Antimicrobial Activity of Ferulic Acid Against Cronobacter sakazakii and Possible Mechanism of Action

Abstract

Cronobacter sakazakii is an opportunistic pathogen transmitted by food that affects mainly newborns, infants, and immune-compromised adults. In this study, the antibacterial activity of ferulic acid was tested against C. sakazakii strains. Minimum inhibitory concentration of ferulic acid against C. sakazakii strains was determined using the agar dilution method. Changes in intracellular pH, membrane potential and intracellular ATP concentration were measured to elucidate the possible antibacterial mechanism. Moreover, SYTO 9 nucleic acid staining was used to assess the effect of ferulic acid on bacterial membrane integrity. Cell morphology changes were observed under a field emission scanning electron microscope. The minimum inhibitory concentrations of ferulic acid against C. sakazakii strains ranged from 2.5 to 5.0 mg/mL. Addition of ferulic acid exerted an immediate and sustained inhibition of C. sakazakii proliferation. Ferulic acid affected the membrane integrity of C. sakazakii, as evidenced by intracellular ATP concentration decrease. Moreover, reduction of intracellular pH and cell membrane hyperpolarization were detected in C. sakazakii after exposure to ferulic acid. Reduction of green fluorescence indicated the injury of cell membrane. Electronic microscopy confirmed that cell membrane of C. sakazakii was damaged by ferulic acid. Our results demonstrate that ferulic acid has moderate antimicrobial activity against C. sakazakii. It exerts its antimicrobial action partly through causing cell membrane dysfunction and changes in cellular morphology. Considering its antimicrobial properties, together with its well-known nutritional functions, ferulic acid has potential to be developed as a supplement in infant formula or other foods to control C. sakazakii.

Introduction

C ronobacter sakazakii is a gram-negative, rod-shaped, non-sporeforming bacterium (Iversen et al., 2004), and it can grow over a wide temperature range (6–47°C) (Harouna et al., 2015). It has been implicated in severe forms of neonatal infections such as bacteraemia, necrotizing enterocolitis (NEC) and infant meningitis (Muytjens and Kollee, 1990) with reported case fatality rates of 50–80% (Healy et al., 2010). In addition to neonates, diseases caused by C. sakazakii infection have been reported for almost every age group. C. sakazakii has already been isolated from milk and fruit powders, cereals, tea leaves, fruits, vegetables, herbs, and spices (Friedemann, 2007). Some C. sakazakii strains are resistant to many antibiotics and a constant increase in antibiotic resistance is observed (Sosnowski et al., 2014). Recently, some authors have proposed several decontamination methods in an effort to reduce levels of C. sakazakii in powdered infant formula, including simultaneous near-infrared radiant heating and ultraviolet radiation (Ha and Kang, 2014), gamma radiation (Lee et al., 2006), electron beam irradiation (Hong et al., 2008), gaseous ozone treatment (Torlak and Sert, 2013), and microwave processing (Pina-Perez et al., 2014). However, these treatments may not kill all of the cells, and surviving cells may proliferate under proper conditions, especially after rehydration. Therefore, it is very important to search for an effective food supplement that controls C. sakazakii in infant formula or other types of food.

Several reports have described the potential use of natural antibacterial substances against C. sakazakii, including blueberry proanthocyanidins and commercial blueberry juice (Joshi et al., 2014), carvacrol and thymol (Lee and Jin, 2008), caprylic acid, citric acid, and vanillin (Choi et al., 2013), trans-cinnamaldehyde (Amalaradjou et al., 2009), water-soluble muscadine seed extracts (Kim et al., 2008), and red muscadine juice (Kim et al., 2010). In most reports, the antimicrobial effects of these plants substances were measured with a rehydrated infant milk formula model (Nair et al., 2004; Amalaradjou et al., 2009). In this method, compounds at different concentrations were added into powdered infant formula to measure the effect of plant compounds on the growth and inhibition of C. sakazakii at different temperatures. But the mechanism of these compounds' inhibitory action has rarely been examined except for trans-cinnamaldehyde (Amalaradjou and Venkitanarayanan, 2011).

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, C₁₀H₁₀O₄; FA) is a phenolic acid (Fig. 1) found in foods such as rice bran, green tea, and coffee beans (Zhao and Moghadasian, 2008; Takahashi et al., 2015) and commonly exists in the hemicelluloses of plant cell walls (Uraji et al., 2013). FA has been demonstrated to have several functions such as antioxidation, arteriosclerosis prevention, and anti-inflammation (Ou and Kwok, 2004). The antimicrobial properties of FA against foodborne pathogen have been recognized for a long time (Lo and Chung, 1999). A previous study demonstrated that FA exhibits a potent antibacterial effect against Gram-positive bacteria but has no effect against Gram-negative bacteria (Takahashi et al., 2013); however, Borges et.al reported that the Gram-positive bacteria were less susceptible to FA than Gram-negative bacteria (Borges et al., 2013). Campos et al. reported that FA affected the cell membrane of lactic acid bacteria, which caused ion leakages and proton influx (Campos et al., 2009). Ganan et al. have demonstrated that FA reduced the viability of Campylobacter jejuni at concentrations of 100 mg/L (Ganan et al., 2009). However, antibacterial activity of FA against C. sakazakii and its possible mechanism have not been reported.

FIG. 1.

Chemical structure of ferulic acid.

Therefore, we evaluated in this study the antimicrobial effects of FA against C. sakazakii strains by determining minimum inhibitory concentrations (MICs). In addition, the effects of ferulic acid on cell membranes of C. sakazakii were examined by measuring changes in the intracellular ATP concentrations, membrane potential, the intracellular pH (pH_in), membrane integrity, and cell morphology.

Materials and Methods

Reagents

Ferulic acid (CAS 1135-24-6) with HPLC purity of at least 98% was obtained from Chengdu Must Bio-technology Co., Ltd (Chengdu, Sichuan,China), and its solution was prepared in ultrapure sterilized water and sterilized by filtration immediately before use to minimize oxidation of the compound. All other chemicals were of analytical grade.

Bacterial strains and culture conditions

C. sakazakii strains ATCC 29544, ATCC 29004, ATCC 12868, and ATCC BAA-894 were purchased from American Type Culture Collection (ATCC, Manassas, VA). Five other C. sakazakii strains (Table 1), which were originally isolated from various infant formula and infant rice cereal in China, were taken from our laboratory strain collection and their antimicrobial susceptibilities were determined. All strains were stored in tryptic soy broth (TSB) with 20% glycerol (v/v) at −80°C. Before each experiment, stock cultures were streaked on tryptic soy agar at 37°C for 18 h. A loopful of each strain was inoculated into 30 mL of TSB and incubated for 18 h at 37°C.

Table 1.

Origin and Antibiotic Resistance Testing of the Five Food Isolate Strains

Strain	Origin	TET	SERE	GAT	CHL	CIP	LEVO	RIF	AMO	NAL	AMP	FOX
12-2	Infant rice cereal	1	2	16	8	0.004	0.032	8	2	2	1	4
14-15	Infant formula	16	64	4	64	0.004	0.032	32	32	64	32	32
18-7	Infant rice cereal	64	256	0.5	8	1	1	16	64	16	32	2
18-8	Infant formula	1	256	0.03	4	8	2	8	2	2	1	2
18-13	Infant formula	1	32	0.5	4	0.25	0.06	8	64	2	64	64

AMO, amoxicillin; AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; FOX, cefoxitin; GAT, gatifloxacin; LEVO, levofloxacin; NAL, nalidixic acid; RIF, rifampin; SERE, streptomycin; TET, tetracycline.

Minimum inhibitory concentration (MIC) determinations

MIC was determined by agar dilution method as described by the European Committee for Antimicrobial Susceptibility Testing (EUCAST, 2000) with some modifications. Escherichia coli ATCC 25922 was used as quality control. During determination of antimicrobial activity of FA, ampicillin was used as a positive reference standard for all test strains. The stock solution of the antibiotic was prepared in sterile water. Those solutions were sterilized through a 0.22 μm Acrodisc filter (Gelman, New York). C. sakazakii chromogenic medium (Beijing Land Bridge Technology Co., Ltd, Beijing, China) was aseptically transferred into sterile 24-well plates containing either FA or the antibiotic. The content (final volume 500 μL) of each well was gently mixed. The final concentrations of FA samples were 0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 mg/mL, whereas that of ampicillin was 100 mg/L. After hardening, the C. sakazakii chromogenic medium was spotted with 2 μL (approximately 10⁴ CFU) of the tested bacterium. The spots were left to dry and then plates were incubated at 37°C for 24 h. The lowest concentration of FA, which did not show any visible growth of test organisms after macroscopic evaluation, was determined as MIC, which was expressed in mg/mL.

Growth curves and kinetic parameters

The growth curves in TSB at 37°C were determined as previously described (Silva-Angulo et al., 2015). C. sakazakii strain ATCC 29544 was grown to an OD₆₀₀ value of 0.1 in TSB. Then 125 μL of the culture was transferred into each well on 96-well microtiter plates. FA was added to the cultures to obtain final concentrations of ¼ MIC, ½ MIC and MIC, and TSB was used as a negative control. Bacteria were further cultured at 37°C, and cell growth was monitored at 600 nm, using a multimode plate reader (Infinite^™ M200 PRO, Tecan, Männedorf, Switzerland).

The model used to fit growth curves to the data obtained comprised the modified Gompertz equation and the following formulas: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{\rm OD}_{\rm t} = A + (B - A) \exp \{ - \exp [ - \mu ( t - { \rm M} ) ] \} \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\lambda = M - ( 1 / \mu ) ,\ { \rm and}\ \mu_{ \max} = ( B - A ) \mu / { \rm e } \end{align*} \end{document}

where OD_t is the optical density at 600 nm (OD₆₀₀) at time t, t is the time (in hours) that has elapsed since incubation, B is the maximum OD₆₀₀, A is the initial OD₆₀₀, M is the time (in hours) of inflexion point in the exponential phase of model function, μ is the relative growth rate at time M, λ is the lag time (i.e., the time until the lag period ends), and μ_max is the maximum growth rate achieved (ΔOD₆₀₀ per hour). The goodness of fit was evaluated by using the coefficient of determination r ².

Measurement of intracellular ATP concentrations

With some modification, the method described by Sanchez et al. (2010) was followed. The overnight culture of C. sakazakii strain ATCC 29544 was centrifuged for 5 min at 5000 × g, and the supernatant was removed. The cell pellets were washed three times with 0.1 mol/L of sodium phosphate buffer (pH 7.0), then cells were collected by centrifugation under the same conditions. A cell suspension (OD₆₀₀ = 0.5, approximately 4 × 10⁸ CFU/mL) was recovered with 50 mL of sodium phosphate buffer and 2 mL of cell solution was taken into a 2.5 mL Eppendorf tube for treating with FA. The FA was then added to each tube resulting in final concentrations of 0 (control), MIC, and 2MIC, respectively. The samples were maintained at 37°C for 30 min.

To extract the ATP from cell suspensions, we applied ultrasound to lyse the cell samples on ice. The ultrasonic instrument model is SCIENTZ-IID (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China), and the ultrasonic instrument created a noncontinuous wave at a frequency of 20 kHz. The samples were exposed to ultrasound for 60 cycles of 3 sec ultrasound with 7 sec intervals (200 W). The Eppendorf tube containing 2 mL of cell solution added to FA was placed on ice and the ultrasonic probe inserted into the solution. When the sonication started, the probe transmits ultrasound waves to break up cells. Then the samples were centrifuged for 5 min at 5000 × g, the top layer was retrieved and stored on ice to prevent ATP loss until measurement. Intracellular ATP was measured by using an ATP assay kit (Beyotime Bioengineering Institute, Shanghai, China). After adding 125 μL of ATP assay mix to 125 μL of supernatant in white, opaque 96-well microtiter plates (Nunc, Copenhagen, Denmark), the supernatant ATP concentration, which represents the intracellular ATP concentration, was measured by applying a microplate reader (InfiniteTM M200 PRO; Tecan).

Intracellular pH_in measurements

Intracellular pH was determined according to a modified method of Breeuwer et al. (1996). To load a fluorescent probe in the sample cells, stock cultures were a loopful of each strain was streaked on tryptic soy agar and incubated at 37°C for 18 h. inoculated into 30 mL of TSB and incubated for 18 h at 37°C. Then, 250 μL of activated cultures of C. sakazakii strain ATCC 29544 was transferred into TSB (30 mL) and incubated at 37°C for 8 h (approximately 0.6 at 600 nm). Cells were then harvested by centrifugation (5000 × g, 10 min) and washed twice with 50 mM HEPES buffer (containing 5 mM EDTA, pH 8). The cell pellet was resuspended in 20 mL of this buffer. Then 3.0 μM of the probe, carboxyfluorescein diacetate succinimidyl ester (cFDA-SE; Molecular Probes, Sigma, St. Louis, MO), was added. Cells were then incubated for 20 min at 37°C, washed once in 50 mM potassium phosphate buffer with 10 mM MgCl₂ (pH 7.0), and resuspended in 10 mL of the same buffer. To eliminate nonconjugated cFDA-SE, glucose (10 mM, final concentration) was added and the cells were incubated for an additional 30 min at 37°C. Lastly, cells were washed twice, then resuspended in 50 mM phosphate buffer (pH 7), and stored on ice.

At three concentrations (0, MIC, and 2MIC), an aliquot (2 mL) of cell suspension labeled by fluorescence was dispensed into a 5 mL tube with FA, then was transferred into black opaque 96-well microtiter plates (Nunc). After treatment for 20 min, fluorescence intensities were measured under two excitation wavelengths (440 nm and 490 nm), rapidly alternating the monochromator between the wavelengths. The emission was collected at 520 nm, where excitation and emission slit widths were 9 nm and 20 nm, respectively. From the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm), we determined the pH_in of the bacteria. All of the measures were made by a microplate reader (Infinite^™ M200 PRO; Tecan) and performed in independent triplicate. During the assay, the system was maintained at 25°C. The fluorescence of the cell-free controls was measured and deducted from values for the treated suspension.

Calibration curves were determined for cFDA-SE loaded cells with different pHs buffers. Buffers consisted of glycine (50 mM), citric acid (50 mM), Na₂HPO₄•2H₂O (50mM), and KCl (50 mM). We adjusted pH with either NaOH or HCl to various pH values (3, 4, 5, 6, 7, 8, 9, and 10). After equilibrating the pH_in and pH_out by addition of valinomycin (10 μM) and nigericin (10 μM), the fluorescence intensity was measured at 25°C. In this assay, a drop in relative fluorescence occurs when the cytoplasmic pH decreases.

Membrane potential determinations

The method described by Sanchez et al. (2010) was followed with minor exceptions. Cells were grown in 30 mL of TSB broth at 37°C to an optical density at 600 nm of 0.5 (approximately 10⁸ CFU/mL), then harvested by centrifugation (5000 × g, 5 min) and washed twice with phosphate-buffered saline. Next, 125 μL of cell suspensions were placed in black, opaque 96-well microtiter plates (Nunc) for 30 min at 37°C. Then, 1 μM of the membrane potential-sensitive fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄ (3); Molecular Probes, Sigma) was added for 30 min at 37°C, followed by addition of FA at three concentrations (0, MIC, and 2MIC). After 5 min, fluorescence was measured at the excitation and emission wavelengths of 492 and 515 nm, using a fluorescence microplate reader (Infinite^™ M200 PRO; Tecan). The excitation and emission slit widths were 3 and 5 nm, respectively. Background fluorescence resulting from the medium was determined and the results corrected.

Assessing bacterial membrane integrity

The LIVE/DEAD^® BacLight^™ Bacterial Viability Kit (Molecular Probes, Eugene, OR) was used to detect the percent of the membrane damage quickly and quantitatively. It has been used for various bacterial species in pure culture. Also, it has been used for bacteria in various environments, such as seawater, drinking water, biofilms, and also different food products (Bunthof et al., 2001). SYTO^® 9 and propidium iodide (PI) are the components of the kit. When used respectively, SYTO 9 (the green-fluorescent nucleic acid) labels all the bacteria, by the contrast, PI (the red-fluorescent nucleic acid) stains bacteria with disrupted membranes. When the two components are added simultaneously, the green fluorescent will be decreased by the red fluorescent. Thus, bacteria with intact cell membranes stain fluorescent green, while bacteria with damaged membranes stain fluorescent red (Bunthof et al., 2001; Campos et al., 2009).

Cell membrane integrity was determined according to a modified method of Alakomi et al. (Alakomi et al., 2005). The overnight culture of C. sakazakii strain ATCC 29544 was harvested by centrifugation (10,000 × g, 15 min). Then, the supernatant was removed and the pellet was re-suspended in 2 mL of 0.85% NaCl. To obtain viable and nonviable cells, 1 mL of the suspension was added to each of two 50 mL centrifuge tubes containing either 20 mL of 0.85% NaCl (for live bacteria) or 20 mL of 70% isopropyl alcohol (for killed bacteria). After incubating both samples at room temperature for 1 hour (mixing every 15 min), both samples were pelleted by centrifugation twice at 10,000 × g for 10 minutes. The optical density was adjusted at 600 nm to 0.5, and viable and nonviable cell suspensions were mixed to achieve five viable cells' proportions (0, 10%, 50%, 90%, and 100%) to obtain standard samples. A 2× working stain solution was prepared by mixing equal volumes of SYTO 9 and propidium iodide (PI) and adding the mixture to 2 mL of filter-sterilized dH₂O.

Finally, suspensions treated with FA (0, MIC, and 2MIC at 37°C, 15 min) were quickly centrifuged (11,000 × g, 1 min), the supernatant was removed and the pellet was resuspended in the same volumes of 0.85% NaCl. Then 100 μL of standard samples and suspension were pipetted in three parallels into black, opaque 96-well microtiter plates (Nunc). Aliquots of 100 μL of the 2× staining solution were added to each well and mixed thoroughly. The plate was then incubated at 25°C in the dark for 15 min and the fluorescence of the bacterial suspensions measured with a fluorescence microplate reader (Infinite^™ M200 PRO; Tecan). The excitation/emission maxima for the dyes are 485/542 nm for SYTO 9 and 485/610 nm for PI. Suspension in the absence of FA was control.

Field emission scanning electron microscope (FESEM) analysis

FESEM assay was carried out following a previous report with some modification (Li et al., 2014). Cells (OD₆₀₀ = 0.5) were treated with FA at 0, MIC, 2MIC, and 4MIC. After incubating at 37°C for 2 h or 4 h, cells were harvested by centrifugation for 10 min at 5000 × g and washed twice with phosphate buffered saline, and then were re-suspended in water containing 2.5% glutaraldeyde and kept at 4°C for 12 h to fix the cells. After centrifuging, the cells were further dehydrated in water-alcohol solutions at various alcohol concentrations (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min each. Finally, the samples were fixed on FESEM support, and then sputter-coated with gold under vacuum, followed by examination under confocal laser scanning microscope (S-4800; Hitachi, Tokyo, Japan).

Statistical analysis

All experiments were performed in triplicate, and each replicate included two technical replicates. Statistical analyses were performed using SPSS software (version 19.0; SPSS, Inc., Chicago, IL). The data were presented as the mean values ± standard deviation (n = 3), and differences between means were tested by Student's t-test. Differences are considered significant at p ≤ 0.05.

Results

MIC of FA on C. sakazakii strains

FA showed inhibitory effects against nine tested C. sakazakii strains (Table 2). The MICs of FA against ATCC 29544, ATCC BAA-894, and isolates 18-7 were 2.5 mg/mL, and the MICs against other test strains were 5 mg/mL. C. sakazakii ATCC 29544 was selected for further studies.

Table 2.

Minimum Inhibitory Concentrations of Ferulic Acid Against Different Cronobacter sakazakii Strains

Strains	Origin	MIC (mg/mL)
ATCC 29544	Child's throat	2.5
ATCC BAA-894	Human clinical specimen	2.5
ATCC 29004	Infant formula	5
ATCC 12868	Infant formula	5
12-2	Infant rice cereal	5
14-15	Infant formula	5
18-7	Infant rice cereal	2.5
18-8	Infant formula	5
18-13	Infant formula	5

MIC, minimum inhibitory concentration.

Growth curves and kinetic parameters

C. sakazakii growth curve in TSB was fitted to the modified Gompertz equation (Fig. 2), the high r ² values (>0.996) confirmed the quality of the models. A comparison of the parameters obtained in this study demonstrated that FA exhibited antimicrobial activity against C. sakazakii and that the observed effects were dependent on the concentration of FA present in the culture medium (P ≤ 0.05). Higher concentrations of FA resulted in a longer lag phase and a lower specific growth rate (Table 3). Therefore, the observed bacteriostatic effect of FA was understood as an increased lag phase accompanied by a reduced growth rate.

FIG. 2.

Growth curves for Cronobacter sakazakii ATCC 29544 cultured in tryptic soy broth with various concentrations of ferulic acid. The lines represent the fit of the experimental data to the modified Gompertz model. The standard deviation associated with each average value is expressed with error bars. CK, control C. sakazakii culture without FA; MIC, minimum inhibitory concentration; OD₆₀₀, optical density/absorbance at 600 nm.

Table 3.

Kinetic Parameters of Cronobacter sakazakii Cells During Growth in Tryptic Soy Broth and the Concentration of Ferulic Acid

Concentration of FA	λ±SE	μ _max ± SE
MIC	2.018 ± 0.204^A	0.009 ± 0.002^E
1/2 MIC	1.468 ± 0.171^B	0.052 ± 0.006^F
1/4 MIC	1.245 ± 0.047^C	0.112 ± 0.003^G
0 (CK)	0.699 ± 0.152^D	0.156 ± 0.014^H

Mean values in the same column followed by different letters are statistically different (p < 0.05) (n = 6).

λ, lag phase (in hours); μ_max, maximum growth rate (in OD per hour); FA, ferulic acid; SE, standard error.

ATP concentrations

There is a decrease of intracellular ATP concentrations of C. sakazakii ATCC 29544 after the treatment with FA (Fig. 3). Comparing control cells with cells treated with FA at MIC and 2MIC, there were significant reductions (p ≤ 0.01) of intracellular ATP in the treated cells. No significant difference (p > 0.05) was observed between cells treated with FA at two different concentrations.

FIG. 3.

Effects of ferulic acid on intracellular ATP production by Cronobacter sakazakii ATCC 29544. Values represent the means of triplicate measurements. Bars represent the standard deviation (n = 3). **p ≤ 0.01.

pH_in

A clear change in intracellular pH was observed after addition of FA (Fig. 4). The pH_in of C. sakazakii ATCC 29544 was 6.13 ± 0.08. The addition of FA at MIC caused a significant (P ≤ 0.01) decrease in pH_in of C. sakazakii from 6.13 ± 0.08 to 4.87 ± 0.08. The addition of FA at 2MIC significantly (p ≤ 0.01) lowered the pH_in from 6.13 ± 0.08 to 2.64 ± 0.21. Moreover, there was a significant (p ≤ 0.01) difference in pH_in when the concentration of FA was increased from MIC to 2MIC.

FIG. 4.

Effects of ferulic acid on the intracellular pH (pHi) of Cronobacter sakazakii ATCC 29544. Values represent the means of triplicate measurements. Bars represent the standard deviation (n = 3). **p ≤ 0.01.

Membrane potential

Cells treated with FA displayed rapid cell membrane hyperpolarization, as evidenced by a decrease in fluorescence (negative values) (Fig. 5). After the addition of FA at MIC or 2MIC, the membrane potential was decreased, and a higher rate was observed by increasing concentrations from MIC to 2MIC.

FIG. 5.

Effects of ferulic acid on the membrane potentials of Cronobacter sakazakii ATCC 29544. Negative (hyperpolarization) values produce a loss of cellular homeostasis. Values represent the means of triplicate measurements. Bars represent the standard deviation (n = 3). **p ≤ 0.01.

Fluorimetric detection of cell membrane injury

The results showed a good linearity between the green fluorescent intensity and the percent of viable bacterial (y = 26031x+13805; r ² = 0.99). FA at two concentrations caused a significant reduction in viable cell fluorescence. FA at MIC caused 63% reduction of cell fluorescence, and FA at 2MIC lead to 76% decrease (Table 4).

Table 4.

Reduction Levels on Fluorescence of Cronobacter sakazakii ATCC 29544 with Various Concentrations of Ferulic Acid

Concentration of FA	Green fluorescence units	Percent viable cell fluorescence	Percent reduction of viable cell fluorescence
0 (CK)	42451	99%	1%
MIC	23381	37%	63%
2MIC	20047	24%	76%

FESEM observation

The morphologic change was investigated by FESEM. The C. sakazakii cells were treated with FA at four concentrations (0, MIC, 2MIC, and 4MIC) for 2 h (Fig. 6A–D) and 4 h (Fig. 6E–H), respectively. The C. sakazakii cells exposed to the FA had a more wrinkled surface compared with the smooth surface of the untreated cells (Fig. 6A–E), and had pores on the cell membrane. Moreover, the cells treated with FA for 4 h showed large surface collapse and breakage, as well as the leakage of intracellular constituents. The number of damaged cells and degree of damage increased with FA concentration. This demonstrated that FA induced pore formation on the cell (Fig. 6).

FIG. 6.

Scanning electron micrographs of Cronobacter sakazakii ATCC 29544 under different conditions: (A) scanning electron micrographs of C. sakazakii ATCC 29544 untreated for 2 h; (B) scanning electron micrographs of C. sakazakii ATCC 29544 treated with ferulic acid at minimum inhibitory concentration (MIC) for 2 h; (C) scanning electron micrographs of C. sakazakii ATCC 29544 treated with ferulic acid at 2MIC for 2 h; (D) scanning electron micrographs of C. sakazakii ATCC 29544 treated with ferulic acid at 4MIC for 2 h; (E) scanning electron micrographs of C. sakazakii ATCC 29544 untreated for 4 h; (F) scanning electron micrographs of C. sakazakii ATCC 29544 treated with ferulic acid at MIC for 4 h; (G) scanning electron micrographs of C. sakazakii ATCC 29544 treated with ferulic acid at 2MIC for 4 h; and (H) scanning electron micrographs of C. sakazakii ATCC 29544 treated with ferulic acid at 4MIC for 4 h.

Discussion

In the present study, the activity and mode of action of FA against C. sakazakii strains with pathogenic potential were examined. Different C. sakazakii strains were tested and MICs varied among different strains. There was no difference between clinical isolates and food-derived isolates in terms of MIC value. Although MIC of ferulic acid against C. sakazakii strains was 2.5 mg/mL using agar dilution method, some growth could still be found under MIC during growth curves analysis using broth dilution. Similar observations have been made during antimicrobial tests of thymol against Staphylococcus aureus ATCC 29213 (Qiu et al., 2010), C. cassia oil against non-O157 strains (Sheng and Zhu, 2014), and Carlinae radix oil against S. aureus strains (Stojanovic-Radic et al., 2012). This may result from the different growth determination by two analyses. The MIC determined by agar dilution methods is the lowest concentration of the agent that completely inhibits visible growth as judged by the naked eye. Even when people use broth dilution to determine MIC, it is a common practice to detect the breakpoint by turbidity visually. A clear broth visually will not equal to completely no growth in broth. While during growth curve analysis, growth was assessed by optical density of cell suspension in broth, which will be more accurate to reflect the bacterial growth. In addition, different inoculum size and media were used for agar dilution and broth dilution methods. In agar dilution method, different concentrations of ferulic acid were incorporated into chromogenic agar plate, followed by the application of cells (10⁴ CFU per spot) onto the surface of solid agar plate. For broth dilution, C. sakazakii (10⁶ CFU/mL) were inoculated into liquid TSB with different concentrations of ferulic acid (Wiegand et al., 2008).

ATP can be used to determine the amount of viable microbial cells present and quantified using a bioluminescence assay comprised of the enzyme luciferase from Photinus pyralis, which has a high sensitivity to ATP and D-luciferin, the enzyme's substrate (Bajpai et al., 2013b). Caillet et al. demonstrated that oregano essential oil decreased cytoplasmic ATP of Escherichia coli O157:H7 due to its effect on cell wall structure (Caillet et al., 2005). They also reported that oregano essential oil affected the permeability of Staphylococcus aureus membranes, leading to a subsequent decrease in cytoplasmic ATP (Caillet et al., 2009). Sanchez et al. reported that white sagebrush and sweet acacia extracts provoked significant decreases in cellular ATP concentrations in V. cholera strains (Sanchez et al., 2010). Similarly, in this work, when intracellular ATP was measured in the presence of FA at MIC and 2MIC, a decrease in the cytoplasmic ATP concentration of C. sakazakii was observed. This might have occurred because of significant impairment in membrane permeability of the tested bacteria, which caused the intracellular ATP leakage through defective cell membrane. On the other hand, the reduction of ATP concentration may also have occurred due to accelerated hydrolysis by the ATPase energy-consuming pump (Herranz et al., 2001).

The carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) technique for measuring the pH_in of bacteria is based on intracellular conjugation of the succinimidyl group of cFSE with the aliphatic amines of intracellular proteins and subsequent elimination of free probe by a short incubation in the presence of glucose (Holyoak et al., 1996). Sanchez et al. reported that four plant extracts changed the pH_in of V. cholera strains due to membrane damage (Sanchez et al., 2010). Similar to that study, FA decreased the pH_in indicating that membrane damage had occurred.

DiBAC₄(3) is an anion fluorescent membrane potential dye that has been used as an indicator of changes in membrane polarization (Suzuki et al., 2003). Sanchez et al. reported that methanolic extracts of basil, white sagebrush, and sweet acacia displayed cell membrane hyperpolarization of the V. cholera strains (Sanchez et al., 2010). Li et al. reported that Staphylococcus aureus cells treated with chlorogenic acid displayed cell membrane hyperpolarization (Li et al., 2014). FA also caused the decrease of fluorescence in the treated cells due to membrane hyperpolarization as a result of a decrease of the membrane potential. Recent studies of this phenomenon have concluded that hyperpolarization occurs first due to a pH change and second due to a hyperpolarization of the cell membrane when K⁺ diffuses outside through K⁺ channels to balance ΔV, which is related to the conductivity of the superficial charges, to get a cellular equilibrium (Bot and Prodan, 2009).

The physical and morphological alterations in cell membrane structure were investigated by FESEM analysis. FA caused severe morphological alterations on the cell membrane of the C. sakazakii leading to disruption of cell integrity. Such morphological alterations have been observed in various types of test organisms when treated with different phenolic acid, bacteriocins and essential oil (Bajpai et al., 2013a; 2013b; 2014; Li et al., 2014; Lu et al., 2014).

There is a good correlation between green/red fluorescence ratio and percentage of viable bacteria. In our study, centrifuging the bacterial suspension as mentioned and resuspending the bacteria could reduce the interference of extracts. On the other hand, during centrifugation, some of nucleic acid from dead cells could be lost. Thus, we used the green fluorescence intensity and percent viable bacteria to establish standard curve. FESEM showed that FA increased bacterial membrane permeability and nucleic acid spill, which caused a reduction in the live cell fluorescence as reported by Campos et al. (2009).

Conclusion

This study demonstrated that FA has antimicrobial activity against C. sakazakii and is likely to act on the cell membrane based on a significant change in intracellular ATP concentrations, a decrease in intracellular pH, cell membrane hyperpolarization, a reduction in bacterial membrane integrity, and morphological alterations. This should lead to potential application of the FA in combination with other preserving techniques to control C. sakazakii in infant formula or other types of food.

Footnotes

Disclosure Statement

No competing financial interests exist.

Acknowledgments

This work was supported in part by New Century Excellent Talent Support Plan (NCET-13-0488), the Twelve-five Science and Technology Support Program (No.2015BAD16B08), National Natural Science Foundation of China (31301498), Fundamental Research Funds in Northwest A&F University (Z109021424), International Collaboration Partner Plan (A213021203) in Northwest A&F University, Special Fund for Sino–U.S. Joint Research Center for Food Safety (A200021501), and Start-Up Funds for Talents in Northwest A&F University (Z111021403).

References

Alakomi

, Matto

, Virkajarvi

, Saarela

. Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations. JMicrobiol Methods, 2005; 62:25–35.

Amalaradjou

MAR

, Hoagland

, Venkitanarayanan

. Inactivation of Enterobacter sakazakii in reconstituted infant formula by trans-cinnamaldehyde. Int J Food Microbiol, 2009; 129:146–149.

Amalaradjou

, Venkitanarayanan

. Proteomic analysis of the mode of antibacterial action of trans-cinnamaldehyde against Cronobacter sakazakii 415. Foodborne Pathog Dis, 2011; 8:1095–1102.

Bajpai

, Sharma

, Baek

. Antibacterial mode of action of Cudrania tricuspidata fruit essential oil, affecting membrane permeability and surface characteristics of food-borne pathogens. Food Control, 2013a;32:582–590.

Bajpai

, Sharma

, Baek

. Antibacterial mode of action of seed essential oil of Eleutherococcus senticosus against foodborne pathogens. Int J Food Sci Tech, 2013b;48:2300–2305.

Bajpai

, Sharma

, Baek

. Antibacterial mode of action of the essential oil obtained from Chamaecyparis obtusa sawdust on the membrane integrity of selected foodborne pathogens. Food Technol Biotech, 2014; 52:109–118.

Borges

, Ferreira

, Saavedra

, Simoes

. Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microb Drug Resist, 2013; 19:256–265.

Bot

, Prodan

. Probing the membrane potential of living cells by dielectric spectroscopy. Eur Biophys J Biophy, 2009; 38:1049–1059.

Breeuwer

, Drocourt

, Rombouts

, Abee

. A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester. Appl Environ Microbiol, 1996; 62:178–183.

10.

Bunthof

, van Schalkwijk

, Meijer

, Abee

, Hugenholtz

. Fluorescent method for monitoring cheese starter permeabilization and lysis. Appl Environ Microbiol, 2001; 67:4264–4271.

11.

Caillet

, Shareck

, Lacroix

. Effect of gamma radiation and oregano essential oil on murein and ATP concentration of Escherichia coli O157:H7. J Food Prot, 2005; 68:2571–2579.

12.

Caillet

, Ursachi

, Shareck

, Lacroix

. Effect of gamma radiation and oregano essential oil on murein and ATP concentration of Staphylococcus aureus . J Food Sci, 2009; 74:M499–M508.

13.

Campos

, Couto

, Figueiredo

, Toth

, Rangel

AOSS

, Hogg

. Cell membrane damage induced by phenolic acids on wine lactic acid bacteria. Int J Food Microbiol, 2009; 135:144–151.

14.

Choi

, Kim

, Lee

, Rhee

. New decontamination method based on caprylic acid in combination with citric acid or vanillin for eliminating Cronobacter sakazakii and Salmonella enterica serovar Typhimurium in reconstituted infant formula. Int J Food Microbiol, 2013; 166:499–507.

15.

[EUCAST] European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases. EUCAST Definitive Document E.DEF 3.1, June 2000: Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clin Microbiol Infect, 2000; 6:509–515.

16.

Friedemann

. Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). Int J Food Microbiol, 2007; 116:1–10.

17.

Ganan

, Martinez-Rodriguez

, Carrascosa

. Antimicrobial activity of phenolic compounds of wine against Campylobacter jejuni . Food Control, 2009; 20:739–742.

18.

, Kang

. Synergistic bactericidal effect of simultaneous near-infrared radiant heating and UV radiation against Cronobacter sakazakii in powdered infant formula. Appl Environ Microb, 2014; 80:1858–1863.

19.

Harouna

, Carraminana

, Navarro

, Perez

, Calvo

, Sanchez

. Antibacterial activity of bovine milk lactoferrin on the emerging foodborne pathogen Cronobacter sakazakii: Effect of media and heat treatment. Food Control, 2015; 47:520–525.

20.

Healy

, Cooney

, O'Brien

, Iversen

, Whyte

, Nally

, Callanan

, Fanning

. Cronobacter (Enterobacter sakazakii): An opportunistic foodborne pathogen. Foodborne Pathog Dis, 2010; 7:339–350.

21.

Herranz

, Chen

, Chung

, Cintas

, Hernandez

, Montville

, Chikindas

. Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136. Appl Environ Microbiol, 2001; 67:1689–1692.

22.

Holyoak

, Stratford

, McMullin

, Cole

, Crimmins

, Brown

, Coote

. Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid. Appl Environ Microbiol, 1996; 62:3158–3164.

23.

Hong

, Park

, Chung

, Kwon

, Chung

, Won

, Song

. Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium in powdered weaning food by electron-beam irradiation. Radiat Phys Chem, 2008; 77:1097–1100.

24.

Iversen

, Waddington

, On

, Forsythe

. Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter Species. J Clin Microbiol, 2004; 42:5368–5370.

25.

Joshi

, Howell

, D'Souza

. Cronobacter sakazakii reduction by blueberry proanthocyanidins. Food microbiology, 2014; 39:127–131.

26.

Kim

, Kim

, Jung

. Antitumor and antioxidant activity of protocatechualdehyde produced from Streptomyces lincolnensis M-20. Arch Pharm Res, 2008; 31:1572–1577.

27.

Kim

, Weng

, Silva

, Jung

, Marshall

. Identification of Natural Antimicrobial Substances in Red Muscadine Juice against Cronobacter sakazakii . J Food Sci, 2010; 75:M150–M154.

28.

Lee

, Oh

, Kim

, Yook

, Byun

. Gamma radiation sensitivity of Enterobacter sakazakii in dehydrated powdered infant formula. J Food Prot, 2006; 69:1434–1437.

29.

Lee

, Jin

. Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii . Lett Appl Microbiol, 2008; 47:315–321.

30.

, Wang

, Xu

, Zhang

, Xia

. Antimicrobial effect and mode of action of chlorogenic acid on Staphylococcus aureus . Eur Food Res Technol, 2014; 238:589–596.

31.

, Chung

. The effects of plant phenolics, caffeic acid, chlorogenic acid and ferulic acid on arylamine N-acetyltransferase activities in human gastrointestinal microflora. Anticancer Res, 1999; 19:133–139.

32.

, Yi

, Dang

, Liu

. Purification of novel bacteriocin produced by Lactobacillus coryniformis MXJ 32 for inhibiting bacterial foodborne pathogens including antibiotic-resistant microorganisms. Food Control, 2014; 46:264–271.

33.

Muytjens

, Kollee

. Enterobacter sakazakii meningitis in neonates: causative role of formula?. Pediatr Infect Dis J, 1990; 9:372–373.

34.

Nair

, Joy

, Venkitanarayanan

. Inactivation of Enterobacter sakazakii in reconstituted infant formula by monocaprylin. J Food Prot, 2004; 67:2815–2819.

35.

, Kwok

. Ferulic acid: Pharmaceutical functions, preparation and applications in foods. J Sci Food Agr, 2004; 84:1261–1269.

36.

Pina-Perez

, Benlloch-Tinoco

, Rodrigo

, Martinez

. Cronobacter sakazakii inactivation by microwave processing. Food Bioprocess Tech, 2014; 7:821–828.

37.

Qiu

, Wang

, Xiang

, Feng

, Jiang

, Xia

, Dong

, Lu

, Yu

, Deng

. Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates. PLoS One, 2010; 5:e9736.

38.

Sanchez

, Garcia

, Heredia

. Extracts of edible and medicinal plants damage membranes of Vibrio cholerae . Appl Environ Microb, 2010; 76:6888–6894.

39.

Sheng

, Zhu

. Inhibitory effect of Cinnamomum cassia oil on non-O157 Shiga toxin-producing Escherichia coli . Food Control, 2014; 46:374–381.

40.

Silva-Angulo

, Zanini

, Rosenthal

, Rodrigo

, Klein

, Martinez

. Comparative Study of the Effects of Citral on the Growth and Injury of Listeria innocua and Listeria monocytogenes Cells. PLoS One, 2015; 10:e0114026.

41.

Sosnowski

, Czubkowska

, Korpysa-Dzirba

, Ostrowska

, Rola

, Osek

. Cronobacter sakazakii—Characteristics and significance in food microbiology. Med Weter, 2014; 70:669–672.

42.

Stojanovic-Radic

, Comic

, Radulovic

, Blagojevic

, Mihajilov-Krstev

, Rajkovic

. Commercial Carlinae radix herbal drug: Botanical identity, chemical composition and antimicrobial properties. Pharm Biol, 2012; 50:933–940.

43.

Suzuki

, Wang

, Yamakoshi

, Kobayashi

, Nozawa

. Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes. Anal Sci, 2003; 19:1239–1242.

44.

Takahashi

, Kashimura

, Koiso

, Kuda

, Kimura

. Use of ferulic acid as a novel candidate of growth inhibiting agent against Listeria monocytogenes in ready-to-eat food. Food Control, 2013; 33:244–248.

45.

Takahashi

, Takada

, Tsuchiya

, Miya

, Kuda

, Kimura

. Listeria monocytogenes develops no resistance to ferulic acid after exposure to low concentrations. Food Control, 2015; 47:560–563.

46.

Torlak

, Sert

. Inactivation of Cronobacter by gaseous ozone in milk powders with different fat contents. Int Dairy J, 2013; 32:121–125.

47.

Uraji

, Kimura

, Inoue

, Kawakami

, Kumagai

, Harazono

, Hatanaka

. Enzymatic production of ferulic acid from defatted rice bran by using a combination of bacterial enzymes. Appl Biochem Biotech, 2013; 171:1085–1093.

48.

Wiegand

, Hilpert

, Hancock

REW

. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc, 2008; 3:163–175.

49.

Zhao

, Moghadasian

. Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: A review. Food Chem, 2008; 109:691–702.

Antimicrobial Activity of Ferulic Acid Against Cronobacter sakazakii and Possible Mechanism of Action

Abstract

Introduction

Materials and Methods

Reagents

Bacterial strains and culture conditions

Minimum inhibitory concentration (MIC) determinations

Growth curves and kinetic parameters

Measurement of intracellular ATP concentrations

Intracellular pHin measurements

Membrane potential determinations

Assessing bacterial membrane integrity

Field emission scanning electron microscope (FESEM) analysis

Statistical analysis

Results

MIC of FA on C. sakazakii strains

Growth curves and kinetic parameters

ATP concentrations

pHin

Membrane potential

Fluorimetric detection of cell membrane injury

FESEM observation

Discussion

Conclusion

Footnotes

Disclosure Statement

Acknowledgments

References

Intracellular pH_in measurements

pH_in