Antibacterial Activities of Plant-Derived Compounds and Essential Oils Toward Cronobacter sakazakii and Cronobacter malonaticus

Introduction

C ronobacter spp. are opportunistic bacterial pathogens that belong to the Enterobacteriaceae family (Iversen et al., 2007). C. sakazakii and C. malonaticus are human pathogens that have been linked to life-threatening infections in neonates and children. Infections in adults are common; however, due to the less severe nature of the reported cases of illness, they represent an indeterminate fraction of actual cases (WHO/FAO, 2008). Published data indicate that Cronobacter spp. are present in a variety of foods. Consequently, an effective method that uses natural antibacterial substances is desirable for reducing the presence of these bacteria in foods.

Several reports have described the effect of plant-derived compounds, including thymol and carvacrol (Lee et al., 2008), trans-cinnamaldehyde (Amalaradjou et al., 2009), vanillin, ethyl vanillin, and vanillic acid (Yemis et al., 2012), against Cronobacter spp. Moreover, essential oils (EOs) and their major components demonstrate significant activity against various pathogenic microorganisms not only in their liquid phase but also as vapors (Lopez et al., 2007).

Reports on plant-derived compounds and essential oils against Cronobacter spp. are very scarce, and no screening study has allowed a direct comparison of their effects. Thus, the aim of the present study was to determine the antibacterial activity of 24 natural agents (Table 1 and Supplementary Data) (Supplementary data are available online at www.liebertpub.com/fpd) against C. sakazakii and C. malonaticus. The antibacterial activities of 24 natural agents (Table 1) were tested in liquid and vapor phase against C. sakazakii ATCC 29544 and C. malonaticus CNCTC 6830 (for details see Supplementary data). Stock solutions (100 mg/mL in 20% dimethyl sulfoxide [wt/vol]) of tested compounds and culture medium containing (g/L) peptone from casein tryptic digest (8.5), soy peptone (1.5), NaCl (2.5), K₂HPO₄ (1.3), and glucose (1.3) (Sigma-Aldrich, Ltd., Prague, Czech Republic) were prepared. Stock solutions (10–500 μL) were injected into 20-mL vials containing sterile culture medium to achieve a final volume of 10 mL and natural agent concentrations of 0.1, 0.2, 0.5, 1, 2, 3, 4, and 5 mg/mL. Controls received an equivalent amount of the solvent (20% dimethyl sulfoxide or ethanol). The flasks were inoculated with 0.2 mL of an overnight culture (10⁸ colony-forming units/mL) and incubated in duplicate at 37°C for 24 h. Residual glucose was determined enzymatically using a commercial Glukosa Liquid 500 S kit from Erba-Lachema (Brno, Czech Republic). The minimum inhibitory concentration (MIC) was the minimum concentration of antibacterial that prevented glucose utilization in the treated cultures (Brander, 1982). The tests were performed in duplicate (in triplicate when parallels differed by more than 10%).

The method described by Kloucek et al. (2012) was used to assess the susceptibility of the tested strains to the volatiles shown in Table 1. In brief, the assay was performed in 4 sections of 90-mm Petri dishes, with 2 sections inoculated with the test strains and the other 2 sections left as the contamination control. Different concentrations of essential oils and pure compounds were applied to the filter paper, creating final vapor concentrations of 0.032, 0.064, 0.128, 0.256, and 0.512 mg/mL in the Petri dishes. The lowest concentration that inhibited bacterial growth after 24 h of incubation at 37°C was considered to be the MIC. The tests were performed in triplicate.

Most of the presented antimicrobials have not been tested against Cronobacter spp. before. Twelve of the tested compounds demonstrated MICs<1 mg/mL (Table 1). Thymol, carvacrol, and trans-cinnamaldehyde were the most effective compounds (MICs ranging from 0.1 to 0.3 mg/mL). Lee (2008) reported the same MICs for thymol and carvacrol (0.19 mg/mL each) and an even lower MIC for eugenol (0.82 mg/mL) for five Cronobacter strains tested in vitro. The MIC of trans-cinnamaldehyde was reported in reconstituted infant milk (Amalaradjou et al., 2009) and ranged from 3 to 5 mg/mL, depending on the treatment time and temperature.

Oregano was the most effective EO (MIC 0.2 mg/mL) and demonstrated a similar effect against C. sakazakii as EO from Ziziphora persica (MIC 0.25 mg/mL), which contains pulegone as the main compound (Ozturk et al., 2007). The C. sakazakii strain was more susceptible to citral, thymoquinone, p-cymene, and thujone compared with C. malonaticus, which was more susceptible to carvacrol and trans-cinnamaldehyde.

The MICs of the tested EOs and their corresponding main active compounds were very similar except for eugenol and clove, which had MICs of 1 mg/mL and 0.5 mg/mL, respectively (Table 1). The higher effectiveness of clove EO could be caused by minor compounds present in the EO because these minor compounds can play important roles in the oil's antibacterial activity. The antibacterial activity of the substances that had not been previously tested against Cronobacter spp. can be compared with the MICs of other natural substances. For example, linalool, camphor, 1,8-cineole, and limonene have similar antibacterial effect as propionic and acetic acid, which have MICs ranging from 1.86 to 3.78 and 1.18 to 2.29 mg/mL, respectively (Back et al., 2009).

To our knowledge, this study is the first to report the MICs of EO vapors against Cronobacter spp. The most effective EOs were cinnamon and oregano and their corresponding major components, trans-cinnamaldehyde and carvacrol, respectively (Table 1). However, the majority of the tested components were not effective even at the highest concentration. The MICs of the antibacterials in vapor and liquid phase were similar; only eugenol and linalool demonstrated a stronger effect in the vapor phase. Moreover, C. sakazakii was more susceptible than C. malonaticus to the EO vapors.

Numerous natural agents have been registered by the European Commission as flavoring substances and are considered safe. Consequently, the results suggest that they can be used directly or indirectly as effective disinfectants or preservatives in the food industry. For example, the EOs could be incorporated in food packaging materials or used to create part of a modified atmosphere, as described elsewhere (Lopez et al., 2007).