Applicability of the EN ISO 11290-1 standard method for Listeria monocytogenes detection in presence of new Listeria species.

During the past six years, new species of the genus Listeria have been isolated from foods and other environmental niches worldwide. The Standard method EN ISO 11290-1 that is currently under revision will include in its scope all Listeria species in addition to L. monocytogenes. The objective of this project was to evaluate the ability of the Standard EN ISO 11290-1 method to detect and identify the newly discovered Listeria spp., and to assess potential over-growth effects of the new species in mixed cultures with L. monocytogenes during each step of the enrichment process. This objective was addressed by the generation of necessary data on the behavior of the new species during the pre-enrichment and the enrichment steps of the reference method as well as data on their phenotypic characteristics on rich and selective media used for isolation and identification. Most of the new Listeria species developed well on selective agar media for Listeria, however the recovery of some species was difficult due to poor growth in Half Fraser and Fraser broth. Good results (consistently positive) were obtained for confirmation at the genus level via the catalase test, the Gram test and the blueish appearance test on non-selective medium, but not with the VP test, as most of the new species yielded a negative result. In the light of results obtained in co-culture experiments and inhibition tests, and considering the growth rates in Half Fraser and Fraser broths, the new species do not seem to interfere with the detection of L. monocytogenes.

Modeling the behavior of Listeria monocytogenes during enrichment in half Fraser broth; impact of pooling and the duration of enrichment on the detection of L. monocytogenes in food.

A stochastic model describing the growth of Listeria monocytogenes during enrichment in half Fraser was developed for the purpose of estimating the effects of modifications to the first enrichment step of the EN ISO 11290-1 detection method. Information pertaining to the variability of growth rates, physiological state of the cell, and the behavior of individual cells contaminating the food were obtained from previously published studies. We used this model to investigate the impact of pooling enrichment broths (wet pooling) on the performance of the standard method. For validation of the model, the numbers of L. monocytogenes occurring in 88 naturally contaminated foods following pre-enrichment were compared to model-simulated microbial counts. The model was then used to perform simulations representative of the natural contamination observed for smoked salmon in the European baseline survey of 2010-2011. The model-estimated L. monocytogenes levels following individual enrichment or following the pooling of five broths where only one would be contaminated were compared. The model indicated a 10% loss of method sensitivity resulting from wet pooling. The model also predicted a 5% decrease in the sensitivity of the method when the duration of the enrichment was reduced from 24 to 22 h.

Evaluation of reduction of Fraser incubation by 24h in the EN ISO 11290-1 standard on detection and diversity of Listeria species.

The EN ISO 11290-1 method for the isolation of Listeria monocytogenes from food is carried out using a double enrichment in Fraser broths. While the method is effective it is also quite long requiring 4-7 days to process a contaminated food, and may be adversely affected by inter-strain and/or inter-species competition in samples containing mixed Listeria populations. Currently, we have little information on the impact of competition on food testing under routine conditions. Food samples (n=130) were analyzed using the standard method and the evolution of Listeria populations in 89 naturally contaminated samples followed over the entire enrichment process. In most instances, maximum increase in L. monocytogenes population occurred over the first 24h following sub-culture in Full Fraser broth and strain recovery was similar at both 24 and 48 h, indicating that the second enrichment step can be reduced by 24h without impacting the recovery of L. monocytogenes or affecting the sensitivity of the method. In approximately 6% of naturally contaminated samples the presence of competing Listeria species adversely impacted L. monocytogenes population levels. Moreover, these effects were more pronounced during the latter 24h of the Fraser enrichment, and potentially could affect or complicate the isolation of these strains.

Sensitive enumeration of Listeria monocytogenes and other Listeria species in various naturally contaminated matrices using a membrane filtration method.

For the enumeration of Listeria monocytogenes (L. monocytogenes) in food, a sensitive enumeration method has been recently developed. This method is based on a membrane filtration of the food suspension followed by transfer of the filter on a selective medium to enumerate L. monocytogenes. An evaluation of this method was performed with several categories of foods naturally contaminated with L. monocytogenes. The results obtained with this technique were compared with those obtained from the modified reference EN ISO 11290-2 method for the enumeration of L. monocytogenes in food, and are found to provide more precise results. In most cases, the filtration method enabled to examine a greater quantity of food thus greatly improving the sensitivity of the enumeration. However, it was hardly applicable to some food categories because of filtration problems and background microbiota interference.