Recovery of Listeria monocytogenes genotypes in challenge tests

Listeria monocytogenes (LMO), a gram-positive zoonotic pathogen that can be transmitted through contaminated food. L. monocytogenes is further subdivided into clonal complexes (CC) and sequence types (ST). LMO strains have adapted to different niches and show growth in different environments such as soil, plants, water, food matrices and food processing facilities. According to EC Regulation 2073/2005 on the food safety criterion of LMOs, food products are classified according to whether LMO growth is possible. Therefore, for products that support growth, there is an additional requirement that L. monocytogenes must not be detectable before the product is placed on the market and must not exceed the limit of 100 cfu/g during shelf life. Mandatory measures to detect the presence and growth of L. monocytogenes include food lot testing, environmental monitoring and challenge testing to estimate the growth potential of L. monocytogenes in different food matrices. The aim of this study was to estimate the growth potential of L. monocytogenes in lettuce samples in collaboration with a private laboratory. An accredited private laboratory prepared the samples for challenge testing and sent the selective agar plates according to ISO 11290-2 for further subtyping. We prepared a strain cocktail of different LMO field isolates (ST121, ST21, ST2) with the reference strain (ST145) available for inoculation of the products. The aim was to determine the potential growth advantage of individual persistent LMO strains over others. Two types of ready-to-eat (RTE) salads were tested for quantitative analysis. Three experiments were performed with the inoculated lettuce samples on days 0, 4, 6 and 8 at three dilutions (1:3, 1:30 and 1:300) at 6°C and 8°C. Experiment 1 was the challenge test with a RTE mixed green salad and experiments 2 and 3 with a monoproduct salad. Reference samples and corresponding success control samples were prepared in triplicates for each sample group. PCR was performed targeting the abcZ, bglA, cat, dapE, dat, IhkA and ldh alleles, which were sent to LGC Genomics for sequencing. The sequencing results were uploaded to the Multi Locus Sequence Typing (MLST) Institute Pasteur database to determine the CC and ST. The results confirmed the hypothesis that the in-house persistent isolates had a growth advantage over the reference strain (ST145). ST121 and ST21 showed a clear growth advantage in all three experiments. There was limited or no detection of ST2 and ST145 in experiments 1 and 2. The results of experiment 3 suggest that the virulent ST2 and the other strains ST121 and ST21 outcompeted each other, explaining the overall reduced recovery of the strains. The inoculation method can be improved in future studies to achieve the target inoculum detection limit of 100 CFU/g. An important factor to consider in challenge testing is the individual plant microbiome, which can influence LMO growth and recovery.