Inactivation of Salmonella in Organic Soil by Cinnamaldehyde,Eugenol,Ecotrol,and Sporan

Abstract

Salmonella can survive in soil for months to years; consequently, soil can be a preharvest source of contamination of produce. Elimination of Salmonella with natural products and processes such as essential oils is important to prevent infection among consumers. Essential oils (distilled extract from plants) have been mainly evaluated in liquid medium and foods in which minimum inhibitory concentration is determined. However, there are no reports describing the impact of essential oils in soil, especially organic soil. We evaluated essential oils for controlling Salmonella enterica serovars in organic soil. Two essential oils (cinnamaldehyde and eugenol), two bio-pesticides (Ecotrol and Sporan), and an organic acid (20% acetic acid) at 0.5%, 1.0%, 1.5%, and 2.0% were mixed with organic sandy soil and inoculated with six different serovars of S. enterica separately. Soils were incubated at room temperature, and samples obtained at 1, 7, and 28 days were enumerated to determine survival. The bactericidal effect of cinnamaldehyde was evident at 0.5%, 1.0%, 1.5%, and 2.0% and during all times of incubation. Overall, Salmonella Negev was the most sensitive strain to oils resulting in significant reductions compared with other strains. Increases in oil concentration resulted in further reduction of Salmonella with all oils used in the study. Up to six log reductions in Salmonella serovars Typhimurium, Negev, and Newport were found after 1 day when cinnamaldehyde, Ecotrol, eugenol, Sporan, or acetic acid was used at 2% level. This study shows the potential use of essential oils to effectively reduce Salmonella populations in soil. The significant reduction of Salmonella could greatly reduce potential contamination of fresh organic produce inadvertently contaminated by soil.

Introduction

A pproximately 1.4 million cases of Salmonella infections occur every year in the United States, resulting in 15,000 hospitalizations and 580 deaths (Shin, 2006). Cost estimates per case of human salmonellosis range from $4.6 to $40 million (WHO, 2005). Salmonellosis in humans is generally contracted through the consumption of contaminated foods of animal origin mainly meat, poultry, eggs, and milk, although other foods, including fresh produce such as lettuce (Gillespie, 2004), tomatoes, cantaloupe, and alfalfa sprouts (Shin, 2006), contaminated with manure or irrigation water have been implicated in its transmission (WHO, 2005). Compared to other bacteria, Salmonella has high survival rates in aquatic environments (Chao et al., 1987; Winfield and Groisman, 2003). Salmonella can be widely disseminated in soil and sediment, even in the absence of active fertilization, as a result of water currents, underground spring, and rain runoff carrying contaminated material (Chao et al., 1987; Abdel-Monem and Dowidar, 1990).

Produce can become contaminated with pathogenic microorganisms at any point during farm to fork continuum. Potential sources of Salmonella contamination on the field could be field fertilized with untreated manure (Beuchat, 2002) or sewage as a soil amendment, field irrigated with water contaminated with animal and human waste, water used to apply fungicides and insecticides, wildlife and domestic animal grazing on or near the fields, dust, equipment exposed to contaminated mud or water, transport vehicles, processing equipment, and workers (Western Growers Association, 2010). Noncomposted manure or improperly composted manure used on the farm, or manure that enters surface waters, may contain these pathogens and subsequently contaminate produce (Millner, 2009). Eliminating pathogens from livestock, for example, by vaccination, may help reduce shedding in manure and consequently reduce the potential for soil and water contamination. However, this method may run into major problems related to adverse immunological reactions in cattle and regulatory issues (Brabban et al., 2004). While such approaches are still being investigated and developed, other researchers are seeking a variety of conventional, novel, and natural alternatives to reduce the risk of produce contamination.

Although a great deal is already known about Salmonella spp., these organisms continue to provide new challenges to food safety, particularly because of the evolution of new strains resulting from the acquisition of genes conferring characteristics such as multiple antibiotic resistance (Blackburn and McClure, 2002). Hence, there is a continued need for research and information concerning the reduction of these bacteria. The use of natural products as antibacterial compounds appeals to growers for controlling pathogenic bacteria in organic systems without resorting to traditional agrichemical fumigants, and pesticides (Conner, 1993; Dorman and Deans, 2000). Public concern about the long-term health and environmental effects of synthetic pesticides has increased interest in use of natural pesticides of both microbial and plant origin in the global market place. Natural insecticides based on essential oils are used by farmers for pest and disease management (Isman, 2006). It has been reported that those plant essential oils not only repel insects, but also have contact and fumigant insecticidal actions against specific pests, and fungicidal actions against plant pathogens (Isman, 2006). However, no study has been done on the impact of those natural pesticides against Salmonella in soil.

Essential oils, also called volatiles, are aromatic oily liquids obtained from plant materials (flowers, buds, seeds, leaves, twig bark, herbs, woods, fruits, and roots), which can be obtained by fermentation, extraction, or distillation (Hili et al., 1997; Burt, 2004). Among these natural antimicrobials are eugenol from cloves, thymol from thyme, carvacrol from oregano, allicin from garlic, cinnamic aldehyde from cinnamon, and allyl isothiocyanate from mustard (Tzortzakis, 2009). Previous studies have demonstrated that leaf essential oils of the cinnamaldehyde had excellent antitermite, antibacterial, antimite, antimildew, antimosquito, and antipathogenic activities (Chang et al., 2001; Cheng et al., 2004; Lee et al., 2005). Cinnamaldehyde also inhibited the growth of Clostridium botulinum (Bowles et al., 1997), Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium (Helander et al., 1998) in liquid media. E. coli O157:H7 and Listeria monocyotogens were inhibited when 1000 ppm eugenol was added in Tryptic soy broth (Blaszyk and Holley, 1998). Ecotrol, based on a concentrated blend of 10% rosemary and 2% peppermint oils, is effective against many insects. It can be applied to agricultural crops, including vegetables and cole, herbs and spices, citrus, pomes and stone fruits, nuts, berries, fruits, and grapes (Anonymous, 2005). Sporan is a fungicide against a broad range of diseases, including blights, molds, scabs, and mildews (Anonymous, 2008). It is composed of rosemary, clove, and thyme oils and is suitable for use on agricultural crops. It disrupts the cell membrane of fungal hyphae and spores resulting in cell death. Sporan and Ecotrol have been approved by Organic Material Review Institute for application on foliar tissues (Anonymous, 2008).

Organic acids and their salts are promising as antimicrobial agents due to their acceptance in food products and low cost (Miller et al., 1996). Organic acids have been used for controlling pathogens in ready-to-eat meats (Patel et al., 2009) and minimally processed fruits and vegetables. The antimicrobial activity of organic acids is due to the pH reduction, depression of internal pH of microbial cells, and disruption of substrate transport by altering cell membrane permeability (Beuchat, 1998). This is the first report on the impact of essential oils in organic soil on survival of Salmonella. This project was performed to determine the effect of essential oils: cinnamaldehyde, eugenol; natural pesticides Ecotrol and Sporan; and acetic acid on organic soil experimentally contaminated with Salmonella.

Materials and Methods

Preparation of bacterial strains

Six S. enterica serovars were used in the study: Thompson 2051H, Tennessee 2053N, and Negev 26 H (Thyme isolates) were provided by Tom Hammack (Food and Drug, College Park, MD), and Braenderup (CDC clinical isolate # 95-682-997), Typhimurium, and Newport (CDC clinical isolate #9113) were used from our Environmental Microbial and Food Safety Laboratory culture collection. Frozen cultures of each strain were partially thawed at room temperature (∼22°C) for 15 min, and streaked onto tryptic soy agar slants (Acumedia, Lansing, MI), and incubated at 37°C for 24 h. Before the experiment, cultures were grown in Tryptic soy broth (Acumedia) at 37°C for 24 h. Cells were centrifuged (7500 g, 10 min, 10°C), washed with phosphate-buffered saline twice, and resuspended in 10 mL sterile peptone water (0.1%). The cell pellets were resuspended in phosphate-buffered saline to obtain an OD₆₀₀ of 1. The cell concentration of individual strain was verified by spiral plating (Microbiology International, Frederick, MD) on XLT4 agar (Acumedia).

Essential oil and soil inoculation

Cinnamaldehyde (Sigma-Aldrich, St Louis, MO), Ecotrol (EcoSMART Tech., Alpharetta, GA), eugenol (Fisher Scientific, Pittsburg, PA), Sporan (EcoSMART Tech), and acetic acid (Fleischmann's, Baltimore, MD) were used in the study. Four individual suspensions with the desired concentrations (0.5%, 1.0%, 1.5%, and 2.0%, v/v) were freshly prepared by dispersing them in sterile distilled water containing 0.5% (w/v) Tween 20 (Fisher Scientific) to dissolve essential oil as reported by Burt (2004). A 20% concentration of acetic acid was prepared using sterile distilled water. Unamended soils were also inoculated to serve as controls.

Organic soil (Downer-Ingleside loamy sand, coarse-loamy, siliceous, semiactive, and mesic Typic Hapludults) was obtained from the U.S. Department of Agriculture Beltsville Agricultural Research Center North Farm. Soil was mixed, screened to remove stones and debris, and stored in a sterile plastic bag before treatment. Soil (10 g) was placed into sterile filter bags (Nasco Whirl-Pak, Fort Atkinson, WI) for each treatment–strain combination, inoculated with 100 μL of 8 log colony forming units (cfu)/mL of designated inocula, and vigorously shaken/massaged to distribute the inoculum thoroughly. Essential oil preparations (0.5%, 1.0%, 1.5%, and 2.0%) were added individually to these filter bags, mixed thoroughly, and incubated at room temperature (22°C) for 28 days.

Enumeration of Salmonella

On days 1, 7, and 28, 10 mL sterile peptone water (0.1%) was added to each soil bag and the bag was pummeled for 2 min (Bagmixer; Interscience, St. Nom, France). Serially diluted soil suspensions were spiral plated on selective agar (XLT4; Acumedia) and incubated at 37°C for 24 h. Typical Salmonella colonies were counted after incubation of 24 h at 37°C. Randomly selected colonies were confirmed by latex agglutination assay (Remel, Lenexa, KS).

Statistical analysis

Colony counts of presumptive Salmonella for each sampling period were converted to log cfu/g. The experiment was performed in triplicate. Data were analyzed by a three-way analysis of variance using the Proc Mixed procedure (SAS 8.2, Cary, NC) for effects of oils, oil concentrations, strains, sampling time, and their interactions. In all cases, the level of statistical significance was p < 0.05.

Results

Inactivation of Salmonella in soil after 24 h

Salmonella was not detected in uninoculated soil used in this study. The impacts of treatment with cinnamaldehyde, Ecotrol, eugenol, Sporan, and acetic acid on Salmonella inoculated in soil are presented in Tables 1 –3. Recovery of Salmonella after 24 h of antimicrobial treatment varied with the serovar. Salmonella populations in untreated soil ranged from 5.92 to 6.36 log cfu/g (Table 1). After 24 h, Salmonella populations in soil treated with cinnamaldehyde at 0.5%, 1.0%, 1.5%, and 2.0% were undetectable (<1 log CFU/g), except for Thompson and Tennessee. Salmonella serovars recovered in soil treated with Ecotrol at 0.5% were not significantly different from those populations recovered in untreated soil. Eugenol at 0.5% concentrations reduced Typhimurium and Tennessee serovars in soil by 4 and 2 log CFU/g, respectively. Only Salmonella Braedenrup was reduced significantly when 0.5% Sporan was used in soil. Overall, Salmonella populations were reduced with increased concentrations of Sporan, acetic acid, eugenol, and Ecotrol. Negev, Newport, and Thompson serovars were significantly reduced when 1% Ecotrol was used, whereas only Braedenrup was significantly reduced in soil treated with 1% eugenol. Likewise, treatment with 1.0% acetic acid reduced (p < 0.05) all serovars used in the study; up to 4.8 log reductions in Negev and Newport serovars were observed with 1% acetic acid. Salmonella serovar populations in soil treated with 1.5% essential oils were significantly lower than corresponding serovars recovered from soil treated with 0.5% oils with the exception of eugenol. Salmonella Negev strain was the most sensitive when treated with 1.5% Ecotrol or eugenol resulting in ∼4.5 log reductions. About 5 log reductions in all but Typhimurium serovars were observed with 1.5% Sporan in soil. Populations of all salmonella strains were undetectable (<1 log CFU/g) in 2% cinnamaldehyde- and acetic acid-treated soil after 24 h. Likewise, complete inhibition of serovars Braedenrup and Typhimurium was observed with 2% Ecotrol, serovar Negev with 2% eugenol, and serovars Typhimurium, Negev, and Newport with 2% Sporan.

Table 1.

Impact of Essential Oils and Vinegar on Salmonella in Soil After 24 h

		Populations of Salmonella serovars in soil treated with oils^*
Treatment	Conc. (%)	Braedenrup	Typhimurium	Negev	Newport	Thompson	Tennessee
Control	0	6.36 ± 0.29^{a x}	6.09 ± 0.24^{a x}	5.92 ± 0.38^{a x}	6.10 ± 0.26^{a x}	6.10 ± 0.30^{a x}	6.07 ± 0.30^{a x}
Cinnamaldehyde	0.5	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}	1.05 ± 1.05^{b x}	0.67 ± 0.67^{c x}
	1	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}
	1.5	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}
Ecotrol	0.5	5.73 ± 0.30^{ab x}	5.94 ± 0.04^{a x}	4.71 ± 0.38^{a x}	4.97 ± 0.30^{a x}	5.37 ± 0.44^{a x}	5.93 ± 0.72^{a x}
	1	5.06 ± 0.37^{a x}	5.09 ± 0.33^{a x}	3.85 ± 0.21^{b x}	4.40 ± 0.32^{b x}	4.34 ± 0.36^{b x}	4.72 ± 0.65^{a x}
	1.5	3.63 ± 0.47^{b x}	2.47 ± 1.37^{b xy}	1.62 ± 0.83^{b y}	3.28 ± 0.64^{b xy}	3.13 ± 0.50^{b xy}	3.38 ± 0.48^{b x}
	2	1.00 ± .0.00^{b xy}	0.00 ± 0.00^{c y}	0.00 ± 0.00^{b y}	1.29 ± 1.29^{bc xy}	2.30 ± 0.30^{bc x}	1.06 ± 1.06^{bc xy}
Eugenol	0.5	5.01 ± 0.11^{ab xy}	2.22 ± 2.22^{b z}	5.14 ± 0.28^{a xy}	5.43 ± 0.75^{a xy}	5.83 ± 0.37^{a x}	4.02 ± 2.09^{b y}
	1	0.00 ± 0.00^{c z}	5.29 ± 0.51^{a x}	4.02 ± 1.42^{ab y}	5.93 ± 0.20^{ab x}	4.79 ± 0.31^{ab x}	5.04 ± 0.04^{a x}
	1.5	2.49 ± 1.25^{bc xy}	2.66 ± 1.33^{b xy}	1.55 ± 1.55^{b y}	3.25 ± 0.78^{b x}	3.39 ± 1.79^{b x}	3.18 ± 1.68^{b xy}
	2	0.83 ± 0.83^{b y}	2.77 ± 0.50^{b x}	0.00 ± 0.00^{b y}	2.90 ± 1.48^{b x}	2.96 ± 1.52^{b x}	2.59 ± 1.30^{b x}
Sporan	0.5	4.52 ± 0.18^{b x}	5.06 ± 0.33^{a x}	4.46 ± 0.88^{a x}	4.71 ± 0.46^{a x}	4.85 ± 0.62^{a x}	5.21 ± 0.20^{ab x}
	1	2.43 ± 1.36^{b xy}	3.20 ± 0.20^{b x}	0.66 ± 0.66^{c z}	2.35 ± 1.19^{c xyz}	1.47 ± 1.47^{c yz}	1.56 ± 1.56^{bc xyz}
	1.5	0.90 ± 0.90^{cd x}	2.08 ± 1.32^{b x}	1.00 ± 1.00^{b x}	0.90 ± 1.55^{c x}	1.27 ± 0.23^{b x}	0.90 ± 0.90^{c x}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.88 ± 0.88^{cd x}	0.56 ± 0.56^{c x}
Vinegar	0.5	5.04 ± 0.46^{ab x}	5.30 ± 0.27^{a x}	5.04 ± 0.11^{a x}	4.96 ± 0.52^{a x}	5.33 ± 0.09^{a x}	4.87 ± 0.75^{ab x}
	1	3.00 ± 0.68^{b xy}	2.44 ± 0.16^{b xyz}	1.01 ± 1.01^{c z}	1.46 ± 0.80^{cd yz}	3.75 ± 0.39^{b x}	2.62 ± 0.26^{b xyz}
	1.5	0.66 ± 0.66^{d xy}	0.00 ± 0.00^{c y}	0.56 ± 0.56^{b xy}	0.00 ± 0.00^{c y}	2.23 ± 0.40^{b x}	0.56 ± 0.56^{c xy}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}

Counts (log cfu/g) ± standard deviation.

abc

Means with different letter in the column within the treatment are significantly different (p < 0.05).

xyz

Means with different letter in the row are significantly different (p < 0.05).

cfu, colony forming units.

Table 2.

Impact of Essential Oils and Vinegar on Salmonella in Soil After 7 Days

		Populations of Salmonella serovars in soil treated with oils*
Treatment	Conc. (%)	Braedenrup	Typhimurium	Negev	Newport	Thompson	Tennessee
Control	0	5.76 ± 0.29^{a x}	5.58 ± 0.23^{a x}	5.16 ± 0.41^{a x}	5.65 ± 0.22^{a x}	5.64 ± 0.10^{a x}	5.82 ± 0.16^{a x}
Cinnamaldehyde	0.5	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}
	1	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}
	1.5	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}
Ecotrol	0.5	5.57 ± 0.30^{a x}	5.09 ± 0.26^{a x}	4.90 ± 0.56^{a x}	4.88 ± 0.23^{ab x}	5.31 ± 0.35^{a x}	5.63 ± 0.20^{a x}
	1	5.43 ± 0.14^{a x}	5.52 ± 0.45^{a x}	5.02 ± 0.64^{a x}	4.92 ± 0.14^{a x}	5.22 ± 0.26^{a x}	5.71 ± 0.15^{a x}
	1.5	4.21 ± 0.66^{ab x}	4.12 ± 0.31^{ab x}	0.00 ± 0.00^{b z}	0.00 ± 0.00^{c z}	2.88 ± 1.45^{b xy}	2.10 ± 1.24^{c y}
	2	1.00 ± 1.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}
Eugenol	0.5	5.30 ± 0.24^{a x}	5.19 ± 0.16^{a x}	2.93 ± 1.52^{b y}	6.22 ± 0.76^{a x}	5.27 ± 0.47^{a x}	5.79 ± 0.14^{a x}
	1	1.49 ± 1.49^{bc y}	4.65 ± 0.41^{a x}	1.31 ± 1.31^{b y}	5.41 ± 0.45^{a x}	4.89 ± 0.25^{a x}	5.36 ± 0.19^{a x}
	1.5	3.88 ± 0.38^{b xy}	2.63 ± 1.35^{b yz}	1.31 ± 2.28^{b z}	4.43 ± 0.59^{a x}	1.70 ± 1.70^{b z}	4.11 ± 0.46^{b xy}
	2	0.82 ± 0.82^{b y}	2.77 ± 0.50^{c x}	0.00 ± 0.00^{b y}	3.69 ± 0.70^{b x}	0.00 ± 0.00^{b y}	2.59 ± 1.30^{b x}
Sporan	0.5	4.52 ± 0.09^{a xy}	5.50 ± 0.22^{a x}	0.00 ± 0.00^{c z}	4.35 ± 0.31^{b xy}	2.90 ± 1.45^{b y}	4.18 ± 0.06^{b xy}
	1	2.95 ± 1.48^{b x}	2.56 ± 1.28^{b xy}	0.88 ± 0.88^{b y}	2.62 ± 1.36^{b x}	2.24 ± 1.21^{b xy}	3.06 ± 1.67^{c x}
	1.5	3.21 ± 1.61^{b x}	0.00 ± 0.00^{c z}	0.00 ± 0.00^{b z}	2.41 ± 1.21^{b xy}	1.29 ± 1.29^{bc yz}	0.00 ± 0.00^{d z}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	1.27 ± 1.27^{c x}	0.96 ± 0.96^{b x}	0.00 ± 0.00^{c x}
Vinegar	0.5	4.69 ± 0.20^{a x}	4.28 ± 0.23^{a x}	2.45 ± 1.28^{b y}	4.27 ± 0.21^{b x}	4.99 ± 0.27^{a x}	3.84 ± 0.55^{b xy}
	1	1.33 ± 1.33^{bc xy}	0.80 ± 0.80^{c xy}	1.01 ± 1.01^{b xy}	0.00 ± 0.00^{c y}	1.08 ± 1.08^{bc xy}	2.42 ± 1.26^{c x}
	1.5	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}

Counts (log cfu/g) ± standard deviation.

abc

Means with different letter in the column within the treatment are significantly different (p < 0.05).

xyz

Means with different letter in the row are significantly different (p < 0.05).

Table 3.

Impact of Essential Oils and Vinegar on Salmonella in Soil After 28 Days

		Populations of Salmonella serovars in soil treated with oils^*
Treatment	Conc. (%)	Braedenrup	Typhimurium	Negev	Newport	Thompson	Tennessee
Control	0	4.91 ± 0.35^{a x}	4.8 ± 0.29^{a x}	4.01 ± 0.94^{a x}	4.63 ± 0.55^{a x}	4.66 ± 0.51^{a x}	4.67 ± 0.48^{a x}
Cinnamaldehyde	0.5	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}
	1	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{d x}
	1.5	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}
Ecotrol	0.5	4.40 ± 0.05^{a x}	4.25 ± 0.20^{a x}	1.96 ± 1.00^{b y}	4.00 ± 0.09^{a x}	4.05 ± 0.34^{a x}	4.09 ± 0.22^{a x}
	1	4.19 ± 0.14^{ab x}	3.74 ± 0.26^{b x}	2.54 ± 0.25^{b x}	3.94 ± 0.06^{b x}	4.42 ± 0.26^{a x}	4.13 ± 0.24^{a x}
	1.5	3.69 ± 0.16^{a x}	2.97 ± 0.23^{b x}	0.56 ± 0.56^{b y}	2.69 ± 0.03^{b x}	3.23 ± 0.88^{a x}	2.15 ± 1.08^{b xy}
	2	0.00 ± 0.00^{b x}	0.72 ± 0.72^{bc x}	0.00 ± 0.00^{b x}	1.02 ± 1.02^{bc x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}
Eugenol	0.5	4.01 ± 0.36^{a x}	4.00 ± 0.73^{a x}	0.90 ± 0.90^{bc y}	4.12 ± 0.64^{a x}	3.94 ± 0.61^{a x}	4.34 ± 0.67^{a x}
	1	3.92 ± 0.22^{b x}	3.92 ± 0.47^{b x}	1.20 ± 1.20^{bcd y}	3.83 ± 0.33^{b x}	4.09 ± 0.35^{a x}	4.30 ± 0.42^{a x}
	1.5	4.21 ± 0.16^{a x}	2.38 ± 1.24^{b yz}	0.90 ± 0.90^{b z}	2.47 ± 1.27^{b yz}	3.88 ± 0.59^{a xy}	2.86 ± 1.47^{b xy}
	2	0.00 ± 0.00^{b y}	2.41 ± 0.24^{b x}	1.46 ± 0.79^{b xy}	2.63 ± 1.40^{b x}	1.30 ± 1.30^{b xy}	1.21 ± 1.21^{b xy}
Sporan	0.5	4.84 ± 0.48^{a x}	4.57 ± 0.48^{a x}	0.00 ± 0.00^{c y}	4.63 ± 0.33^{a x}	4.87 ± 0.38^{a x}	4.79 ± 0.46^{a x}
	1	2.18 ± 0.48^{c xyz}	0.90 ± 0.90^{c z}	1.82 ± 0.91^{bc yz}	3.32 ± 0.00^{b xy}	1.70 ± 0.00^{b yz}	3.68 ± 0.00^{b x}
	1.5	0.56 ± 0.56^{b xy}	0.00 ± 0.00^{c y}	0.00 ± 0.00^{b y}	2.22 ± 1.11^{b x}	0.00 ± 0.00^{b y}	0.00 ± 0.00^{c y}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}
Vinegar	0.5	4.11 ± 0.05^{a x}	3.23 ± 0.42^{a x}	0.96 ± 0.96^{bc y}	3.79 ± 0.04^{a x}	4.40 ± 0.23^{a x}	4.02 ± 0.54^{a x}
	1	1.28 ± 1.28^{cd xy}	1.43 ± 1.43^{c xy}	0.82 ± 0.82^{cd xy}	0.00 ± 0.00^{c y}	0.00 ± 0.00^{c y}	1.69 ± 1.69^{c x}
	1.5	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}
	2	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{c x}	0.00 ± 0.00^{b x}	0.00 ± 0.00^{b x}

Counts (log cfu/g) ± standard deviation.

abc

Means with different letter in the column within the treatment are significantly different (p < 0.05).

xyz

Means with different letter in the row are significantly different (p < 0.05).

Inactivation of Salmonella in soil after 7 days

After 7 days, Salmonella populations remained the same or decreased in most treated samples. Salmonella Negev recovered after 7 days from soil treated with 0.5% eugenol, Sporan, or acetic acid were significantly lower than their corresponding populations recovered after 24 h. Conversely, the occasional significant increase was observed in Salmonella populations after 7 days, such as increase in Salmonella Typhimurium with eugenol at 0.5%, and Braedenrup and Newport with 1.5% Sporan treatment. Salmonella strains in soil treated with cinnamaldehyde were still undetectable at 7 days with concentrations of 0.5%, 1.0%, 1.5%, and 2.0%. Salmonella Negev recovered after 7 days in soil treated with 0.5% eugenol, Sporan, and acetic acid (2.93, <1, and 2.45 log CFU/g) were significantly lower than those recovered after 24 h (5.44, 4.71, and 4.96 log CFU/g), respectively. Similarly, Salmonella Thompson strain recovered in soil treated with 0.5% Sporan after 7 days (2.9 log CFU/g) were lower (p < 0.05) than the Thompson strain recovered at 24 h (4.85 log CFU/g). After 7 days of incubation, all Salmonella serovars were undetectable in soil treated with 1.5% or 2% acetic acid, or with 2.0% Ecotrol except Salmonella Braedenrup. Likewise, Salmonella Braedenrup, Salmonella Typhimurium, Salmonella Negev, and Salmonella Tennessee were undetectable in soil treated with 2.0% Sporan; Salmonella Negev and Salmonella Thompson were not detectable in soil treated with 2% eugenol.

Inactivation of Salmonella in soil after 28 days

In general, Salmonella populations were reduced further with nearly all treatments after 28 days of incubation at room temperature (22°C). Salmonella Negev populations detected after 28 days in soil treated with 0.5% Ecotrol, eugenol, or acetic acid were significantly lower than those detected after 7 days. Likewise, populations of Tennessee and Typhimurium serovars detected in soil treated with 1% Ecotrol, eugenol, or Sporan were significantly lower than those recovered after 7 days. Occasional increase in populations of some serovars was observed such as of Salmonella Braedenrup with 1% eugenol, and Salmonella Newport with 1.5% Ecotrol treatment. Overall, the increased concentration of the essential oils in the soil was associated with increased bacterial inhibition. Salmonella populations were undetectable in soil treated with 1.5% or 2% acetic acid, 2% Sporan, or 0.5%–2% cinnamaldehyde. All but Newport serovars were undetectable in soil treated with 2% Ecotrol, whereas only Salmonella Braedenrup populations were undetectable when soil was treated with 2% eugenol.

Discussion

Salmonella is an enteric bacterium; animals shed the bacteria in their feces, and soil that contains fresh or incompletely composted manure from wild or domesticated animals can act as a reservoir for the bacteria. Islam et al. (2004) found in their study that the survival profiles of Salmonella on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with Salmonella for several months. Multiple studies have shown that Salmonella can be isolated from fresh produce, and the prevalence of Salmonella in healthy whole fresh vegetables can be as high as 8% (Beuchat, 1996; Doyle, 2000). Therefore, it is of great importance to observe some measures of safety during the preharvest. This is the first study that demonstrates the efficacy of essential oils against salmonella in organic soil.

The inhibitory effect of cinnamaldehyde against Salmonella at room temperature was greater than the inhibitory effect of other oils used in this study, exhibiting up to 6 log reduction in Salmonella at all concentrations (0.5%, 1.0%, 1.5%, and 2.0%), and at all times (24 h, 7 days, and 28 days). Obaidat and Frank (2009) reported that cinnamaldehyde inactivated Salmonella and E. coli O157:H7 on sliced tomato at 4°C. Raybaudi-Massilia et al. (2009) reported that 0.7% cinnamon oil on fresh cut melons reduced Salmonella Enteritidis by >4 log in 21 days. Helander and others (1998) concluded that trans-cinnamaldehyde gained access to the periplasm and to the deeper parts of the bacterial cell, resulting in cell death. Gill and Holley (2004) indicated that cinnamaldehyde produced a decrease in the intracellular adenosine triphosphate (ATP) by ATPase activity, resulting in enough disruption of cell membrane to disperse the proton motive force by leakage of small ions (Raybaudi-Massilia et al., 2009).

The effect of Eugenol on Salmonella was inconsistent during the trial. For example, up to ∼4 log reduction in Salmonella Typhimurium was observed within 24 h in soil treated at 0.5% eugenol; however, its population increased after 7 days. The similar results were observed with Braedenrup serovar in soil treated with 1.5% eugenol. Increase in Salmonella populations after 7 days could be due to the repair of injured cells. Smith-Palmer et al. (2001) also reported initial inhibition of Salmonella Enteritidis with clove oil followed by recovery of this pathogen during the subsequent storage period. Kim and others (1995) also found that eugenol could kill initial bacterial populations by affecting the cellular structures or biochemical reactions of the growing bacterial cells, but once the bacteria overcame the inhibitory effect they multiplied rapidly. The active compound in Ecotrol, rosemary, is known to possess antimicrobial effect. Some researchers have shown that essential oils of rosemary, sage, and thyme were the most active against E. coli (Ouattara et al., 1997; Smith-Palmer et al., 1998).

Sporan was superior to Ecotrol and eugenol in reducing Salmonella in soil. This could be attributed to the synergetic effects of the active compounds such as rosemary oil, clove oil, and thymol present in Sporan. The bactericidal effect of Sporan on Salmonella serovars was noticeable after 24 h when 1.0% Sporan was used. Juven et al. (1994) suggested that the inhibition of Salmonella Typhimurium and Staphylococcus aureus by thyme oil was due to the hydrophobic and hydrogen bonding of its phenolic constituents to cell membrane proteins, thereby altering the membrane permeability. Thymol dissolves in the hydrophobic domain of cytoplasmic membrane and increases the permeability to ATP that results in lethal damage to bacterial cell (Ultee et al., 1999; Burt, 2004).

Salmonella populations reduced over time with increasing concentrations of the various treatments. The study showed a strong correlation between the oil concentration and the antimicrobial efficiency. Acetic acid, Sporan, Ecotrol, and eugenol showed dose-related increases in reducing Salmonella in soil samples. Organic soil, due to their complexity and their composition in nutrients, might explain the necessity of high concentration of essential oils. Smith-Palmer et al. (2001) found that higher concentrations of oils were needed to completely inhibit Salmonella Enteritidis in high-fat cheese. The complex nature of foods compared to laboratory media may allow rapid recovery of injured bacteria (Gill et al., 2002; Rasooli, 2007). Therefore, greater concentration of essential oils are needed in food, and possibly in soil to achieve the same effect of bacterial inhibition (Smid and Gorris, 1999; Rasooli, 2007).

Although Tween 20 was used in the preparation of the essential oil solution to increase the solubility of the hydrophobic compound and to aid its penetration into bacterial cell wall and membrane, the low efficacy of some of these treatments could be due to the lack of solution homogeneity. Zaika (1988) reported that test medium (i.e., water content, liquid medium, solid medium, food, or beverage), oil, and its active components (i.e., the process of oil extraction, concentration, geographic origin, and climate) and microorganisms tested (inoculation size, origin of culture, strain difference, and spore forming) influenced the antimicrobial activity of spices and their extracts, essential oils, or active components.

In the absence of antimicrobials, Salmonellae were reduced in organic soil by ∼1 log cfu/g (p < 0.05) in 28 days, which is not uncommon, as it has been reported to survive up to 968 days in soil (Jones, 1986). Other studies have indicated that soil is a possible reservoir for enteric pathogens (Santamaria and Toranzos, 2003), demonstrating that soil can be a possible source of contamination of agricultural products.

The widespread use of pesticides have significant drawbacks, including increased cost, handling hazards, concern about pesticide residues on food, and threat to human health and environment (Paster and Bullerman, 1988). Public demand of safer produce has increased interest on alternative soil preservative to replace synthetic chemical pesticides and to have a synergetic effect with compost. One such alternative is the use of essential oils with pesticidal activity, as well as they tend to have low mammalian toxicity, less environmental effects, and wide public acceptance (Paranagama et al., 2003). Soil provides a wealth of nutrients that can be utilized by a variety of microorganisms. Association with soil particles can provide bacteria with high concentration nutrients, due to the release of both organic molecules from attached algal cells, and protection against predation (Fish and Pettibone, 1995). For example, adhesion of Salmonella cells to soil particles correlates with cell surface hydrophobicity (Stenstrom, 1989), which is manifested by the modification of the bacterial outer membrane in response to changes in environmental conditions (Winfield and Groisman, 2003). Therefore, it is not surprising that enteric bacteria are capable to survive in soil. Many times, cells growing in soils are in a viable but not cultivable state and can easily be resuscitated by internalizing in vegetables, by earthworms, or by coming across a mammalian host (Williams et al., 2006). Therefore, it is important to find a natural solution for the treatment of organic soil to reduce enteric pathogens. From our results, the use of essential oils, their synergistic effects, and their application in soil should be further evaluated.

Conclusion

The antimicrobial activity of essential oils in controlling pathogens on fresh produce and other foods has been demonstrated. Soil is one of the major sources of fresh produce contamination at the farm level. In the absence of pathogen kill step in fresh produce processing, it is necessary to minimize its contamination at the preharvest level. This study indicated that essential oils can be exploited as a technique for future good agricultural practices. Use of these oils will significantly reduce potential transfer of pathogens from soil to fresh produce and, consequently, will help reduce fresh produce-related outbreaks and recalls.

Footnotes

Disclosure Statement

No competing financial interests exist.

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