The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.

Aims:  To investigate the presence of viable but non‐culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. Methods and Results:  Under low RH (47–69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (109 L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3–4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. Conclusions:  Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. Significance and Impact of Study:  Enumeration on culture media presumably under‐estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.

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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.

The saprophytic Paenibacillus and Bacillus spp. found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products. Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C. botulinum type A, proteolytic type B, and type E strains in a well diffusion assay. Nineteen strains were positive against C. botulinum. Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C. botulinum strains. The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P. polymyxa strains. The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purées under aerobic or anaerobic conditions. In co-cultures with the positive strain of P. polymyxa in nutrient broth and vegetable purées, a C. botulinum type E strain was inhibited whenever P. polymyxa reached stationary phase and produced its antimicrobial activity before C. botulinum began its exponential growth phase. The antimicrobial activity of P. polymyxa against C. botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity. Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides.

Aims:  To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). Methods and Results:  The addition of GB (≥25 mmol l−1) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0·05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. Conclusions:  Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. Significance and Impact of the Study:  Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.