European survey and evaluation of sampling methods recommended by the standard EN ISO 18593 for the detection of Listeria monocytogenes and Pseudomonas fluorescens on industrial surfaces.

The ready-to-eat products can be contaminated during processing by pathogen or spoilage bacteria, which persist in the industrial environment. Some bacterial species are able to form biofilms which protect them from environmental conditions. To check the bacterial contamination of the surfaces in the food industries, the professionals must regularly use surface sampling methods to detect the pathogen such as Listeria monocytogenes or the spoilage such as Pseudomonas fluorescens. In 2010, we designed and carried out a European survey to collect surface sampling information to detect or enumerate L. monocytogenes in food processing plants. A total of 137 questionnaires from 14 European Union Member States were returned. The outcome of this survey showed that the professionals preferred friction sampling methods with gauze pad, swab and sponges versus contact sampling methods. After this survey, we compared the effectiveness of these three friction sampling methods and the contact plates, as recommended in the standard EN ISO 18593 that was revised in 2018, on the recovery of L. monocytogenes and of P. fluorescens in mono-specie biofilms. This study showed no significant difference between the effectiveness of the four sampling methods to detach the viable and culturable bacterial population of theses mono-specie biofilms.

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.

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.

The overgrowth of Listeria monocytogenes by other Listeria spp. in food samples undergoing enrichment cultivation has a nutritional basis.

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. In cases where multiple Listeria species are present within the original sample, L. monocytogenes can be overgrown during enrichment by other species of listeria present in the original sample. From a practical perspective, this can result in a false negative or complicate the ability of public health investigators to match food and clinical isolates. We have further investigated this phenomenon by analysing the growth kinetics of single species and pairs of different species over the ISO 11290-1 enrichment process. The overgrowth of a strain of L. monocytogenes by a strain of Listeria innocua resulted primarily from interactions which occurred in late exponential phase, where it was observed that growth of both strains stopped when the dominant strain reached stationary phase. In a second mixed culture, the dominant L. monocytogenes strain suppressed the exponential growth rate of the second Listeria welshimeri strain. Both findings suggest that the overgrowth could partially be explained in terms of a nutritional competition. Multi-factor analysis of Fraser broth constituents and growth temperatures using both stressed and non-stressed inoculants failed to identify any single factor in the ISO 11290-1 methodology which would contribute to the overgrowth phenomenon in our model system. Furthermore, species was not a significant factor in observed differences in growth parameters among a wider array of strains which had been stressed or not stressed prior to grown in Fraser broths, even though some strains had significantly faster growth rates than others. Limiting diffusion in Fraser broth through the addition of agar significantly reduced the extent of the overgrowth in experiments using mixtures of strains originally isolated from foods where overgrowth had been previously observed. Taken together, these findings support that the overgrowth phenomenon in most instances has a nutritional basis.

Effect of the inoculum size on Listeria monocytogenes growth in structured media.

Obtaining quantitative data concerning the relative impact of various factors that may influence bacterial growth is of great importance for microbial risk assessment and predictive microbiology. The objective of this work was to investigate the effect of the initial Listeria monocytogenes density on all the growth parameters of this pathogen (lag phase duration, growth rate and maximum population density attained) on a sterile solid model system mimicking smoked fishery products, and in real cold-smoked salmon, a product likely to be contaminated with L. monocytogenes. Growth of the pathogen was monitored using a sensitive enumeration method, recently developed, based on membrane filtration followed by the transfer of the filter on a selective media [Gnanou Besse, N., Audinet, N., Beaufort, A., Colin, P., Cornu, M. and Lombard, B., 2004. A contribution to the improvement of Listeria monocytogenes enumeration in smoked salmon. International Journal of Food Microbiology, 91, 119-127.]. Depending on the experimental conditions, we found a significant effect of the inoculum size, both on lag phase duration, and on the maximal population attained. Moreover, the effect of the inoculum size on the growth of L. monocytogenes was dependent on a complex set of interactions. Factors which have appeared to impact on this effect include the cells physiological state, the background microflora, the texture of the media and the packaging system. It is important to understand how these interactions affect the growth of Listeria in order to predict and control its development in food.