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Keywords:

  • desiccation;
  • field culture;
  • fresh produce;
  • Listeria;
  • parsley;
  • relative humidity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To investigate the population dynamics of Listeria monocytogenes and Listeria innocua on the aerial surfaces of parsley.

Methods and Results:  Under 100% relative humidity (RH) in laboratory and regardless of the inoculum tested (103–108 CFU per leaf), counts of L. monocytogenes EGDe, LO28, LmP60 and L. innocua CIP 80-12 tended towards approx. 105 CFU per leaf. Under low RH, Listeria spp. populations declined regardless to the inoculum size (104–108 CFU per leaf). L. innocua CIP 80-12 survived slightly better than L. monocytogenes in the laboratory and was used in field cultures. Under field cultures, counts of L. innocua decreased more rapidly than in the laboratory, representing a decrease of 9 log10 in 2 days in field conditions compared to a decrease of 4·5 log10 in 8 days in the laboratory. Counts of L. innocua on tunnel parsley cultures were always higher (at least by 100 times) than those on unprotected parsley culture.

Conclusions:  Even with a high inoculum and under protected conditions (i.e. plastic tunnels), population of L. monocytogenes on the surface of parsley on the field would decrease by several log10 scales within 2 days.

Significance and Impact of the Study:  Direct contamination of aerial surfaces of parsley with L. monocytogenes (i.e. through contaminated irrigation water) will not lead to contaminated produce unless it occurs very shortly before harvest.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Listeria monocytogenes is responsible for approx. 26% and 43% of all deaths related to food-borne illness, respectively, in France in the 1990s and in United States in the period 1998–2002 (Vaillant et al. 2003; Lynch et al. 2006). Several outbreaks of listeriosis have been linked to the consumption of contaminated fresh produce and botanical preparations (Schlech et al. 1983; Ho et al. 1986; Farber et al. 1990). In particular, in 1981, contaminated coleslaw caused 41 cases of listeriosis and 18 deaths (Schlech et al. 1983). The contamination had been linked to a cabbage field fertilized with sheep faeces contaminated with L. monocytogenes. The survival of Listeria spp. during several weeks in soil fertilized with organic amendments and the transfer of Listeria from soil to aerial plant surfaces by splashing water was demonstrated (Van Renterghem et al. 1991; Girardin et al. 2005). Similarly, Salmonella enterica and Escherichia coli O157: H7 survived for 100–250 days in soil fertilized with organic amendments and transferred to fresh produce grown (Natvig et al. 2002; Islam et al. 2004a,b). However, in the above works, the survival of the food-borne pathogenic bacteria on the aerial fresh produce surfaces was not quantified independently of survival in the soil.

To fully evaluate the risk linked to preharvest contamination, it is necessary to evaluate the persistence of foodborne pathogenic bacteria on aerial fresh produce surfaces under field conditions. The survival and the growth of L. monocytogenes have been assessed on a broad range of postharvest fruits and vegetables such as endive, tomato, melon, watermelon, papaya, coleslaw, parsley and lettuce (George and Levett 1990; Beuchat and Brackett 1991; Carlin et al. 1996; Delaquis et al. 2002; Lang et al. 2004; Penteado and Leitao 2004), but no studies have examined the ability of L. monocytogenes to grow or/and to survive on the aerial surface of growing plants.

The objective of this study was to assess the population dynamics of L. monocytogenes and L. innocua on the leaf surfaces of a growing edible plant. Parsley was used as a model because it has been found to be contaminated with L. monocytogenes (i.e. presence in 25 g of parsley) (Porto and Eiroa 2001) and it is a common ingredient in ready-to-eat foods. First, we compared in laboratory parsley cultures the behaviour of L. innocua and L. monocytogenes considering various inoculum densities and various relative humidities. Then, experiments were conducted on field parsley cultures with L. innocua, including the situation of plants protected by agricultural plastic tunnel, a common practice for fresh produce production.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains, plant material and culture conditions

Listeria monocytogenes and L. innocua strains used in this study are listed in Table 1. The strains LmP60 and LiP60 were isolated in our laboratory from organic amendments used on horticultural crops and were identified as L. monocytogenes and L. innocua, respectively, according to the French standard AFNOR V08-055 (Anon 1996). The serovar of L. monocytogenes LmP60 was 1/2b (Kerouanton-Le Gall, AFSSA, Maisons-Alfort, France, unpublished data).

Table 1. Listeria spp. used in this study
StrainsSources
  1. *Strains were isolated at 2004 in INRA, Avignon, France.

  2. †Unité des interactions bactéries-cellules, Institut Pasteur Paris, France.

  3. ‡Department of Microbiology and Alimentary Pharmabiotic Centre, University College, Cork, Ireland.

L. innocua
 CIP 80-12Pasteur Institute Collection
 LiP60Isolated from amendments*
L. monocytogenes
 EGDe (serotype 1/2a)H. Bierne†
 LO28 (serotype 1/2c)R.D. Sleator‡
 LmP60 (serotype 1/2b)Isolated from amendments*

Seeds of parsley (Petroselinum sativum var. ‘Géant d’Italie’) were sown and raised in greenhouses on commercial substrate (TREF, Vulaines, France) for 3–4 weeks before being transplanted at the three-leaf stage in the field or in individual pots for laboratory experiments.

Under field conditions, experimental plots of parsley were set up at INRA research centre (Avignon, Vaucluse, France) during two periods in April and May 2005. Two rows of each ten parsley seedlings, spaced at 1 m were planted in 4-m2 field plots surrounded by a 2-m wide buffer zone. For experiments under plastic tunnels, plots were covered with agricultural plastic tunnels (transparent plastic, 30 μm thick, Triplast T30, tunnels 50 cm high). The irrigation method applied for parsley in field conditions was drip irrigation.

In the laboratory, parsley plants were placed in small greenhouses (57 × 42 × 26 cm). The average temperature was 20°C (±2), and the greenhouses lids were opened by 10 cm to obtain low relative humidity (RH) or closed to obtain a 100% RH. The soil was watered using a pipette whenever necessary during the experiment.

RH and temperature in the plant canopy were recorded with a datalogger (Almemo 2290-8, Ahlborn, France) at least three times a day. In field conditions for experiments under plastic tunnels, measures were performed both in the plant canopy and outside the tunnels.

Listeria spp. inoculation on parsley

To prepare the inoculum, a Listeria spp. culture grown on TSA (TSB + 15 g l−1 of agar, Oxoid, Basingstoke, UK) for 48 h at 30°C was inoculated into 10 ml of TSB for 24 h at 30°C and then was inoculated again into the same broth. After an overnight incubation at 30°C under shaking, bacterial cells in stationary phase were harvested after centrifugation (8000 g; 10 min), washed twice in a sterile saline solution (0·9% NaCl w/v) and suspended again in the same solution at the appropriate concentration.

Each parsley leaf was inoculated with 45 μl of the Listeria spp. suspension, deposited with a micropipette in three 15-μl drops. A total of 60 leaves in each greenhouse or each field plot were inoculated. Fifteen hours after inoculation, no free water was visible on the surface of inoculated leaves, even under 100% RH. Each experiment was performed twice at an interval of several weeks using independently prepared Listeria inocula and parsley plants. Noninoculated leaves incubated under the same conditions as inoculated leaves were used as controls.

The fate of parsley leaves under low RH of an adapted inoculum was compared with that of an inoculum grown in TSB. The adapted inoculum corresponded to L. monocytogenes inoculated on parsley leaves as described above and grown on parsley leaves under saturated humidity during 7 days. Then, the humidity was shifted from saturated to low RH and the culture in TSB, as described earlier, was inoculated on leaves on the same batch of parsley.

Bacterial counts

At each sampling time and for each treatment, three samples of three inoculated leaves each were randomly collected with sterile instruments. The average weight of each three leaf-samples was approx. 0·5 g. The three drops of the inoculum approx. covered a surface of 0·6 cm2 as one droplet had a diametre of 0·5 cm on the leaf). Leaf samples were blended in 10 ml of phosphate buffer (0·1 mol l−1, pH 6·8) with a Stomacher® (Merk Eurolab, Strasbourg, France) at full speed for 1 min. One hundred microlitres of appropriate dilutions in phosphate buffer of the leaf homogenate were spread on plates of Oxford media (Oxoid).

Listeria populations lower than the limit of detection of plating (i.e. 100 CFU per leaf), were determined using a MPN method (Anon 1996). The leaf homogenate prepared as previously described was mixed with the same volume of Fraser broth (Oxoid) with a Stomacher, and 1 ml of this mixture was used for serial ten-fold dilutions in half-diluted Fraser broth. After incubation at 30°C for 24 h, 100 μl of the enrichment cultures were transferred to 10 ml Fraser broth for a second enrichment. After 48 h of incubation at 37°C, the presence of Listeria spp. in the enrichment tubes was confirmed by streaking on Oxford agar. MPN calculations were performed with the MPN Calculator software available at http://members.ync.net/mcuriale/mpn.

Statistical analysis

Results were expressed as means ± SE of the replicate samples (n = 3) of the log10 of CFU or MPN per leaf. The effects of factors were tested by anova at 95% significance using Systat software version 9 (SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effect of inoculum size on the fate of Listeria spp. on laboratory parsley cultures exposed to different humidities

In order to determine the effect of the inoculum concentration on subsequent survival of cells of L. monocytogenes on laboratory parsley cultures, suspensions with different concentrations were inoculated. Under 100% RH and regardless of the inoculum size tested (103, 107 and 108 CFU per leaf), counts of L. monocytogenes EGDe converged to approx. 105 CFU per leaf in 5 days after inoculation (Fig. 1a).

image

Figure 1.  Fate of a Listeria monocytogenes EGDe inoculum at different concentrations on parsley leaves, under 100% RH (a) and under 45% (±3) RH (b) in laboratory cultures. Counts of L. monocytogenes were done by plating on Oxford agar for populations above 102 CFU per leaf, and by MPN in Fraser broth for lower populations. The horizontal dashed line corresponds to the limit of detection of culturable bacteria by MPN in Fraser broth. Symbols below the horizontal dashed line correspond to negative results for L. monocytogenes. bsl00041, bsl00001 and bsl00066 represent different sizes of inoculum. Bars represent the standard error (n = 3) for each sampling point (error bars smaller than symbols are not shown).

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Under nonsaturated RH (45% ± 3) and with a high inoculum dose (108 CFU per leaf), the counts of L. monocytogenes EGDe dropped by about 3 log10 within the first 2 days, then the remaining fraction of the population slowly declined (Fig. 1b). Finally, the L. monocytogenes populations declined by 5 log10 over 8 days. With an initial inoculum of 107 CFU per leaf, L. monocytogenes populations presented a similar biphasic pattern of decline (Fig. 1b). With an initial inoculum of approx. 104 CFU per leaf, L. monocytogenes dropped to the detection threshold (i.e. a decline of 4·5 log10) within 1 day and was not detected thereafter (Fig. 1b). Therefore, under low RH, L. monocytogenes populations declined regardless of the inoculum size. Over five experiments performed under nonsaturated humidity with L. monocytogenes EGDe and an inoculum size of approx. 108 CFU per leaf, no relation between the RH and the survival of L. monocytogenes was observed, within the range of RH tested (from 34% ± 8 to 60% ± 8).

Two other strains of L. monocytogenes LO28 and LmP60 were compared with EGDe on parsley leaves. Under low RH (59% ± 15 and 34% ± 8, respectively, for experiments with LO28 vs EGDe and LmP60 vs EGDe) and with an inoculum of 108 CFU per leaf, no significant difference was observed between these two strains and EGDe (P > 0·05). In both experiments after 8 days, counts of LO28 and LmP60 declined on parsley leaves by, respectively, 5 and 4·5 log10 (data not shown). Under 100% RH and with an inoculum of 103 CFU per leaf, counts of L. monocytogenes LO28 and LmP60 increased up to a level of approx. 105 CFU per leaf. Therefore, these two strains of L. monocytogenes tested on parsley leaves behaved as L. monocytogenes EGDe on parsley leaves under 100% and low RH.

In order to determine if Listeria adapted to parsley leaves could survive better than freshly cultured cells, we compared the survival on parsley leaves under low RH of L. monocytogenes grown on parsley leaves under saturated humidity (adapted inoculum) with that of the same strain grown overnight in a laboratory medium (nonadapted inoculum). No significant difference in survival of both inoculum was observed (P > 0·05). After 3 days under low RH on parsley leaves, the adapted and nonadapted inoculum lost, respectively, 3·9 and 3·4 log10 CFU per leaf (Fig. 2). Therefore, only nonadapted inoculum was used under field cultures.

image

Figure 2.  Fate of Listeria monocytogenes adapted (bsl00041) and non-adapted (bsl00043) on parsley leaves under 60% (±6), in laboratory cultures. The adapted inoculum corresponds to L. monocytogenes grown on parsley leaves under saturated humidity (first 7 days on the graph) and the non-adapted corresponds to L. monocytogenes grown overnight in a laboratory medium (inoculated on day 7). See Fig. 1 for keys of the figure.

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Listeria innocua strain CIP 80-12 was tested as a nonpathogenic Listeria surrogate of L. monocytogenes on parsley leaves. Under low RH (49% ± 10) and with a high inoculum of 108 CFU per leaf, counts of L. innocua CIP 80-12 decreased by 4·4 log10 within 8 days (Fig. 3b). For the lower cell-density applied on parsley leaves under low RH, L. innocua CIP 80-12 declined more slowly than L. monocytogenes EGDe (Fig. 1b), LO28 and LmP60, showing a slightly higher resistance of L. innocua on parsley leaves. Under 100% RH, with cells-density of 103, 105 and 108 CFU per leaf, counts of L. innocua CIP 80-12 converged to a level of approx. of 105 CFU per leaf (Fig. 3a) as observed for L. monocytogenes EGDe (Fig. 1a), LO28 and LmP60. In conclusion, under 100% and low RH, L. innocua survived similarly or slightly better on parsley leaves than L. monocytogenes.

image

Figure 3.  Fate of a Listeria innocua CIP 80-12 inoculum at different concentrations on parsley leaves, under 100% RH (a) and under 49% (±10) RH (b) in laboratory cultures. See Fig. 1 for keys of the figure.

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Fate of Listeria innocua on parsley leaves in field cultures

For safety reasons, L. monocytogenes was not used to inoculate field parsley cultures to avoid handling high concentrations of pathogenic bacteria in an open environment. In the laboratory, L. innocua CIP 80-12 showed a similar behaviour on parsley leaves under 100% RH and a similar or slightly better survival than L. monocytogenes EGDe, LO28, LmP60 under low RH. Therefore, using L. innocua instead of L. monocytogenes for experiments in the field will not lead to an underestimation of the risk. Whatever cells-density applied on parsley leaves under field conditions, L. innocua CIP 80-12 populations declined as observed for low RH in the laboratory, but the rate of decline was much higher under field conditions than in the laboratory: approx. 9 log10 in 2 days (Fig. 4) compared with 4–5 log10 in 8 days (Figs 1b and 3b). Listeria innocua was not detected after 3 days except with the highest inoculum, for which a small number of L. innocua seemed to persist on leaves. Experiments were performed in April. Average minimal RH and maximal temperatures (during daytime) were, respectively, 38% (±3) and 20°C (±1); average maximal RH and minimal temperatures (during night) were, respectively, saturation and 5°C (±2).

image

Figure 4.  Fate of a Listeria innocua CIP 80-12 inoculum at different concentrations on field parsley cultures. During the daytime, average minimal RH and maximal temperatures were, respectively, 38% (±3) and 20°C (±1), average maximal RH and minimal temperatures during the night were, respectively, saturation and 5°C (±2). See Fig. 1 for keys of the figure.

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The behaviour of L. innocua LiP60 was also assessed under field conditions and was compared with L. innocua CIP 80-12. During the first 2 days after inoculation and for two experiments, numbers of L. innocua LiP60 and CIP 80-12 on parsley leaves were not significantly different (P > 0·05, data not shown).

The effect of protecting the parsley crop with agricultural plastic tunnel was tested in May. Inside tunnels, the RH and the temperature given by datalogger were, respectively, 55% (±7) and 29°C (±9) during daytime. Outside tunnels, the RH was 38% (±13) and the temperature was 29°C (±9) during daytime. In both outside and inside tunnels during night, temperature dropped to 16 ± 1°C and RH reached saturation. Two days after inoculation on parsley leaves, numbers of L. innocua CIP 80-12 was detected only after enrichment in Fraser broth (MPN method; Anon 1996) and decreased from 109 to 102 MPN per leaf on tunnel parsley cultures and from 109 to 3 MPN per leaf on unprotected parsley cultures (Fig. 5). In this experiment, persistence of a small amount of Listeria cells was also observed on tunnel parsley leaves. Survival of L. innocua LiP60 was also higher on tunnel parsley cultures than on unprotected parsley cultures. Over four experiments, the survival of L. innocua (CIP 80-12 and LiP60) on parsley tunnel was always approx. 100 times higher than that on unprotected culture (data not shown).

image

Figure 5.  Fate of Listeria innocua CIP 80-12 on tunnel parsley cultures (bsl00000) and on unprotected parsley cultures (bsl00001). See Fig. 1 for keys of the figure. RH and the temperature were, respectively, 55% (±7) and 29°C (±9). Outside tunnel’s RH was 38% (±13) and temperature was 29°C (±9).

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Figures 4 and 5 present the survival of L. innocua on parsley leaves, respectively, in April and May. Their comparison shows that, for an initial inoculum of 109 CFU per leaf, L. innocua declined to 9 log10 in 2 days in both May and April, although temperatures were different (between 5 and 20°C in April and between 16 and 29°C in May).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Under 100% RH, the levels of populations of L. monocytogenes tended towards 105 CFU per leaf, presumably corresponding to the maximal carrying capacity of parsley leaves for L. monocytogenes. The decrease in counts of L. monocytogenes observed under 100% RH for inoculum higher than the carrying capacity of parsley leaves is presumably because of resource depletion. On laboratory parsley cultures under low RH, L. monocytogenes populations on leaves decreased, whatever the levels of inoculation, in particular for the lower inoculation level which was below the carrying capacity of the leaves existing under 100% RH. The decrease in L. monocytogenes populations under low RH is presumably because of the stress caused by reduced water availability. Low RH has been proposed as the main factor limiting survival of bacteria on plant surfaces. For instance, Salmonella Typhimurium populations decline rapidly under low RH on cilantro, bean and corn plants whereas they are able to grow under humid conditions on cilantro leaves (O’Brien and Lindow 1989; Brandl and Mandrell 2002). Epiphytic bacterial populations usually decrease after prolonged periods of dry weather but increase following rain and irrigation (Hirano and Upper 2000). Under low RH and high inoculum, the rate of decline of L. monocytogenes populations decreased after a few days. This may be explained either by the settlement of L. monocytogenes in rare and more protected sites on the leaf surface, as suggested by Wilson et al. for Pseudomonas syringae (Wilson and Lindow 1994), and/or by the presence in the inoculum of a small proportion of cells better adapted to the stress conditions encountered on parsley leaves. This behaviour on parsley leaves was observed for several strains of L. monocytogenes with different serotypes, EGDe, LO28 and LmP60, a strain isolated from organic amendments. The use of freshly cultured cells of Listeria may explain their poor persistence on parsley leaves under laboratory. However, in our study under laboratory conditions, the preadaptation of Listeria to the leaf environment (growth on leaves under saturated humidity) did not improve its survival on parsley leaves under low RH. Under 100% RH, L. innocua tended towards the same maximal carrying capacity of parsley leaves as the three strains of L. monocytogenes tested, and the rate of decline of L. innocua under low RH was slightly lower (<4·5 log10 in 8 days) than that of the L. monocytogenes strains (4·5–5 log10 in 8 days). Therefore, L. innocua CIP 80-12 was used on field parsley cultures.

On field parsley cultures, the populations of the two strains of L. innocua used declined faster than L. monocytogenes in laboratory cultures. As L. innocua survived slightly better than L. monocytogenes on laboratory parsley cultures, we can assume that L. monocytogenes would also decline faster in the field than in the laboratory. The same behaviour was observed with a laboratory-maintained strain (CIP 80-12) and a strain recently isolated from organic amendments (LiP60). On field parsley cultures, L. innocua populations on leaves decreased rapidly whatever the levels of inoculum, as observed under low RH in laboratory parsley cultures. In a previous work, parsley was grown in soil containing L. innocua, and in spite of high transfer from soil to parsley leaves, L. innocua was detected on parsley leaves only in low numbers and disappeared from leaves 30 days before it disappeared from the soil (Girardin et al. 2005). This is consistent with the low survival of L. innocua on parsley leaves we observed in the work.

In sewage water, which would represent an extremely worst case for irrigation water, maximal concentrations of L. monocytogenes were around 103 CFU l−1 (Watkins and Sleath 1981). In our work, the lowest initial inoculum we tested, 104 L. monocytogenes per leaf, was undetectable after 2 days on parsley leaves in the laboratory under nonsaturated humidity. Protected cultures and inoculum density increased survival of L. monocytogenes. Even with the highest inoculum tested on parsley, culture protected by tunnels, populations of L. monocytogenes on parsley leaves was reduced by at least 7 log10 in 2 days. The probability for L. monocytogenes to persist for more than 2 days on parsley leaves surfaces is presumably very low. However, experiments performed under field conditions with high inoculum showed that a small residual population of L. innocua remained stable on parsley leaves for at least 2–3 days. Therefore, it cannot be excluded that a very small fraction of L. monocytogenes populations survived for longer periods. Moreover, L. monocytogenes survives for several weeks in the soil and the soil can contaminate parsley leaves (Girardin et al. 2005).

Under nonsaturated RH, L. monocytogenes declined on parsley leaves, but we could not observe a clear relationship between RH and the rate of decline. In particular, in the field under tunnel, L. monocytogenes declined much faster than in the laboratory although the range of RH was similar during the day. In the field, UV irradiation could explain the rapid decline of L. innocua. Pedersen et al. showed that in day light UV irradiation could cause a 1 log10 decrease in 1 h of an Enterobacter cloacae on bean leaves. This is consistent with 6–7 log10 decrease in 5–6 h we observed on Fig. 5 (Pedersen and Leser 1992).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work received support from the French Ministry of Agriculture under an ‘Aliment-Qualité-Sécurité’ Programme, Contract AQS-R02/07. This work was performed by Nicolas Dreux in the course of his PhD studies, for which he received fellowships from the ‘Institut National de la Recherche Agronomique’ and from the ‘Conseil Régional Provence-Alpes-Côte d’Azur’.

We are grateful to Dr. Annaëlle Kerouanton-Le Gall (AFSSA, Maisons-Alfort, France) for strain LmP60 confirmation and serotyping.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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