Assessment of the relationship between the MLST genetic diversity of Listeria monocytogenes and growth under selective and non-selective conditions.

Listeria monocytogenes can grow under stressful conditions and contaminate various food categories. Progresss in DNA sequencing-based identification methods, such as multi-locus sequence typing (MLST) now allow for more accurate characterization of pathogens. L. monocytogenes MLST genetic diversity is reflected by the different prevalence of the "clonal complexes" (CCs) in foods or infections. Better understanding of the growth potentials of L. monocytogenes is essential for quantitative risk assessment and efficient detection across CCs genetic diversity. Using optical density measurements taken with an automated spectrophotometer, we compared the maximal growth rate and lag phase of 39 strains from 13 different CCs and various food origins, in 3 broths mimicking stresful food conditions (8 °C, aw 0.95 and pH5) and in ISO Standard enrichment broths (Half Fraser and Fraser). This is important as growth could influence risk through pathogen multiplication in food. Besides, enrichment problems could lead to a lack of detection of some CCs. Despite small differences highlighting natural intraspecific variability, our results show that growth performances of L. monocytogenes strains under the conditions tested in selective and non-selective broth do not appear to be strongly correlated to CCs and cannot explain higher CC "virulence" or prevalence.

Lack of correlation between growth rate and sequence type among Cronobacter sakazakii.

Species identification and growth rates for a collection of Cronobacter strains from clinical and non-clinical sources have been previously reported. However, advancements in DNA sequencing-based identification methods now allow for more accurate identification. Here we report the sequence types (STs) for 24 strains of Cronobacter sakazakii and examine any possible correlation between sequence type and growth rate, which could influence risk through greater pathogen multiplication and reach of infectious doses during time between formula preparation and feeding. The most common clonal complexes (CCs) identified were C. sakazakii CC1 and CC4. CC1 strains belonged to ST1 (n = 8) and ST391 (n = 1), while CC4 included ST4 (n = 4), ST255 (n = 1) and ST295 (n = 1). Three strains were found to belong to CC100 and two were found to belong to ST64. The remaining STs identified were represented by single strains. CC4 strains have a slightly not significant tendency for faster growth rates at 25 °C; however, the small sample size suggests that more strains need to be analysed to determine if this is a true result. In conclusion, the growth rates of C. sakazakii strains do not appear to be strongly correlated to ST.

Validation of standard method EN ISO 11290 - Part 1 - Detection of Listeria monocytogenes in food.

The reference method for the detection and enumeration of L. monocytogenes in food (Standards EN ISO 11290-1&2) has been validated by inter-laboratory studies in the frame of the Mandate M381 from European Commission to CEN. In this paper, the collaborative studies led in 2013 on 5 matrices (cold-smoked salmon, milk powdered infant food formula, vegetables, environment, and cheese) to validate the recently revised Standard EN ISO 11290-Part 1 are reported. According to the results obtained, the revised Standard EN ISO 11290-1 can be considered as a good method for the detection of L. monocytogenes in foods and food processing environments, in particular for the matrices included in the study. According to the matrices, the sensitivity rate varied from 91.1% to 100%, and the specificity rate varied from 97.6% to 100%. Positive samples were most often detected after 24 h half-Fraser enrichment.

Validation of standard method EN ISO 11290 - Part 2 for the enumeration of Listeria monocytogenes in food.

The reference method for the detection and enumeration of L. monocytogenes in food (Standards EN ISO 11290-1&2) have been validated by inter-laboratory studies in the frame of the Mandate M381 from European Commission to CEN. In this paper, the inter-laboratory studies led in 2013 on 5 matrices (cold-smoked salmon, milk powdered infant food formula, vegetables, environment, and cheese) to validate Standard EN ISO 11290-2 are reported. According to the results obtained, the method of the revised Standard EN ISO 11290-2 can be considered as a good method for the enumeration of L. monocytogenes in foods and food processing environment, in particular for the matrices included in the study. Values of repeatability and reproducibility standard deviations can be considered satisfactory for this type of method with a confirmation stage, since most of them were below 0.3 log10, also at low levels, close to the regulatory limit of 100 CFU/g.

Validation of standard method EN ISO 22964:2017 - Microbiology of the food chain - Horizontal method for the detection of Cronobacter spp.

The new version of the ISO standard method for detection of Cronobacter spp. (EN ISO 22964:2017) was validated in the frame of the European Commission Mandate M381 to CEN. Seventeen laboratories from nine countries participated in the interlaboratory studies to determine the performance characteristics of the method. The performance of the method was evaluated using matrices for which the presence of Cronobacter spp. is considered to be of serious concern, such as infant formula and its ingredients and representatives of categories cited in the EC Regulation 2073/2005 on microbiological criteria for foodstuffs for Cronobacter spp. The five matrices included in the validation were: two types of powdered infant food formulas (with and without probiotics); lactose; starch and environmental samples (swabs). The samples were each tested at two different levels of contamination, plus a negative control. Inoculation levels ranged from 4 to 95 CFU/sample. Each participant examined eight replicates of each level of inoculation, a total of 24 samples per matrix type. Specificity was calculated for each matrix used in the validation, with results ranging between 99 and 100%. Sensitivity of the method was calculated for samples in which no fractional recovery was expected and the values that were obtained ranged between 65 and 100%, depending on the matrix, the inoculation level and the interfering microbiota present in the samples. LOD50 value was calculated for three food items (the two powdered infant formulas and the starch) with values between 0.8 and 1.1 CFU/sample.

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.

Validation of a stochastic modelling approach for Listeria monocytogenes growth in refrigerated foods.

A stochastic modelling approach was developed to describe the distribution of Listeria monocytogenes contamination in foods throughout their shelf life. This model was designed to include the main sources of variability leading to a scattering of natural contaminations observed in food portions: the variability of the initial contamination, the variability of the biological parameters such as cardinal values and growth parameters, the variability of individual cell behaviours, the variability of pH and water activity of food as well as portion size, and the variability of storage temperatures. Simulated distributions of contamination were compared to observed distributions obtained on 5 day-old and 11 day-old cheese curd surfaces artificially contaminated with between 10 and 80 stressed cells and stored at 14°C, to a distribution observed in cold smoked salmon artificially contaminated with approximately 13 stressed cells and stored at 8°C, and to contaminations observed in naturally contaminated batches of smoked salmon processed by 10 manufacturers and stored for 10 days a 4°C and then for 20 days at 8°C. The variability of simulated contaminations was close to that observed for artificially and naturally contaminated foods leading to simulated statistical distributions properly describing the observed distributions. This model seems relevant to take into consideration the natural variability of processes governing the microbial behaviour in foods and is an effective approach to assess, for instance, the probability to exceed a critical threshold during the storage of foods like the limit of 100 CFU/g in the case of L. monocytogenes.

Evaluation of an enumeration method for Listeria monocytogenes at low contamination levels in cold-smoked salmon.

For the enumeration of Listeria monocytogenes in cold-smoked salmon, a sensitive enumeration method, based on membrane filtration followed by transfer of the filter on a selective medium has been recently developed (Gnanou Besse et al., 2004, A contribution to the improvement of L. monocytogenes enumeration in cold-smoked salmon. International Journal of Food Microbiology, 91, 119-127). The aim of the study was to assess the performance of this enumeration method through an inter-laboratory study, using cold-smoked salmon artificially contaminated at 2 different levels (approximately 0.6 and 1.6 log10 CFU g(-1)). A reproducibility standard deviation of 0.23 log10 CFU g(-1)and 0.15 log10 CFU g(-1) was obtained for the method respectively at the lower level and the higher level. Under certain conditions, the uncertainty of measurement can be derived from the method reproducibility standard deviation and was calculated to be 0.46 log10 CFU g(-1) for the lower contamination level and 0.30 log10 CFU g(-1) for the higher contamination level. These values can be considered as satisfactory for such low contamination levels.

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.

Evolution of Listeria populations in food samples undergoing enrichment culturing.

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. It is believed that the double enrichment can allow the overgrowth of Listeria innocua in samples where both species are present. In this study, we have evaluated the impact of overgrowth between Listeria species and strains during each step of the enrichment process. The effect of factors minimizing interactions between strains or phage inhibitory effects has also been estimated. In an artificially contaminated food undergoing enrichment, overgrowth could result from competitive interactions between Listeria spp. resulting from the production of bacteriocins and bacteriophage at high initial contamination levels (>10(4) cfu/g), but not at lower levels (50-100 cfu/g) as generally found in contaminated foods. At high levels of inoculation, the competitive effect could be reduced by solidification of the selective broths, to limit the diffusion of the inhibitors. Overgrowth resulting from differences in growth rate occurred independent of the initial contamination level. However, in naturally contaminated foods undergoing enrichment, there were no absolute correlations between growth rates or inhibitory profiles in terms of strain evolution during enrichment. In fact, Listeria strains which were predominant in the original sample in most cases remained the dominant strains at the end of the enrichment, although the relative proportion of any given strain could change significantly over the enrichment process. Additional factors which have yet to be identified impact on the evolution of Listeria in the two-step enrichment process. Analysis of strain evolution in eight naturally contaminated foods has indicated that the second enrichment step in Fraser broth can be reduced from 48 to 24 h without impacting on the recovery of L. monocytogenes. Our limited survey of naturally contaminated foods also demonstrated that maximum recovery of L. monocytogenes and other Listeria strains was found following 24 h incubation in 1/2 Fraser Broth. This finding suggests that it may be possible to shorten the current two-step isolation method further without reducing method sensitivity.

Combining fuzzy querying of imprecise data and predictive microbiology using category-based reasoning for prediction of the possible microbial spoilage in foods: application to Listeria monocytogenes.

Various predictive models of microbial behavior have been created and extensive data collection has been done by numerous private or public laboratories. However, significant differences between predicted and observed values in foods have been observed and need to be stressed, understood and explained as much as possible. In this paper, we present a software tool (currently at the level of a prototype) able: (i) to store in a database all relevant information expressed on one hand as qualitative or quantitative data and on the other hand as precise or imprecise data; (ii) to retrieve the more relevant information from the database using queries where criteria may be expressed as fuzzy values in order to enhance the flexibility of the search: (iii) to compute, in addition to the nearest data, an estimation of searched values using statistical models. The architecture of this software tool is structured as a category-based reasoning system. Example queries about Listeria monocytogenes (L. monocytogenes) illustrate the functionalities of this tool.