Background: Listeria monocytogenes is a serious food-borne pathogen of global significance responsible for listeriosis in humans, generally associated with Ready-To-Eat foods (RTE). This study investigated the inhibitory potential of extracts from Terminalia chebula and Embelia ribes against L. monocytogenes in-vitro, in seafood and on food processing surfaces.

Materials and methods: Aqueous and ethanolic extracts were prepared from the fruits of Terminalia chebula and Embelia ribes. The antimicrobial activities the extracts were tested by disc diffusion and agar well diffusion assays. The Minimum Bactericidal Concentration (MBC) was determined using a microplate dilution assay and the minimum bactericidal concentration was also determined. The extracts were tested for their anti-biofilm activities on steel and plastic surfaces. Tuna meat artificially spiked with L. monocytogenes was dip-treated with plant extract to determine the effect on L. monocytogenes populations during storage at 2°C.

Results: Ethanolic extract of E. ribes and aqueous extract of T. chebula showed the highest anti-L. monocytogenes activity with a Minimum Bactericidal Concentration (MBC) of 12.5 mg/ml. Significant reductions in the biofilm-forming ability of L. monocytogenes on both plastic and stainless steel surfaces were achieved with ethanolic extract E.  ribes and aqueous extract of T. chebula. This extract, applied to tuna slices as a dip treatment, could reduce L. monocytogenes counts by over 2.6 log CFU.

Conclusion: This study provides strong evidence for the anti- L. monocytogenes activities of plant extracts, which can be potentially useful in controlling L. monocytogenes in foods and food processing environments. 

Keywords: Listeria monocytogenes; Terminalia chebula; Embelia ribes; Biofilm; Antimicrobial; Pathogen Control

Keywords: Listeria monocytogenes; Terminalia chebula; Embelia ribes; Biofilm; Antimicrobial; Pathogen Control

Introduction

L. monocytogenes is a Gram-positive, facultatively anaerobic, non-spore-forming rod-shaped bacterium, a natural inhabitant of the soil, water and decaying plant materials [1,2]. L. monocytogenes is also an important food-borne pathogen causing infections of varying intensities in the elderly, pregnant women, neonates and immunocompromised individuals [3]. Listeria infections manifest as septicemia, meningitis, encephalitis and in pregnant women, listeriosis may result in miscarriages or stillbirths [4-6]. In the United States alone, L. monocytogenes is responsible for about 1,600 illnesses each year, with over 94% hospitalization and 15-20% death [7]. In 2010, the global burden of listeriosis was estimated at 23,150 cases and 5,463 deaths [8]. Listeria outbreaks are most frequently linked to Ready-To-Eat (RTE) foods, including deli meats, scalded sausages, hot dogs, RTE salads, soft cheeses (particularly those made from unpasteurized milk), smoked seafood and contaminated fruits and vegetables [9,10]. RTE fishery products have been the sources of Listeria outbreaks, including cold-smoked and gravad fish, which are modified atmosphere packaged [11-13]. The incidence of L. monocytogenes in fish and fishery products has been reported from India [14-18].

Due to its ubiquitous nature, broad host range and the ability to proliferate at different environmental conditions, including refrigerated temperatures, L. monocytogenes is a successful foodborne pathogen and a serious threat to food safety. The bacterium is able to grow at broad ranges of temperatures (0.4°C to 45°C) and pH conditions (4.3-9.5) and survive at relatively low water activity (as low as 0.90) and high salt concentrations (up to 10%) [5,19]. The capacity of L. monocytogenes to withstand harsh environmental conditions, along with its ability to colonize, multiply and persist on processing equipment, poses a significant challenge to the food industry. Listeria has a strong ability to attach to stainless steel, glass, plastic and rubber to produce biofilms, which are resistant to commonly employed sanitizers and biocides, posing a threat to food safety [20-22]. L. monocytogenes has been isolated from various food processing surfaces and equipment, including saws and tables in slaughterhouses, the deck of fishing vessels, the floor of fish landing centers, plate freezers and food handlers from fish processing plants [23,24].

Considering the numerous outbreaks, product recalls and fatalities associated with L. monocytogenes contamination, preventing the entry, growth and proliferation of this pathogen in foods is of critical importance. Natural antimicrobials of bacterial and plant origin are potentially useful in controlling L. monocytogenes in foods and food contact surfaces. The interest in natural compounds as food preservatives is increasing, which has led to research efforts focused on identification, extraction and food applications of compounds from natural origins that have better bacteriostatic/bactericidal activities and offer consumer safety than their synthetic counterparts. Many compounds from plant sources have been effectively used as natural food preservatives to produce a microbiologically safe product with retained quality attributes [25,26]. Several natural compounds, like bacteriocins, organic acids, essential oils, chitosan, etc., have antimicrobial properties against Listeria spp. In this context, the goal of the present study was to identify new antimicrobials of plant origin for the control of Listeria monocytogenes in fish and processing surfaces.

Materials and Methods

Plant Materials and Preparation

Terminalia chebula and Embelia ribes used in this study were collected from Dakshina Kannada District, Karnataka, India. Based on their applications in traditional Indian medicine, the fruits of these plants were used for solvent extraction. The fruits were cleaned, dried under shade at room temperature for 24 hours and ground in a mixer into a fine powder.

Preparation of Aqueous Extracts of Plants

One-gram of powdered plant material was soaked in 10 ml of distilled water and incubated at room temperature (28 ± 2°C) for one week with intermittent shaking. The extracts were then centrifuged at 5000 rpm and the resulting supernatant was filtered using a 0.2 µm syringe filter. The final extract was used at a 100 mg/ml concentration for subsequent testing.

Preparation of Ethanol Extracts

Ethanol extracts were prepared by soaking powdered plant materials in 95% ethanol (1 g per 10 ml) for one week with continuous shaking. Afterwards, the crude ethanolic extracts were obtained by evaporating the solvent to dryness at 50°C under reduced pressure using a rotary evaporator (Steroglass, Germany). The resulting residues were resuspended in Dimethylsulphoxide (DMSO) to achieve a final concentration of 100 mg/ml (w/v), filtered through a 0.2 µm syringe filter and stored at room temperature until further use [27].

Antimicrobial Susceptibility Testing

Bacterial Strains and Culture Conditions

Listeria monocytogenes strains MTCC 1143, MTCC 657, ATCC 19111 were used in this study. The isolates were preserved on Brain Heart Infusion (BHI) agar slants at 4°C (HiMedia, Mumbai, India) and broth cultures were subsequently prepared in Tryptic Soy Broth (TSB).

Agar Well Diffusion Method

The agar well diffusion method was performed as described previously [28]. Briefly, L. monocytogenes cultures were grown in TSB (HiMedia, Mumbai, India) to 0.5 McFarland units. The liquid culture was uniformly spread on Tryptone Soya Agar (TSA) plates using sterile cotton swabs. The plates were allowed to dry for 5 min inside a laminar flow. Wells measuring 6-8 mm in diameter were cut into the agar surface and 100 µl of aqueous and ethanol plant extracts were dispensed into each well. The plates were incubated upright at 37°C for 16-18 hours. Antibacterial activity was assessed by measuring the diameter of the inhibition zones around the wells, with a gentamicin (10 µg) disc (HiMedia, Mumbai, India) serving as the positive control.

Agar Disc Diffusion Method

The inhibitory activity of plant extracts against L. monocytogenes was determined by agar disc diffusion method as described previously, with minor modifications [29]. TSA plates were inoculated with L. monocytogenes as described in the previous section. Sterile 10-mm filter paper discs were loaded with 50 μl of plant extract and placed on the inoculated plates. The final concentration of extract tested was 5 mg/disc. Each disc was pressed down to ensure complete contact with the agar surface and distributed evenly so that they were at least 24 mm apart. The agar plates were then incubated at 37°C. After 16 to 18 hours of incubation, the diameters of the zones of inhibition were measured. Gentamycin was used as a control.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)     

The MIC was determined by the micro broth dilution method [28]. Bacterial strains were grown to an OD₆₀₀ of approximately 0.5. The test antimicrobial solution was prepared and diluted to a 2X concentration. A volume of 100 µL of 2X BHI broth was dispensed into all wells of a sterile microtiter plate. Subsequently, 100 µL of the 2X antimicrobial solution was added to the first well in column 1 and mixed thoroughly by pipetting up and down 5-8 times. A serial two-fold dilution was performed across columns 1 to 10 by transferring 100 µL from one column to the next, mixing in each step and discarding 100 µL from column [10]. Sterile water (100 µL) was added to columns 11 and 12 to serve as controls. A Listeria monocytogenes inoculum was prepared at a concentration of 10⁴ to 10⁵ CFU/mL by dilution with broth or saline. Each well in columns 1 to 11 was inoculated with 5 µL of the bacterial suspension, while column 12 was left uninoculated to act as a sterility control and blank for plate reading. The plates were incubated at 37°C for 12-18 hours. Bacterial growth was recorded either manually or spectrophotometrically using an ELISA reader. The microbroth dilution assay used to determine the MIC of the plant extracts was also employed to assess their Minimum Bactericidal Concentration (MBC). Aliquots from each well of the microtiter plate were transferred onto TSA and incubated at 37°C for 24 h. The lowest concentration at which no visible bacterial colony growth was observed was recorded as the MBC of the extract against L. monocytogenes. This procedure was performed in triplicate to ensure reproducibility of the results.

Biofilm Assay Using Polystyrene Microtiter Plates

The ability of various Listeria monocytogenes strains to adhere to polystyrene surfaces was assessed using a microtiter plate assay as previously described [30]. Bacterial strains were cultured overnight in tryptic soy broth with 0.6% yeast extract (TSB-YE) without agitation. Overnight cultures were diluted 1:40 in fresh TSB-YE, vortexed and 100 µl of the suspension was dispensed into six wells of a microtiter plate. Wells containing only fresh medium served as blanks. The plates were incubated at 30°C for 40 hours. After incubation, wells were washed five times with sterile distilled water and air-dried for 30 min. Biofilm cells were stained with 1% freshly prepared crystal violet for 30 min at room temperature. Wells were then washed five times with 150 µl sterile distilled water, air-dried and destained with 200 µl of 95% ethanol at 8°C for 45 min. Finally, 100 µl of the destained solution was transferred to a fresh microtiter plate and absorbance was measured at 595 nm to quantify biofilm formation [22].

Inhibition of Biofilm Formation on Stainless Steel and Plastic Surfaces 

The effect of plant extracts on biofilm formation on stainless steel and plastic surfaces was evaluated using a previously described procedure with minor modifications [31]. Stainless steel and plastic surface chips measuring 10 × 10 mm were used to investigate biofilm formation by Listeria monocytogenes (MTCC 1143). Before use, each chip was individually cleaned and sanitized by immersion in a neutral detergent for 1 hour, followed by rinsing with sterile distilled water, drying and subsequent cleaning with 70% (v/v) alcohol. After the sanitation process, the stainless steel chips underwent an additional sterilization step by autoclaving at 121°C for 15 minutes to ensure complete sterility before biofilm assays. Listeria monocytogenes (MTCC 1143) culture was used as the inoculum after establishing a viable cell concentration of 10⁵ CFU/mL, determined through a growth curve constructed by correlating optical density with plate counts. Stainless steel and plastic chips (three chips per tube) were immersed in 5 mL of BHI broth. Plant extracts were added to each tube at concentrations equivalent to half of their respective MBCs to evaluate their inhibitory effects on biofilm formation by L. monocytogenes. Control tubes, containing chips without any plant extract, were prepared separately for each surface type. The broths were inoculated with 125 µL of the bacterial suspension (corresponding to a 1:40 dilution of the overnight culture) and incubated at 37°C for 48 hours without shaking to facilitate biofilm development.

After the 2-day incubation, the chips were carefully removed from each tube using sterile forceps and rinsed with sterile saline. Biofilms were scraped from the chips using sterile swabs, which, along with the chips, were transferred into tubes containing 9 ml physiological saline and vortexed for 2 min. Serial dilutions of the suspension were plated in duplicate on PCA and incubated at 37°C for 24 h. Colony-forming units (CFUs) were then enumerated to determine biofilm cell counts.

Storage Study of Tuna Slices Dip Treated with Extracts

Yellowfin tuna (Thunnus albacares), purchased from the local fish market in Versova, Mumbai, was cut into uniform slices weighing approximately 10g each and pasteurized by dipping in sterile water maintained at 90°C for 1 min to eliminate contaminating bacteria. Two extracts, which demonstrated the highest antimicrobial activity against Listeria monocytogenes, were selected for preparing the dip solutions. Aqueous solutions of the Terminalia chebula fruit extract and ethanolic extract of Embelia ribes fruit were prepared to dip the tuna slices. The concentration of the dip solution was adjusted to 12.5 mg/mL for both extracts, corresponding to their MBCs. The 10 g fish slices were then immersed in 100 mL of the prepared solution. After treatment, the slices were inoculated with L. monocytogenes by dipping them into a 100 mL cell suspension (10⁸ CFU/mL) and placed in separate sterile plastic pouches. Control slices were treated with sterile water and spiked with the same bacterial suspension. All slices were stored at 2°C and sampling was performed at regular intervals to determine the counts of L. monocytogenes. For this, 10 g of tuna slice was homogenized with 90 ml sterile physiological saline (0.85% NaCl w/v) and 10-fold serially diluted in physiological saline. One-hundred microliters from each dilution was spread plated in duplicates on TSA, incubated at 37°C for 24 hours and the colony counts were determined as CFU/g. 

Results

Preparation of Extracts

All the extracts were naturally coloured. Ethanol extracts were brightly coloured compared to their aqueous counterparts. Ethanol extracts could be easily resuspended in 100% DMSO, but this resulted in preparations that were more turbid compared to the original ethanol preparations. Filteration using 0.2 µm filter reduced the turbidity of aqueous extracts, but extracts in DMSO remained turbid. The extracts were active even after a month when stored at ambient temperature.

Antimicrobial Activities of Extracts

The extracts were tested for their antibacterial activity against L. monocytogenes (MTCC 1143) using both well and disc diffusion methods (Fig. 1). Disc diffusion test revealed maximum activity by E. ribes Ethanolic Extract (ERE), followed by T. chebula Water Extract (TCW). By the well diffusion method, the maximum Inhibition was exhibited by the extract TCW, followed by ERE. The details are shown in Table 1.

Minimum Bactericidal Concentrations (MBC) of Extracts Against L. monocytogenes

Both TCW and ERE exhibited an MBC of 12.5 mg/ml, whereas TCE was bactericidal at 25 mg/ml (Table 2). All three L. monocytogenes strains tested were inhibited at comparable MBCs, indicating no significant variation in strain sensitivity. Due to the color and turbidity of the extracts, visual determination of MIC was unreliable; therefore, subsequent experiments were conducted using MBC or ½ MBC concentrations of the extracts.

Effect on Biofilm-Forming Ability of L. monoctyogenes

Three strains of L. monocytogenes, viz., MTCC 1143, MTCC 657 and MTCC 1143, were tested for their ability to form biofilms on the polystyrene surface. MTCC 1143 exhibited better biofilm-forming ability compared to the other two strains (data not shown). Based on this, MTCC 1143 was selected to study further the effect of TCW and ERE on the biofilm-forming ability of this strain on steel and plastic surfaces.

Biofilm Formation on Plastic and Steel Surfaces and Its Inhibition

L. monocytogenes (MTCC 1143) formed biofilms on steel and plastic chips after 40 hours of incubation. Treatment with extracts at their ½ MBC significantly reduced the biofilm cell counts on both surfaces compared to the control. Both the extracts were effective against biofilm formation, with more than one log reduction in the biofilm cell counts (Table 3). A higher reduction of biofilm quantity was observed on the plastic surface than on steel (Fig. 2).

Dip Treatment of Tuna Slices with Plant Extracts

The extracts ERE and TCW, which showed maximum bactericidal activity, were chosen to give a dip treatment on Yellowfin tuna slices spiked with L. monocytogenes. The dip solutions were prepared with a final 12.5 mg/ml concentration for both extracts. A one-minute dip significantly (p=0.05) reduced the L. monocytogenes counts in both the treatments (Table 4). The decreasing trend in counts continued for an additional day (day 3) of storage at 2°C (Fig. 3). From day 3 onwards, the bacterial counts increased steadily in both treatments. Both the extracts were effective in reducing the count significantly compared to the control. Tuna slices treated with extract ERE showed fewer survivors than TCW, while the control slices did not show much difference in count throughout the storage period (Fig. 3).