2018 Volume 24 Issue 5 Pages 911-920
Outbreaks of foodborne illness caused by Listeria monocytogenes in or on fresh produce have been reported. Several contamination routes have been proposed and the cultivation process is one of the suspected routes. Leaf lettuce is one of the most important fresh produce products. In the present paper, we investigated the possible routes of L. monocytogenes contamination in leaf lettuce during cultivation. Leaf lettuce was cultivated in soils inoculated with cocktails of three isolates of L. monocytogenes belonging to different serotypes (1/2a, 1/2b, and 4b). The viability and injury state of L. monocytogenes in soils, and bacteria survival in or on leaf lettuce were investigated during 10 weeks of cultivation. Soils were artificially contaminated with L. monocytogenes at levels of 4, 6 or 8 log CFU/g, followed by cultivation of leaf lettuce in the contaminated soils. Populations of L. monocytogenes in the soil decreased to less than the detection limit (< 2 log CFU/g) at 8–10 weeks after the start of cultivation. L. monocytogenes was detected in some harvested leaf lettuce leaves at low levels, almost equivalent to the detection limit. As L. monocytogenes was not detected in the leaves of leaf lettuce plants cultivated in highly-contaminated soils after surface disinfection, the possibility of internalization of L. monocytogenes into leaf lettuce plants was considered low. Spraying of water contaminated with bacteria at greater than 3.2 log CFU/plant led to the survival of bacteria on the leaf lettuce leaves even after seven days. Furthermore, leaf damage prolonged the survival period of bacteria on the leaves.
Outbreaks of foodborne illnesses associated with fresh produce and minimally processed fruits or vegetables frequently occur in developed countries (Kozak et al., 2013; Sivapalasingam et al., 2004). In 2011, outbreaks caused by the unusual enteroaggregative verocytotoxin-producing Escherichia coli O104:H4 were reported, and the incriminated food items were bean and seed sprouts (ii). Furthermore, a Salmonella Agona outbreak linked to papayas from Mexico (iii) and outbreaks caused by Salmonella Poona infection in cucumber (i) were reported in the United States in 2011 and 2015, respectively. Furthermore, in 2011, Listeria monocytogenes infection associated with cantaloupes was also reported in the USA (McCollum et al., 2013). In Japan, outbreaks of E. coli O157:H7, which were linked to minimally processed foods originating from Chinese cabbage and cucumbers, occurred in 2012 (iv) and 2014 (Tabuchi et al., 2015), respectively.
In some outbreaks caused by fresh produce or minimally processed vegetables, cultivation processes have been suspected as points of contamination by pathogens (Beuchat, 1996; Tauxe et al., 1997). During cultivation, inappropriate handling by workers using contaminated water may lead to contamination of fresh produce by pathogenic bacteria. Thus, we and several other researchers have attempted to clarify the possible contamination routes of pathogens in fresh produce. In particular, water, which is used for irrigation, mixing with pesticides, washing produce, or making processed foods, was proposed as one of the contamination sources (Beuchat, 1996). If pathogens contaminate the edible parts of fresh produce plants, they might trigger a food-poisoning incident.
In 1979, an outbreak of listeriosis in Boston was suspected to be caused by the consumption of raw vegetables such as lettuce (Ho et al., 1986). As described above, the causative food of the listeriosis outbreak in the USA in 2011 was cantaloupes (McCollum et al., 2013). L. monocytogenes is widely distributed in the environment; thus, it is possible that L. monocytogenes can contaminate food materials including fresh produce at several points from farm to table. In fact, L. monocytogenes contamination of fresh produce, which was sold at the supermarket or recently harvested at the farm, such as cabbage, lettuce, radish, tomato, and cucumber were reported (Arumugaswamy et al., 1994; Heisick et al., 1989; Prazak et al., 2002; Vahidy et al., 1992).
Several points, such as cultivation at the farm, transportation, and handling during packaging etc., were considered to be possible critical sources for contamination of fresh produce. As shown by Prazak et al. (2002), careless cultivation, i.e., use of contaminated soil or water, might lead to contamination of fresh produce with pathogens such as L. monocytogenes. The cultivation step should be considered as one of the important contamination points. Several researchers have investigated the mechanisms of L. monocytogenes contamination in fresh produce during cultivation (Chitarra et al., 2014; Honjoh et al., 2016; Van Renterghem et al., 1991). We previously reported the possibility of contamination of tomato fruits by L. monocytogenes via contaminated soil or water (Honjoh et al., 2016). Although tomato fruits are not typically in close proximity to the soil, the edible parts of leafy vegetables are. Thus, the risk of soil contamination by certain pathogenic bacteria is greater in leafy vegetable such as lettuce.
On the other hand, several months are required to cultivate fresh produce. If a pathogen contaminates fresh produce, the pathogens might be exposed to low nutrients or water, or other adverse physiological conditions for extended periods (Honjoh et al., 2016). There are several kinds of cultivation methods in vegetable production, and are dependent on the kind of vegetable. In other words, bacteria live in varied circumstances (temperature, humidity and so on), which are dependent on the cultivation methods. Thus, the survival rate and injury state of bacteria on fresh produce will be dependent on the various vegetables. As is well known, injured bacteria can recover after improvement in their surrounding environments, leading to food poisoning. Thus, it is important to study the survival period or injured states of contaminated bacteria in produce.
In the present paper, we attempted to clarify the mechanism of L. monocytogenes contamination in leaf lettuce plants, via soil and water, and to determine the viability or injury state of the bacteria during cultivation. Furthermore, bacterial survival on the surface of leaf lettuce was also investigated.
Bacterial strains and culture conditions Isolates of Listeria monocytogenes belonging to 1/2a, 1/2b, or 4b serotypes in our laboratory stock (Liu et al., 2012) were used. Three strains of L. monocytogenes were separately inoculated to 100 mL of Luria broth (BD Diagnostics, Sparks, MD, USA) and incubated with shaking at 37 °C overnight. The cultured bacterial cells were harvested by centrifugation at 8,000 × g for 5 min, and resuspended in phosphate buffered saline (PBS) at concentrations of approximately 9 log CFU/mL by adjusting the OD600 to 0.3. The cell suspensions of three strains were mixed at equal volumes. Then, the mixed suspension was diluted with PBS to cell concentrations of 3, 5, or 7 log CFU/mL and used for further experiments.
Preparation of soil contaminated with L. monocytogenes Commercially available soil (peat-moss/perlite/vermiculite) was purchased from Oishi Bussan Co., Ltd., Tokyo, Japan. About 5 kg of soil was autoclaved at 121 °C for 20 min before inoculation. Approximately 100 g of dry soil was mixed with 50 mL of the L. monocytogenes mixture at 5, 7, or 9 log CFU/mL, respectively, resulting in 150 g of wet soil. The soil concentrations of L. monocytogenes were approximately 4, 6, or 8 log CFU/g after mixing. These contaminated soils were then placed in individual plastic pots (diameter: 7.5 cm).
Disinfection of seeds Seeds of Lactuca sativa var. crispa (Banchu Red Fire) were obtained from Takii Seed Co., Ltd., Kyoto, Japan. For disinfection, the seeds were dipped in 70% ethanol for 10–15 s, and then in 0.2% sodium hypochlorite solution for 10 min. After disinfection, the seeds were rinsed three times with sterile water for germination.
Growth condition of leaf lettuce plants The disinfected seeds were sown in pots with the contaminated soil, and leaf lettuce plants were grown at 20 °C under a photosynthetic photon flux density of 40 µmol m−2 s−1 with a photoperiod consisting of 16-h light in the growth chamber (Model CLE-303; TOMY SEIKO Co., Ltd., Tokyo, Japan). As regular maintenance, 20 to 30 mL of sterile tap water was added directly into the soil without contacting the leaves or stems every two or three days and 20–30 mL of nutrient solution (0.2% Hyponex; Hyponex, Osaka, Japan) was applied to leaf lettuce plants once per week.
Sampling methods for soil and leaf lettuce During cultivation, approximately 5 g of the soil samples was analyzed for L. monocytogenes population every two weeks. After 10 weeks of cultivation, lettuce plants were harvested to determine viable bacteria and L. monocytogenes counts. For microbial analysis, three leaves from the outside of the plants were harvested as a sample set.
Surface disinfection of leaf lettuce To determine whether L. monocytogenes was internalized to the leaf lettuce or attached to the surface of leaves, harvested leaf lettuce was disinfected with 500 mL of 70% ethanol for 1 min, then rinsed twice in 500 mL of sterile water for 1 min. Next, leaf samples were further disinfected in 500 mL of 10% of sodium hypochlorite for 1 min and rinsed twice in 500 mL of sterile water for 1 min. All disinfection steps were carried out in a 500 mL beaker with a stirring bar at room temperature. In order to confirm the success of disinfection of the surface of leaves, the rinse water at the final step was subjected to a qualitative test for L. monocytogenes.
Irrigation with contaminated water to maintain high soil levels of L. monocytogenes To examine the prevalence of L. monocytogenes on growing leaf lettuce plants in 150 g of wet soil, which had been prepared at 8 log CFU/g as described above, 20 mL of approximately 9 log CFU/mL of the L. monocytogenes cell suspension in sterile water was prepared and added to the soil by irrigation every two weeks. The number of L. monocytogenes in the soil was determined from the collected soil (5 g) just before irrigation every two weeks, except for at the start of the investigation. The initial level of L. monocytogenes was determined from soil that was collected after contamination. Leaf lettuce plants were cultivated in the soil for 10 weeks and leaves were harvested for detection of L. monocytogenes.
Determination of L. monocytogenes viable counts in soil and leaf lettuce Soil and leaf lettuce samples were respectively transferred to Stomacher bags. Next, a ten-fold dilution of the sample was made by adding PBS, and the samples were homogenized for 30 s using a Stomacher. Homogenized samples were serially diluted with PBS, and then the diluted samples (100 µL) were spread onto tryptic soy agar (TSA; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 0.6% dried yeas t extract (YE; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), CHROMagar listeria (CHROM; Kanto Chemical Co., Inc., Tokyo, Japan), CHROM containing 4% NaCl (CHROM+NaCl) in duplicate and incubated at 37 °C for 48 h. All colonies on TSA containing 0.6% YE (TSAYE) were counted as total viable bacterial counts. On the other hand, colonies of L. monocytogenes exhibiting light blue with halos on CHROM were recognized as positive. Positive colonies on CHROM were counted as the sum of the number of injured and normal (non-injured) cells, and positive colonies on CHROM+NaCl were counted as non-injured normal cells of L. monocytogenes as previously reported (Honjoh et al., 2016). Thus, the values obtained by subtracting the numbers of positive colonies grown on CHROM+NaCl from those on CHROM were judged as the numbers of L. monocytogenes injured cells.
Qualitative detection of L. monocytogenes in soil or leaf lettuce For qualitative analysis, samples were subjected to primary or secondary enrichment procedures before plating onto CHROM. Half Fraser Broth (HFB; Oxoid, Thermo Fisher Scientific Inc., Tokyo, Japan) was used as a primary enrichment broth. Samples (e.g., 1 g) were suspended in 9 volumes (e.g., 9 mL) of HFB and homogenized for 30 s using a Stomacher, a hand-type homogenizer, or a vortex mixer. Next, the homogenized samples were incubated at 30 °C for 48 h. Then, one loopful of the culture in HFB was spread onto CHROM for confirmation of L. monocytogenes growth. In secondary enrichment, 1 mL of the culture in HFB was inoculated into 9 mL of Fraser Broth (FB; Oxoid) and the inoculated samples were incubated at 37 °C for 48 h. Then, one loopful of the culture in FB was spread onto CHROM for confirmation of L. monocytogenes growth. Positive colonies were subjected to colony direct PCR with hlyA primer set 7 (forward primer: 5′-AAATCATCGACGGCAACCT, reverse primer: 5′-GGACGATGTGAAATGAGC) as described in the method of Liu et al. (2012) for confirmation of L. monocytogenes.
Survival of L. monocytogenes on leaf lettuce To simulate contamination by sprinkler irrigation, a suspension of L. monocytogenes at various cell concentrations was sprayed onto leaf lettuce plants. Concentrations of L. monocytogenes were determined as CFU/mL by directly plating the bacterial suspension onto CHROM. By spraying once, an approximately 0.5-mL volume was released from the spray bottle. A volume of 4 mL was directly sprayed onto leaf lettuce plants. After 7 days, whole leaf lettuce plants were used for qualitative detection of L. monocytogenes.
Effect of leaf damage on the survival of L. monocytogenes To investigate the survival of L. monocytogenes on the surface of leaf lettuce, 10 µL of L. monocytogenes suspensions (approximately 6 or 8 log CFU/mL) was surface spotted on the veins of leaves at 10 weeks of cultivation. In order to investigate the effect of leaf damages on the survival of L. monocytogenes, a shallow cut was made in the main vein of leaves with a knife before inoculation. For the control, undamaged leaves were used as intact leaves. Cultivation of leaf lettuce plants was continued as normal. Just after inoculation and on the 3rd, 6th, 9th, and 12th day after inoculation, only the inoculated parts (in a 2 cm square) of the leaves that were cut with the knife were harvested for qualitative detection of L. monocytogenes.
Viability and injury of L. monocytogenes in initially- and highly-contaminated soils Survival of L. monocytogenes in soil during cultivation of leaf lettuce plants was investigated. As shown in Fig. 1, the population of L. monocytogenes at an initial inoculum of 4 and 6 log CFU/g in soil increased slightly once within two weeks, and then decreased gradually to the detection limit (2 log CFU/g) or less after 8 weeks. L. monocytogenes counts in 8 log CFU/g-inoculated soils decreased gradually to the detection limit after 10 weeks. However, qualitative analysis of L. monocytogenes showed positive results in all samples (data not shown).
Changes in viable counts of L. monocytogenes in initially-contaminated soils during leaf lettuce cultivation. ○, Viable bacterial counts on TSAYE; ●, L. monocytogenes counts on CHROM; ▲, L. monocytogenes counts on CHROM+NaCl. Data are means ± standard deviations (SD) (n=3).
To maintain contamination levels in soils, L. monocytogenes-contaminated water was used to irrigate the soil every two weeks. Then, L. monocytogenes counts in soils were confirmed just before and one day after irrigation with the contaminated water (Fig. 2). Although L. monocytogenes counts were around 6 log CFU/g one day after irrigation, the counts gradually decreased at a rate of 1–2 log CFU/g per two weeks.
Changes in L. monocytogenes and viable bacterial counts in highly-contaminated soil during leaf lettuce cultivation. Contaminated water with L. monocytogenes was irrigated to soils every two weeks as indicated by arrows. ○, Total viable bacterial counts on TSAYE; ●, L. monocytogenes counts on CHROM; ▲, L. monocytogenes counts on CHROM+NaCl. Data are means ± SD (n=3).
We previously reported on the viability of Salmonella Typhimurium in soils during leaf lettuce cultivation (Honjoh et al., 2014). Salmonella was detected at levels of 3–6 log CFU/g during 10 weeks of cultivation. However, as shown in Fig. 1, L. monocytogenes counts decreased to the lower limit of detection (2 log CFU/g) by 8–10 weeks. Although not a direct comparison, the viability of L. monocytogenes in soils is likely to be lower than that of Salmonella during leaf lettuce cultivation. A similar tendency was found for the viabilities of Salmonella and L. monocytogenes in soils during tomato cultivation in our previous studies (Mishima et al., 2012; Honjoh et al., 2016). At present, although the reason for the difference between Salmonella and L. monocytogenes survival in soil is unknown, L. monocytogenes seems to be more sensitive to exposure to the soil environment than Salmonella.
In our previous study (Honjoh et al., 2016), L. monocytogenes counts in soils during cultivation of tomato plants decreased to the detection limit (2 log CFU/g) by 6–8 weeks. However, in the present paper, L. monocytogenes counts in soils during cultivation of leaf lettuce plants decreased to the detection limit by 8–10 weeks. Thus, L. monocytogenes in soil for leaf lettuce cultivation seemed to survive longer than in soil for tomato cultivation. The growth conditions of L. monocytogenes during cultivation of leaf lettuce and tomato plants differed in plant species and cultivation temperatures. Differences in plant species would influence the rate of L. monocytogenes decrease. Chitarra et al. (2014) investigated the effects of several leafy vegetables on the viability of L. monocytogenes in soils. They reported that L. monocytogenes survival in soils varied according to the species of cultivated vegetables, and suggested the possibility that basil plants extrude antimicrobial compounds from the root. Thus, the plant species might also have an influence on the survival of L. monocytogenes in soil. Furthermore, leaf lettuce and tomato plants were cultivated at 20 °C and 25 °C, respectively. Since L. monocytogenes are psychrophilic bacteria, the lower temperature (a temperature difference of 5 °C) of their surrounding environment might influence bacterial viability with long-term cultivation of the vegetable. Chitarra et al. (2014) investigated the viability of L. monocytogenes in a growth substrate during cultivation of lettuce at 24 and 30 °C. In the case of cultivation at 24 °C, L. monocytogenes survived in the growth substrate during 80−100 days after contamination. However, in the case of cultivation at 30 °C, L. monocytogenes was not detected in the growth substrate. Viability was obviously affected by the 6 °C temperature difference (between 24 and 30 °C). Thus, the lower temperature of lettuce cultivation might be one of the factors responsible for the delay in the rate of decrease in viable counts of L. monocytogenes in soils.
The injury of L. monocytogenes was also investigated. In Fig. 1, at any inoculation level, differences in colony numbers between CHROM and CHROM+NaCl were approximately 0.5–1.0 log CFU/g from just after the start of cultivation until the time of harvest, suggesting that the majority of detected L. monocytogenes cells were injured. Furthermore, even in highly-contaminated soils (Fig. 2), most of the detected L. monocytogenes cells were considered to be in an injured state, the same as in the case of the initially-contaminated soil.
Injured L. monocytogenes were observed just after the start of cultivation of leaf lettuce. This injury was thought to be caused by some stress during the culture of L. monocytogenes, preparation of the bacterial suspension, or preparation of the contaminated soil. However, since the proportion of injured L. monocytogenes increased even during cultivation of leaf lettuce, the soil environment was considered to be one of the stress conditions for L. monocytogenes. Of the L. monocytogenes cells detected, approximately 0.5–1.0 log CFU/g cells were estimated to be injured, similar to our previous investigation of tomato cultivation (Honjoh et al., 2016). Differences in vegetables between tomato and lettuce did not influence the appearance of injured L. monocytogenes cells. There are few papers focusing on the recovery of injured L. monocytogenes (Busch and Donnelly, 1992; Dreux et al., 2007). Of these, Dreux et al. (2007) reported that viable, but non-culturable (VBNC)-state L. monocytogenes cells, induced by dry conditions, on parsley did not recover growth, even after transfer to saturated humid conditions. As Busch and Donnelly (1992) demonstrated, heat-injured L. monocytogenes are likely to recover in enrichment media. However, in the present paper, L. monocytogenes injury was suggested to be induced by other stresses, such as low nutrient condition, drought, and so on. Since not all of the injured L. monocytogenes in soils are VBNC and the mechanisms of injury of L. monocytogenes in soils are complicated, it might be necessary to study the possibility of recovery of injured L. monocytogenes after improvement in bacterial environmental conditions.
L. monocytogenes counts in leaf lettuce plants cultivated in initially- and highly-contaminated soils After cultivation, counts of L. monocytogenes in sets of 3 leaves from the outside of leaf lettuce plants were investigated (Table 1). Out of all samples, three set samples were used for L. monocytogenes counts, which were around 2 log CFU/g. L. monocytogenes was detected and counted in two set samples.
| Initial inoculum (log CFU/g) | Sample No. | Leaves from outside to inside | L. monocytogenes counts (log CFU/g) |
|---|---|---|---|
| 4 | 1 | 1st 3 leaves | < 2.00 |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 2 | 1st 3 leaves | < 2.00 | |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 3 | 1st 3 leaves | < 2.00 | |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 6 | 1 | 1st 3 leaves | < 2.00 |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 2 | 1st 3 leaves | < 2.00 | |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 3 | 1st 3 leaves | < 2.00 | |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 8 | 1 | 1st 3 leaves | < 2.00 |
| 2nd 3 leaves | 2.30 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 2 | 1st 3 leaves | < 2.00 | |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | < 2.00 | ||
| 4th 3 leaves | < 2.00 | ||
| 3 | 1st 3 leaves | < 2.00 | |
| 2nd 3 leaves | < 2.00 | ||
| 3rd 3 leaves | 2.30 | ||
| 4th 3 leaves | < 2.00 |
To investigate the contamination and possible internalization of L. monocytogenes in leaves, leaf lettuce plants were cultivated in highly-contaminated soil where the concentration of L. monocytogenes was maintained at greater than 4 log CFU/g during cultivation (Fig. 2). To confirm contamination, the leaves of lettuce plants were harvested and quantitatively analyzed after 10 weeks of cultivation (Table 2). L. monocytogenes was quantitatively detected from either 3 leaves of each sample plant, although detected levels were not obviously high (approximately 2 log CFU/g). Furthermore, to investigate the internalization of L. monocytogenes from soils and via roots into the leaves, harvested leaves were disinfected and qualitatively analyzed. Although the data is not shown, L. monocytogenes was not detected from any of the disinfected leaves.
| Sample No. | Leaves from outside to inside | Viable bacterial counts (log CFU/g) | L. monocytogenes counts (log CFU/g) |
|---|---|---|---|
| 1 | 1st 3 leaves | 6.43 | 2.30 |
| 2nd 3 leaves | 6.48 | <2.00 | |
| 3rd 3 leaves | 6.43 | <2.00 | |
| 4th 3 leaves | 6.38 | <2.00 | |
| 2 | 1st 3 leaves | 6.35 | <2.00 |
| 2nd 3 leaves | 6.35 | <2.00 | |
| 3rd 3 leaves | 6.46 | 2.00 | |
| 4th 3 leaves | 6.42 | <2.00 | |
| 3 | 1st 3 leaves | 6.54 | 2.90 |
| 2nd 3 leaves | 6.84 | <2.00 | |
| 3rd 3 leaves | 6.67 | <2.00 | |
| 4th 3 leaves | 6.37 | <2.00 |
As shown in Tables 1 and 2, L. monocytogenes counts in the leaves (edible parts) of leaf lettuce plants, which had been cultivated in initially- and highly-contaminated soils, were quantitatively determined. Although contamination to the edible parts of most leaf lettuce plants cultivated in both soils was confirmed, the contamination levels were not obviously high. Because the edible parts of leaf lettuce are close to the soil during cultivation, the possibility of contamination of the edible parts from contaminated soils was suggested to be high. However, the contamination levels were quantitatively determined to be under or around the lower limit of detection. These results suggest that even if L. monocytogenes contaminated the edible parts of leaf lettuce plants, the viability of bacteria on the edible parts was not thought to be high.
Internalization of L. monocytogenes into leaf lettuce plants from soils and via roots during cultivation was investigated (data not shown). After disinfection with 10% sodium hypochlorite solution, L. monocytogenes was not detected in the edible parts of leaf lettuce plants, suggesting that the possibility of internalization of the bacteria into the leaves would be very low. Jablasone et al. (2005) inoculated L. monocytogenes to lettuce seeds and then investigated internalization of the bacteria. They did not confirm bacterial internalization after 49-day cultivation, which supports our results. Thus, even if the soil used in lettuce cultivation was contaminated with L. monocytogenes and the bacteria might contaminate the exterior of the lettuce, the bacteria were not likely to be internalized into the lettuce. On the other hand, Chitarra et al. (2014) reported that L. monocytogenes was internalized into lettuce leaves via the growth substrate, which was composed of a soil-less mixture (1:3 v/v, peat-perlite). Since we used commercially available soils for the cultivation of lettuce, conditions in their study, such as water content or nutrient composition, differed from those of the present study. Recently, Shenoy et al. (2017) also reported that L. monocytogenes was internalized into 20-day old romaine lettuce seedlings via artificially pre-contaminated seeds. Differences in media, contamination points, or the growth stages investigated among experimental conditions might lead to different results regarding internalization. In the present paper, internalization of L. monocytogenes into leaf lettuce leaves was not confirmed.
Survival of L. monocytogenes in leaves after spray inoculation To simulate the irrigation of leaf lettuce plants with contaminated water, spray inoculation was carried out. As shown in Table 3, on the 7th day after spray inoculation, L. monocytogenes was detected in leaf lettuce plants inoculated at greater than 1,600 CFU/plant.
| Inoculum (CFU/plant) | L. monocytogenes positive/Total | |
|---|---|---|
| Just after inoculation | 7 days after inoculation | |
| 68,800 | 3/3 | 3/3 |
| 24,200 | 3/3 | 1/3 |
| 10,400 | 3/3 | 3/3 |
| 4,960 | 3/3 | 3/3 |
| 1,620 | 3/3 | 1/3 |
| 1,600 | 3/3 | 0/3 |
| 800 | 3/3 | 0/3 |
| 160 | 3/3 | 0/3 |
| 80 | 1/3 | 0/3 |
| 16 | 0/3 | 0/3 |
| 4 | 0/3 | 0/3 |
| 0 | 0/3 | 0/3 |
Several contamination routes are considered during cultivation (Beuchat, 1996). One of the routes is pathogen contaminated-irrigation water. Assuming contamination by irrigation water, survival of L. monocytogenes on the surface of leaf lettuce leaves was investigated (Tables 3 and 4). As shown in Table 3, when leaf lettuce plants were contaminated with over 1,600 CFU/plant (3.2 log CFU/plant) of L. monocytogenes, bacteria were qualitatively detected even on the 7th day after inoculation. On the other hand, L. monocytogenes was not detected in leaf lettuce plants inoculated with bacteria at 160 (2.2 log CFU/plant) or 800 (2.9 log CFU/plant) CFU/plant. Likotrafiti et al. (2013) studied the changes in counts of L. monocytogenes on lettuce leaves during storage, and showed the effects of storage temperatures at 10, 20, and 30 °C on bacterial survival. They showed that L. monocytogenes did not grow on the leaf surface, and L. monocytogenes counts on lettuce stored at 20 °C decreased from 4 log CFU/cm2 to 2–3 log CFU/cm2 on the 5th day after inoculation. In their study, L. monocytogenes cells were suspended in Ringer's solution before inoculation. As Ringer's solution contains only several minerals (sodium chloride, potassium chloride, calcium chloride and sodium bicarbonate and so on.), it is unclear if the nutrient conditions are sufficient for the survival of L. monocytogenes on the leaf surface. This situation mirrors that of the present paper, and the nutrient conditions would not be conducive for the survival of L. monocytogenes on the leaf surface of lettuce stored or cultivated at 20 °C. Our results showed that if the irrigation water is contaminated with bacteria and plants are contaminated at greater than a certain level (e.g., 3.2 log CFU/plant), the contamination might to lead to food poisoning, depending on the remaining viable bacteria and cultivation or storage conditions.
| Inoculum (log CFU/spot) | Surface state of leaves | Enrichment culture | L. monocytogenes positive/Total | ||||
|---|---|---|---|---|---|---|---|
| Days after inoculation | |||||||
| 0 | 3 | 6 | 9 | 12 | |||
| 4 | Intact | Primary | 3/3 | 2/3 | 3/3 | 0/3 | 0/3 |
| Secondary | — | 2/3 | 3/3 | 0/3 | 0/3 | ||
| Damaged | Primary | 3/3 | 3/3 | 2/3 | 3/3 | 3/3 | |
| Secondary | — | 3/3 | 2/3 | 3/3 | 3/3 | ||
| 6 | Intact | Primary | 3/3 | 3/3 | 2/3 | 1/3 | 0/3 |
| Secondary | — | 3/3 | 2/3 | 1/3 | 0/3 | ||
| Damaged | Primary | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | |
| Secondary | — | 3/3 | 3/3 | 3/3 | 3/3 | ||
Effect of leaf damage on the survival of L. monocytogenes The effect of crop damage induced by workers during cultivation on the survival of contaminating L. monocytogenes was investigated. As shown in Table 4, L. monocytogenes was not detected in intact leaves inoculated at 4 and 6 log CFU/spot on the 9th and 12th day after inoculation, respectively. However, in damaged leaves, L. monocytogenes was detected even on the 12th day after inoculation.
Furthermore, assuming scarring of leaves during cultivation, L. monocytogenes was inoculated on the damaged parts (Table 4). When L. monocytogenes was inoculated onto intact leaves at 4 and 6 log CFU/spot, the bacteria were detected by the 6th and 9th day after inoculation, respectively. On the other hand, when the bacteria were inoculated onto damaged leaves, they were detected even on the 12th day after inoculation, regardless of inoculation level. There are few papers studying the effect of lettuce leaf damage on the survival of L. monocytogenes. However, the fate of L. monocytogenes on minimally processed, cut, or shredded lettuce during storage was investigated (Francis and O'Beirne, 2001; Takeuchi et al., 2000; Tian et al., 2012). Viable counts of L. monocytogenes on the cut edge of lettuce leaves were higher than those on the surface of leaves after incubation at 4 °C for 18 h (Takeuchi et al., 2000). Minimally processed lettuce leaves with L. monocytogenes were stored at 4 or 15 °C for 7 or 15 days, respectively (Tian et al., 2012). Furthermore, shredded lettuce leaves with L. monocytogenes were stored at 4 or 8 °C for 12 days (Francis and O'Beirne, 2001). In these papers, L. monocytogenes counts remained at the same levels or increased during storage. The storage temperatures (4, 8, 15 °C) of lettuce leaves were lower than the cultivation temperature (20 °C) of the present study. Thus, the temperature was considered to be one of the important factors in bacterial survival. Furthermore, Francis and O'Beirne (2001) investigated the survival and growth of L. monocytogenes on dry coleslaw mix (80% shredded cabbage and 20% shredded carrot), and reported that the population of L. monocytogenes on the dry coleslaw mix decreased during storage. Thus, dry conditions are also likely to have an influence on the survival and growth of L. monocytogenes. Nutrients and water extruded from damaged parts of lettuce leaves were utilized by L. monocytogenes for growth or survival. Especially, water would be critically important for the survival and growth of L. monocytogenes on vegetables. In addition, L. monocytogenes might internalize into the leaf via a damaged conduit and spread within the leaf. Since we investigated a limited area of the damaged leaf (Table 4), internalization of L. monocytogenes was not confirmed. However, it is conceivable that roots or stems are damaged by agricultural workers during the cultivation process; thus, it will be necessary to confirm internalization of L. monocytogenes via damaged regions and investigate the growth behavior of L. monocytogenes following internalization in the future.
Likotrafiti et al. (2013) investigated the effects of temperature and relative humidity on changes in the viable counts of artificially attached L. monocytogenes on lettuce, cucumber, and parsley. They reported that the viability of L. monocytogenes on the respective vegetables was affected by the surface state of the vegetables, storage temperature, and environmental humidity. Viable counts of L. monocytogenes on lettuce and parsley gradually decreased during 5-day storage at 20 °C. On the other hand, the bacterial counts on cucumber increased from 4 to 5 log CFU/cm2. Although they used three vegetables stored at the same temperatures, the growth or survival of the bacteria differed depending on the respective vegetable. In addition to the temperature effect on bacterial viability, they showed that low humidity influenced the survival of L. monocytogenes on cucumber epidermis during storage. Thus, surface state and humidity would be important factors for bacterial survival on lettuce.
In conclusion, we showed that L. monocytogenes counts in the soils gradually decreased during 10 weeks of cultivation of leaf lettuce plants. Bacterial contamination of the edible parts was confirmed, although the contamination level was quite low. However, internalization of the bacteria into leaf lettuce was not confirmed. Contamination of leaf lettuce leaves by L. monocytogenes at greater than a certain level would lead to bacterial survival greater than one week and leaf damage prolongs the survival period of the bacteria.
Acknowledgements This study was financially supported by the Ministry of Agriculture, Forestry and Fisheries of Japan under the title for improvement of food safety and animal hygiene [Elucidation of the emergence mechanism of injured bacteria, and development of technology for detection and control of the bacteria; Project No.11].
