Potential Bio-Control Agent from Rhodomyrtus tomentosa against Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen implicated in many outbreaks of listeriosis. This study aimed at screening for the potential use of Rhodomyrtus tomentosa ethanolic leaf extract as a bio-control agent against L. monocytogenes. Twenty-two L. monocytogenes isolates were checked with 16 commercial antibiotics and isolates displayed resistance to 10 antibiotics. All the tested isolates were sensitive to the extract with inhibition zones ranging from 14 to 16 mm. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged from 16 to 32 µg/mL and 128 to 512 µg/mL, respectively. Time-kill assay showed that the extract had remarkable bactericidal effects on L. monocytogenes. The extract at a concentration of 16 µg/mL reduced tolerance to 10% NaCl in L. monocytogenes in 4 h. Stationary phase L. monocytogenes cells were rapidly inactivated by greater than 3-log units within 30 min of contact time with R. tomentosa extract at 128 µg/mL. Electron microscopy revealed fragmentary bacteria with changes in the physical and morphological properties. Our study demonstrates the potential of the extract for further development into a bio-control agent in food to prevent the incidence of L. monocytogenes contamination.


Plant Extract Preparation
The classified reference voucher specimen of Rhodomyrtus tomentosa leaves (NPRC0057) deposited in the Faculty of Traditional Thai Medicine, Prince of Songkla University, Thailand, was extracted according to the previously published methodology [12]. Briefly, R. tomentosa leaves were dried, grounded, extracted with 95% ethanol, and subsequently evaporated of the ethanol using a rotary evaporator. The extract was dissolved in 100% dimethyl sulfoxide (DMSO; Bangkok, Thailand) to obtain a concentration of 100 g/L and kept at´20˝C until use.

Antibiotic Susceptibility Screening of Isolates
Antibiotic susceptibility patterns of L. monocytogenes were determined using 16 commercial antibiotics commonly used in human and veterinary medicine, following a standard agar disc susceptibility assay proposed by the Clinical and Laboratory Standards Institute (CLSI) [18] with a slight modification. Three to five bacterial colonies from an 18 h culture were transferred into TSB and incubated at 37˝C for 4 h. The bacterial suspension was adjusted to McFarland standard turbidity of No. 0.5 and aliquot was spread plated on the surface of a well-solidified Mueller-Hinton agar (MHA; Difco Le Port de claix, France) plate, using a sterile cotton swab. The inoculated plate was air dried in a biosafety cabinet for 10 min and subsequently seeded with antibiotic discs (Oxoid, Hampshire, England) consisting of penicillin G (10 units), ampicillin (10 µg), gentamycin (10 µg), sulfamethoxazole-trimethoprim (25 µg), erythromycin (15 µg), chloramphenicol (30 µg), amikacin (30 µg), streptomycin (10 µg), vancomycin (30 µg), ciprofloxacin (5 µg), norfloxacin (10 µg), teicoplanin (30 µg), fusidic acid (10 µg), kanamycin (30 µg), imipenem (10 µg), and ceftazidime (30 µg). The plate was incubated at 37˝C for 18 to 24 h and zones of inhibition were measured using a vernier caliper. The means of inhibition zones and standard errors from two independent studies were recorded and compared with the respective antibiotic interpretative standards for Staphylococci as outlined in CLSI, except for ampicillin and penicillin G, for which interpretative standards were specifically provided for L. monocytogenes [18].

Antibacterial Activity of Rhodomyrtus tomentosa Ethanolic Leaf Extract against Listeria monocytogenes Using Agar Disc Diffusion Assay
Preliminary screening of L. monocytogenes isolates with ethanolic leaf extract of R. tomentosa was set up using the agar disc diffusion assay [18] with a slight modification. Adjusted bacterial suspension was spread plated on MHA following the procedure described in Section 2.3 above. Paper discs containing 2.5 mg of the extract were prepared by pipetting 10 µL aliquot of a 250 mg/mL solution of the extract onto sterile 6 mm Whatman filter paper discs and dried at 37˝C overnight. Discs containing 1% DMSO were used as control. Discs were seeded onto the surface of MHA followed by incubation at 37˝C for 18 to 24 h. The experiment was set up in two independent studies and means of inhibition zones˘standard error were calculated.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Rhodomyrtus tomentosa Ethanolic Leaf Extract against Listeria monocytogenes
Antibacterial activity of R. tomentosa ethanolic leaf extract was determined against L. monocytogenes isolates following a slightly modified broth microdilution method outlined by CLSI [18]. A four-hour culture of L. monocytogenes was grown in TSB as described in Section 2.3 above and adjusted to McFarland standard turbidity of No. 0.5. The suspension was diluted 1:100 in 0.85% normal saline solution (NSS) to a final concentration of approximately 10 6 CFU/mL. One hundred microliters of R. tomentosa ethanolic leaf extract, prepared in four folds of the highest concentration, were serially diluted to two folds of varying concentrations (512 µg/mL to 8 µg/mL) in a sterile 96-well flat bottom microtitre plate and subsequently inoculated with 100 µL of the bacterial cells in triplicates. The plant extract at various concentrations without the bacterial cells, 1% DMSO, and TSB media alone were included as controls followed by incubation at 37˝C for 24 h. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration that produced a complete inhibition of visible growth in the microtitre plate. Ten microliter aliquots were spotted on TSA plates from wells displaying no visible growth and the plates were incubated at 37˝C for 24 h. The minimum bactericidal concentration (MBC) of the extract was recorded as the lowest concentration completely preventing bacterial growth on TSA plates after incubation at 37˝C for 24 h. Penicillin G (Sigma, St. Louis, MO, USA) was used as reference antibiotic. All experiments were set up in triplicates for two independent studies.

Time-Kill Assay
The rate of killing by R. tomentosa ethanolic leaf extract against L. monocytogenes actively dividing cells in nutrient medium was investigated using a time-kill assay [14,19]. Multidrug-resistant food isolates including L. monocytogenes PSU-KV-133LM, L. monocytogenes FSL R2-574, and L. monocytogenes Scott A were selected for the study. Bacterial inoculum (10 6 CFU/mL) from growing culture in TSB prepared as described in Section 2.5 above was supplemented with the extract at 1 /2 MIC, MIC, 2 MIC, and 4 MIC and subsequently incubated at 37˝C for 24 h. Surviving cells were enumerated at time 0, 2, 4, 8, 12, and 24 h using a serial dilution and plate count method. Control cultures with 0.1% DMSO were incubated under the same condition. The experiment was set up in duplicates for three independent studies.

Test for Salt Tolerance
The inability of L. monocytogenes cells treated with R. tomentosa ethanolic leaf extract in the presence of 10% sodium chloride (NaCl; Emsure, Billerica, MA, USA) to grow on TSA was tested following Carson et al.'s [20] method with slight modifications. L. monocytogenes representatives used in Section 2.6 above were used in the study. Bacterial culture (10 6 CFU/mL) was prepared in TSB as described in Section 2.5 above. This was treated with 1 /2 MIC, MIC, 2 MIC, and 4 MIC of the extract and 0.1% DMSO in two groups consisting of either presence or absence of 10% NaCl in 96-well microtitre plates. Plates were incubated at 37˝C in a shaker (Forma Scientific, Inc., Ohio, OH, USA) at 150 rpm. One hundred microliter aliquot was sampled at 0, 2, 4, 8, 12, and 24 h and surviving cells were enumerated on TSA plates by serial dilution and plate count method. The study was set up in duplicates for three independent studies.

Determination of Bacterial Inactivation Using Rhodomyrtus tomentosa Ethanolic Leaf Extract
Bacterial inactivation assay was set up to study the behavior of L. monocytogenes cells in the stationary phase of growth when treated with R. tomentosa ethanolic leaf extract in normal saline solution, following the protocol by Pattanayaiying et al. [21] with slight modifications. The stationary phase of L. monocytogenes was pre-determined from a bacterial growth curve monitored for 24 h. Cells attained stationary phase from about 8 to 24 h and mid-stationary phase cells, cultured for 16 h, was used in the study. L. monocytogenes representatives used in Section 2.6 above were grown in TSB supplemented with 0.6% yeast extract (LabM, Lancashire, UK) (TSB-YE) overnight for 16 h. Cells were harvested by centrifugation at 8000 rpm for 15 min, washed five times using sterile NSS, and adjusted to a final concentration of approximately 10 8 CFU/mL in NSS. Cell suspensions were supplemented with the extract at 1 /2 MIC, MIC, 2 MIC, and 4 MIC and the control with 0.1% DMSO in flat bottom 96-well microtitre plates, followed by incubation at 37˝C for 12 h. Samples were taken at time 0, 0.5, 1, 2, 4, and 12 h and the remaining bacterial population was determined by serial dilution and plate count method. All experiments were set up in duplicates for three independent studies.

Scanning Electron Microscopy (SEM)
The effects of R. tomentosa ethanolic leaf extract on L. monocytogenes Scott A cells were observed using electron microscopy. Cells were grown in TSB-YE medium for 4 h and adjusted to approximately 10 6 CFU/mL. The culture was supplemented with the extract at MIC and 2 MIC. The control consisted of bacterial culture incorporated with 0.1% DMSO. The tubes were incubated at 37˝C for 12 h in a shaker at 150 rpm. After incubation, cells were harvested by centrifugation at 8000 rpm for 5 min, washed three times with NSS, subsequently coated onto slides and fixed with a 3% glutaraldehyde solution (Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The slides were washed three times with NSS and dehydrated at room temperature with various ethanol concentrations consisting of 30%, 50%, 70%, 90%, and 100% for 15 min at each step. Cells were dried further and sputter-coated with gold palladium under vacuum and the morphology was observed and photographed with FEI Quanta 400 FEG Scanning Electron Microscope.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Rhodomyrtus tomentosa Ethanolic Leaf Extract against Listeria monocytogenes Isolates from Ready-to-Eat Food and Food Processing Plant Environments
Minimum inhibitory concentrations and minimum bactericidal concentrations of the extract against isolates ranged from 16 to 32 µg/mL and 128 to 512 µg/mL, respectively (Table 3). MIC 50 (MIC at which 50% of the isolates were inhibited) and MIC 90 (MIC at which 90% of the isolates were inhibited) were 16 µg/mL and 32 µg/mL, respectively. MBC 50 (MBC at which 50% of the isolates were killed) and MBC 90 (MBC at which 90% of the isolates were killed) were both 256 µg/mL. In addition, the extract displayed similar MIC and MBC values of 32 µg/mL and 128 µg/mL, respectively, against the three reference strains investigated. The MIC value of penicillin G against isolates ranged from 0.125 to 1 µg/mL, while MIC 50 and MIC 90 were 0.250 µg/mL and 1 µg/mL, respectively. The MBC values of penicillin G against the isolates ranged from 1 to 4 µg/mL and MBC 50 and MBC 90 values were 2 µg/mL and 4 µg/mL, respectively. The MIC and MBC values of Penicillin G against L. monocytogenes Scott A were 2 µg/mL and >4 µg/mL, respectively, while these values against both FSL R2-574 and FSL F2-501 were 0.25 µg/mL and 1 µg/mL, respectively.

Time-Kill Curve of Listeria monocytogenes Treated with Rhodomyrtus tomentosa Ethanolic Leaf Extract
A reference strain, L. monocytogenes Scott A, widely used for ascertaining the efficacy of food processing and preservation techniques, one food strain, FSL R2-574 and one food isolate, PSU-KV-133LM, showing resistance to two antibiotics, were selected for time-kill studies (Figure 1

Reduction of Salt Tolerance in Listeria monocytogenes by Rhodomyrtus tomentosa Ethanolic Leaf Extract
R. tomentosa ethanolic leaf extract reduced salt tolerance in L. monocytogenes at 1 /2 MIC (16 µg/mL) in 4 h ( Figure 2). However, cells in the control group survived in the presence of 10% NaCl for 24 h in all tested isolates. Furthermore, cells were killed rapidly and more effectively with the plant extract in the presence of 10% NaCl than in the absence of salt.
Nutrients 2015, 7 7460 R. tomentosa ethanolic leaf extract reduced salt tolerance in L. monocytogenes at ½ MIC (16 µg/mL) in 4 h ( Figure 2). However, cells in the control group survived in the presence of 10% NaCl for 24 h in all tested isolates. Furthermore, cells were killed rapidly and more effectively with the plant extract in the presence of 10% NaCl than in the absence of salt. and PSU-KV-133 (F) after treatment with the extract at various concentrations + 10% NaCl. The detection limit was 10 2 CFU/mL.

Inactivation of Listeria monocytogenes Treated with Rhodomyrtus tomentosa Ethanolic Leaf Extract
Stationary phase cells were treated with the plant extract in suspension at different concentrations. At 1 /2 MIC, cells were inactivated by greater than 4-log units within 12 h of incubation in all tested isolates (Figure 3). At the MIC, a greater than 3-log reduction was observed within 4 h of treatment in L. monocytogenes Scott A and PSU-KV-133LM, while reduction was beyond detection limit within 2 h in FSL R2-574. Similarly, at 2 MIC, Scott A and PSU-KV-133LM were inactivated by greater than 3-log units in 2 h, while FSL R2-574 was inactivated beyond detection limit in 2 h. At the MBC, cells were inactivated beyond the detection limit (more than 6-log reduction) in 1 h, 2 h, and 4 h of incubation in FSL R2-574, PSU-KV-133LM, and Scott A, respectively. The number of cells in the control sample remained approximately the same within 12 h of incubation.
PSU-KV-133 (C), able to form colonies on tryptic soy agar after treatment with Rhodomyrtus tomentosa ethanolic leaf extract at various concentrations, and Scott A (D); FSL R2-574 (E); and PSU-KV-133 (F) after treatment with the extract at various concentrations + 10% NaCl. The detection limit was 10 2 CFU/mL.

Effects of Rhodomyrtus tomentosa Ethanolic Leaf Extract on Cells of Listeria monocytogenes Scott A, Determined by Scanning Electron Microscopy
After 12 h of treatment with the plant extract at 32 µg/mL and 64 µg/mL, few to no cells were left in the treated samples (Figure 4). Treated cells displayed reduced sizes when compared with the longer cells observed in the control sample with the incorporation of 0.1% DMSO. Incomplete separation of cells or abnormal cell division was displayed by dying cells. Treated cells were found to come close to one another, which may suggest an attempt to increase cell survival. Surfaces of treated cells appeared rough and distorted with cracking degenerative changes, while the surfaces of cells in the control sample appeared smooth with no changes. Nutrients 2015, 7 12

Discussion
Plant extracts are rich in aromatic substances and secondary metabolites, which have potencies as food preservatives. The present study was aimed at screening for a potential bio-control agent from Rhodomyrtus tomentosa against the foodborne pathogen L. monocytogenes. As observed in our study, antibiotic resistance in L. monocytogenes has been documented previously. Conter et al. [24] found resistance to 12 antibiotics in L. monocytogenes strains from food and food processing plant environments using the automated VITEK2 system. Our results revealed resistance to one or more commercial antibiotics as well as intermediate susceptibility to L. monocytogenes isolates from ready-to-eat food and food processing plant environments. Resistance genes in these isolates could be potentially transferable among strains, which may lead to increased therapeutic failure and constitute potential health hazards to consumers.
The results obtained from the time-kill analysis showed that the extract demonstrated an overall bactericidal activity against L. monocytogenes. Some studies have documented the bactericidal activity of R. tomentosa extracts against other pathogens using the time-kill assay. Vegetative cells and endospores of Bacillus cereus were reduced by at least 3-log units within 6 to 8 h and 2 h, respectively, after incubation with R. tomentosa ethanolic leaf extract at 2 MIC and 4 MIC [14]. Also, a bioactive compound isolated from R. tomentosa leaf (rhodomyrtone) at 4 MIC reduced 99.9% of the initial cell numbers of Staphylococcus aureus in 3 h [15].
In addition, NaCl can constitute an osmotic stress condition to contaminating microbial cells in food and this is a commonly used hurdle in the food industry. However, L. monocytogenes is capable of surviving and proliferating in the presence of high salt concentrations, which has lead to long-term adaptation to sublethal and stress conditions. In the present study, tolerance of L. monocytogenes to 10% NaCl was highly reduced. A number of genes upregulated by the alternate sigma (σ) factor (σ B ) are linked to the survival of L. monocytogenes in harsh environments. These genes were hypothesized to biosynthesize exopolisaccharides, which function in the modification of cell wall structure against high osmolarity [30]. Sublethal injury in microbial cells can distort the normal osmoregulating potential of the cell membrane and the ability of cells to exclude toxic substances. In our study, the reduction of salt tolerance in L. monocytogenes could be indicative of membrane damage in the cells, caused by the activity of the plant extract, thereby altering the cell membrane function as an osmotic barrier. A similar observation was reported by Carson et al. [20]. Treatments with tea tree oil and its compounds induced sublethal injury in the cell membrane of Staphylococcus aureus, causing loss of survival in growth medium incorporated with 5% and 7.5% (w/v) NaCl. The observations in our study may be a valuable insight into the effectiveness of multiple hurdle technology, using the plant extract in the presence of certain salt concentrations to control L. monocytogenes in food.
Microbial cells in the stationary phase of growth are generally less sensitive to injury from antibiotics than those in the exponential phase of growth [31]. In the present study, R. tomentosa ethanolic leaf extract demonstrated a remarkable activity against L. monocytogenes cells in the stationary phase of growth. Antimicrobial agents that inhibit the macromolecular synthetic process in bacteria, such as beta lactams and quinolones, often have minute effects on organisms in the stationary phase of growth. However, scanning electron micrographs of L. monocytogenes cells treated with R. tomentosa extract showed reduced cell sizes with abnormal cell division. This mechanism appears to be closely related to the mechanism of aminoglycosides' action, majorly involving the inhibition of protein synthesis in microbial cells [32]. The activity of the extract on stationary phase L. monocytogenes cells may therefore be an effect of several bioactive components in the extract with different mechanisms of action. Micrographs of treated cells showed that exposure to plant extract had rigorous effects on the viability of L. monocytogenes cells. Treated cells were positioned close together, which may be a strategy to increase survival [33], and cell surfaces appeared rough and distorted with cracking degenerative changes which were absent in untreated cells. These observations correlated well with salt tolerance inhibition results. The disruption of cell membrane integrity may have resulted in the loss of osmoregulation potential [20], thereby causing cell degeneration [34]. A previous study reported abnormal binary fission and irregular shapes in Streptococcus pyogenes cells treated with R. tomentosa ethanolic leaf extract. However, treated cells maintained cell wall structures similar to untreated cells in the study [35]. Sianglum et al. [13] observed damage to cell wall-and membrane-related proteins in Staphylococcus aureus by the subinhibitory concentration of rhodomyrtone, a pure compound isolated from R. tomentosa leaves. Interestingly, the observations were comparable to results found in our study. Furthermore, essential oil from the leaves of Macleaya cordata altered cell wall and membrane integrity in Ralstonia solanacearum [34], with a cell appearance similar to that of treated L. monocytogenes observed in our study.
R. tomentosa leaves, roots, buds, and fruits have been used as traditional treatments for inflammatory and infectious conditions such as diarrhea, dysentery, colitis, abscesses, and hemorrhages in Vietnam, China and Malaysia [36], and Thailand and Bangladesh [37,38] over a long period of time. Also, the extract has no effect on Gram-negative organisms which represent the majority of the intestinal microflora. The extract may therefore pose no significant risk to indigenous gut flora. Earlier published data indicated that toxicity of R. tomentosa ethanolic leaf extract on skin cells is very low. The IC 50 of Nutrients 2015, 7 7465 the extract was 476 µg/mL, which is approximately 15 to 30 folds of the MIC values (16 to 32 µg/mL) in the present study [39]. Reports on the cytotoxicity of compounds extracted from R. tomentosa leaves, tested on human embryonic kidney cells, demonstrated no toxicity up to the highest dose [40]. Overall, R. tomentosa ethanolic leaf extract appears to interfere with cell growth and cell division as well as disrupt cell membrane integrity in L. monocytogenes. The extract shows promise as a natural alternative to synthetic food antimicrobials to prevent the incidence of L. monocytogenes contamination.

Conclusions
The development of control measures for L. monocytogenes food contamination is important and still ongoing. Our study demonstrated the remarkable activity of R. tomentosa ethanolic leaf extract in inactivating this pathogen. The extract shows promise for further development into a bio-control agent against L. monocytogenes. R. tomentosa extract could be applied as a natural food additive to guard against hazards associated with L. monocytogenes food contamination without compromising food safety.