid at 1 or 2X MICs led to 1.5-2.4 ppm and 2.9-4.1 ppm K + release within 2 h, respectively. As shown in Figure 5, asiatic acid treatments caused bacterial nucleotide leakage (P < 0.05). Asiatic acid at 1X MIC increased 1-2.2 folds nucleotide leakage; and at 2X MIC increased 2.7-4.3 folds nucleotide leakage in test bacteria within 2 h.
The antibacterial effects of asiatic acid in ground beef are presented in Table 3. After 3 days of storage at 25°C, the addition of asiatic acid dose-dependently inhibited the growth of test bacteria in ground beef (P < 0.05), in which 8 mg of asiatic acid treatments led to bacterial levels in ground beef lower than 2 log 10 .

Discussion
The antibacterial activity of asiatic acid against E. coli, B. subtilis and S. sonnei has been reported before [13]. The results of our present study extend the inhibitory effects of this agent toward other Gram-negative and Gram-positive foodborne bacterial pathogens including S. Typhimurium DT104, P. aeruginosa, L. monocytogenes, S. aureus, E. faecalis, and B. cereus. The MICs of this agent against those bacteria were ≤ 40 µg/ml. Furthermore, we used ground beef as a food model to evaluate the antibacterial potency of asiatic acid. The results reveal that the addition of asiatic acid markedly inhibits bacterial growth in ground beef. These findings indicate that asiatic acid is an effective wide spectrum antibacterial agent against seven foodborne bacterial pathogens in media and ground beef. Thus, this agent could be      considered as a potent additive in foods to prevent bacterial contamination. In addition, we found that heat treatments of up to 100°C did not affect the inhibitory effects of asiatic acid against test bacteria. This heat-resistant property benefits its application for foods requiring a heating process of 100°C or lower. That is, this compound is applicable for raw and cold foods, as well as food products treated with high temperature, short time pasteurization. Bacterial membrane integrity is important not only for bacteria's self-protection but also for the functions of membrane-associated enzymes responsible for energy generation, respiration, and redox balance [24,25]. Thus, bacterial membrane rupture easily impairs these critical functions, which in turn affects the bacteria's survival and growth. In our present study, asiatic acid destroyed the membrane integrity of test bacteria, which subsequently interfered with these above functions, and caused bacterial apoptosis. Potassium ions are the most abundant cations in bacteria such as E. coli, and their homeostasis is regulated by K + transporters such as Kdp [26]. These cations are involved in many aspects of bacterial physiological actions including growth, survival, and virulence [27]. Thus, the loss of K + as we observed was definitely detrimental upon the bacteria's growth and survival. We found that asiatic acid treatments increased K + release from cytoplasm and/or mitochondria in test bacteria. These results indicate that asiatic acid induces irreversible damage of the cytoplasmic membranes, and disturbed K + homeostasis in test bacteria. It is well known that nucleotides such as DNA and RNA are crucial factors responsible for cell repair and replication [28]. The release of nucleotides and their derived compounds including DNA and RNA from bacteria can be quantified by monitoring their absorbance at 260 nm because these substances possess strong UV absorption at this wavelength [29,30]. In our present study, asiatic acid treatments effectively promoted nucleotides release from the intracellular compartments of test bacteria, which was reflected in their increased absorbance at 260 nm. Since nucleotides were released, the observed death in asiatic acid treated bacteria can be explained. These findings imply that asiatic acid is able penetrate into bacteria, cause damage in DNA-containing organelles like  mitochondria or nuclei, which in turn alters nuclear stability. Our above findings indicate that asiatic acid exerts its antibacterial actions through causing membrane damage, and increasing K + and nucleotides release in test bacteria. Asiatic acid is a triterpenoid naturally occurring in many edible plant foods [31]. Based on its natural, tasteless, and odorless properties, this agent might be safe and not affect food flavor. In our present study, 8 mg of asiatic acid in 100 g ground beef was equal to 8 ppm, and exhibited markedly anti-bacterial effects. This dosage is not considered high. Thus, the application of asiatic acid to prevent bacterial contamination in foods seems feasible. Our results support the idea that this agent could be applied to foods against bacterial contamination; or used as a bactericide in farms and/or slaughter houses to enhance environmental sanitation. However, further study regarding its safety and possible side effects is necessary before it is used for food preservation. In addition, several animal studies have reported that the dietary intake of asiatic acid could provide anti-diabetic, anti-hyperlipidemic, and hepatic protection via its anti-oxidative activity [32]. It is highly possible that using this triterpene as an antioxidant also improves foods' oxidative stability and benefits food preservation.
In conclusion, asiatic acid dose-dependently inhibited the growth of E. coli O157:H7, S. Typhimurium DT104, P. aeruginosa, L. monocytogenes, S. aureus, E. faecalis, and B. cereus in medium and in ground beef. Asiatic acid could impair membrane integrity, increase the release of potassium ions and nucleotides in test bacteria. These findings support the contention that asiatic acid is a potent agent to prevent foods from being contaminated by the aforementioned bacteria.