Antimicrobial efficacy of Lippia citriodora natural extract against Escherichia coli and Enterococcus faecalis in “Piel de Sapo” melon juice

Abstract Background The minimal inhibitory concentration (MIC) of an aqueous extract of Lippia citriodora with reported functional properties (PLX®) was determined on two strains of Escherichia coli (E. coli) belonging to serogroups commonly associated with foodborne illnesses (E. coli O157:H7 ATCC 700728 and E. coli O111 isolate 172) in vegetable products and two control strains for antimicrobial tests assays (E. coli ATCC 25922 and Enterococcus–En. faecalis ATCC 29212). Results Mean MIC values at standard pH (7.4) in broth for the E. coli strains tested ranged from 4,444 µg/ml (35ºC) to 1,250 µg/ml (10ºC) and to 182 µg/ml (4ºC). At pH 5.5, conditions resembling those of melon juice, MIC was about 2 times higher at 35 and 10ºC compared with 4ºC. The MIC of En. faecalis was similar or slightly lower than those of E. coli at the conditions tested. In melon juice fortified with PLX® (2,500 µg/ml, maximum sensorial acceptable limit), the three strains of E. coli maintained their viability although none showed growth potential after 4 days at 4ºC. Conclusions PLX® could be added to melon juice to control E. coli O157:H7 and E. coli O111 during refrigerated storage, reducing the risk of microbiological contamination in this food.

1990s (Castillo, Martínez-Téllez, & Rodríguez-García, 2014) because of their growing involvement in foodborne disease outbreaks (0.5 outbreaks per year during 1973-1991 to 1.3 during 1992-2011, considering only outbreaks linked to a single variety of melon; Walsh, Bennett, Mahovic, & Gould, 2014). Therefore, it could be expected that the increase in melon outbreaks could be even higher considering not only all notified outbreaks, but also including those caused by fruit salads in which various types of melon, and taking into account the limited shelf life of fresh melon products (Walsh et al., 2014).
Melons may, occasionally, become contaminated with pathogenic microorganisms (including verotoxin-producing Escherichia coli-VTEC) during the preharvest process, harvest, and postharvest treatments (EFSA (European Food Safety Authority), 2013; USDA-FDA, 2018). These pathogens may, consequently, be transferred into the fresh products obtained from melon. Recently available data indicate that VTEC ranks second, after Salmonella spp., among the bacterial agents more frequently responsible for melon outbreaks in the United States during the period 1973-2011.
To date, as far as we know, there have been no VTEC outbreaks associated with low-acid melon juice (pH > 4.6); however, other acidic fruit juices (pH ≤ 4.6), such as fresh apple juice/cider, have been linked to a number of serious incidents related to VTEC infection in the United States during the 1990s (mostly serotypes O157:H7 and O111) (see Salomão (2018)). Both serogroups are among the VTEC most frequently associated with severe illnesses in the United States and EU and are characterized by low infective dose (Croxen et al., 2013). As a result, fruit juices with pH > 3 have to be considered potential sources for pathogenic strains of Enterobacteriaceae (Reinders, Biesterveld, & Bijker, 2001). In order to control the presence of possible pathogenic microorganisms in fresh juices, refrigeration must be used as an additional barrier to the more or less acidic pH of nonpasteurized fruit juices (ICMSF, 2011). Furthermore, the application of naturally occurring antimicrobial compounds, such as herbal extracts, has recently been proposed to increase the safety of fresh juices (Shahbaz, Kim, Kim, & Park, 2018). These natural products could even improve shelf life and functional properties of the original juice. In this sense, we recently proposed the use of an aqueous extract of Lippia citriodora (PLX ® ) in melon juice (MJ) to improve its functional properties and antioxidant capacity (Rúa et al., 2018).
For this, the aim of the presented research was to investigate the potential effect of PLX ® in inactivating E. coli and En. faecalis in broth under standard pH and temperature conditions, as well as those resembling refrigerated preservation of melon juices. Finally, we investigated the survival of these microorganisms in melon juice, fortified with PLX ® (PLX ® FMJ) at 2,500 µg/ml, stored under ordinary market preservation conditions (4ºC for 4 days; Rúa et al., 2018).

| Bacterial strains and growth conditions
Two verotoxigenic E. coli strains (O157:H7 and O111) and two con-

| MIC and MBC assays
The minimum inhibitory concentration (MIC) of PLX ® against four strains was estimated using the antimicrobial microdilution assay de- colonies and determine the appropriate dilution that corresponds to a concentration of 5.0 log 10 CFU/ml (initial inoculum), according to ICMSF and International (1978).
The MIC value was considered to be the minimum concentration of antimicrobial compound that inhibits visible growth of the test strain (Barry, 1976). In another set of experiments, we used LSM broth or Mueller-Hinton at pH value 5.5 by adjusting both broths with 1 mol/L HCl. Prior to the assay, the effect of the concentration of ethanol used was investigated, and we did not find any inhibitory effect of the growth of bacteria. Minimal bactericidal concentration (MBC) was determined by using aliquots from wells corresponding to the MIC values and from those with higher concentration and observing the lacking of growth on TSA. MBC was defined as ≥ 99.9% (3 log 10 ) decrease in viable cells (Barry, 1976). At least two independent tests were performed in duplicate with each strain.

| Survival and growth of E. coli and En. faecalis in "Piel de Sapo" melon juice at 4 º C
Melon juice was prepared as we have previously reported (Rúa et al., 2018). Freshly nonsterilized extracted MJ (25 ml) and PLX ® FMJ at a final concentration of PLX ® of 2,500 µg/ml were inoculated separately with 10 3 CFU/ml for E. coli and 10 5 CFU/ml for En. faecalis.
Survival and growth of the strains were assessed at 0, 1, 2, 3, and 4 days at 4ºC. At each sampling time, aliquots of 1 ml were taken, diluted in peptone water (0.1% w/v), and plated on TSA + 0.6% yeast extract (YE) (w/v) (TSAYE) (for En. faecalis) and on Tryptone Bile Agar with X-glucuronide, Biokar (TBX agar), for E. coli for enumeration. As the TSBYE medium for En. faecalis is not selective, the counts were corrected for the possible presence of this bacterium in MJ, using a control of juice without inoculation and taking into account the morphology of this bacterium. The plates were incubated for 24 hr at 35ºC, and the results were presented as log 10 CFU per milliliter of juice. The experiments were done in two batches of melon juice, each consisting of two conditions (MJ and PLX ® FMJ). In each condition included within the batch, samples were taken in duplicate each day to evaluate microbial growth. For each strain and for each day of storage, the difference between the log 10 CFU/ml at the evaluation day and the log 10 CFU/ml at the beginning of the experiment (t = 0) was calculated for the two lots. Growth potential (δ) was defined as the highest value obtained between two lots. The results were interpreted considering that a value δ > 0.5 log 10 indicates that the melon juice is able to support the growth of the bacteria tested (Beaufort, Cornu, Bergis, Lardeux, & Lombard, 2014).

| Statistical analysis
All data analyses were performed using the SPSS 24.0 package (SPSS software available at the University of León). One-way ANOVA with Tukey's multiple comparison test was used for the analysis of parametric data.

| Antimicrobial activity of PLX ® against E. coli and En. faecalis in broth under different temperature and pH conditions
The antibacterial activity (MICs and MBCs) of PLX ® for the four strains studied (Table 1)    a Data are expressed as means ± SD. In each column, different letters mean significant differences (p < .05). 24 hr of culture at 35ºC, 120 hr of culture at 10ºC, and 240 hr of culture at 4ºC. b It is the lowest concentration used in the assay. std pH, standard pH (7.4 for Escherichia coli or 6.7 for Enterococcus faecalis).
differences in cell wall composition of Gram-negative and Grampositive bacteria (Nikaido & Neidhardt, 1996;Nikaido, 2003). In the same way, E. coli was more resistant to malic acid (MBCs three times higher) than Listeria monocytogenes at 5ºC than at 20 or 35ºC for 24 hr in mango, pineapple, and papaya juices (Rathnayaka, 2013).

| Viability of E. coli and En. faecalis in refrigerated "Piel de Sapo" melon juice
When the three strains of E. coli and the strain of En. faecalis were grown in both MJ and PLX ® FMJ (Rúa et al., 2018) (Table 4) et al., 2015).
The effectiveness of natural antimicrobials, such as plant-derived compounds, has been demonstrated against foodborne pathogens (Holley & Patel, 2005;Tajkarimi, Ibrahim, & Cliver, 2010). Therefore, the increased occurrence of these microorganisms could serve as motivation to find effective natural antimicrobials to use as food preservatives. The results of the present study indicate that Lippia citriodora extract (PLX ® ) might be used as an alternative hurdle to reducing the risk associated with foodborne pathogen infection in fruit juice. temperature (4ºC) conditions. These results emphasize the need to check the effect of antimicrobial activity of plant extracts, such as PLX ® , on the food itself to assess possible interaction with other intrinsic factors of the food (e.g., composition in sugars and vitamins).

| CON CLUDING REMARK S
This work also underlines the importance of performing an individualized study of the spoilage and pathogen strains to determine their growth variability in food.

ACK N OWLED G M ENTS
María Rosario García-Armesto and Javier Rúa contributed with equal responsibility to conducting this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing, and written informed consent was obtained for all study participants.

Bacterial strain
Highest growth potential (δ) (log 10 CFU/ml) Note: δ ≤ 0.5 log 10 , the melon juice is not able to support the growth of the bacteria. δ > 0.5log 10 , the melon juice is able to support the growth of the bacteria (in bold). Growth potential (log 10 CFU/ ml) is estimated as the difference in the media of results at one day of storage at 4ºC and the media of the results at the onset of the storage (0 days). Abbreviation: MJ, plain melon juice; PLX ® FMJ, plain melon juice fortified with PLX ® .

MJ PLX ® FMJ
TA B L E 4 Highest growth potential (δ) (log 10 CFU/ml) among the batches for Escherichia coli and Enterococcus. faecalis strains used in this study, in "Piel de Sapo" plain melon juice and fortified with PLX ® (2,500 µg/ml) stored for 4 days at 4ºC