International Journal of Food Microbiology 136 (2010) 345351

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

The overgrowth of Listeria monocytogenes by other Listeria spp. in food samples undergoing enrichment cultivation has a nutritional basis
Nathalie Gnanou Besse a,, Lena Barre a, Colin Buhariwalla b, Marie Lone Vignaud a, Elissa Khamissi a, Emilie Decourseulles a, Marjorie Nirsimloo a, Minyar Chelly a, Martin Kalmokoff b
a b

Agence franaise de scurit sanitaire des aliments Laboratoire d'Etudes et de Recherches sur la Qualit des Aliments et sur les Procds agro-alimentaires (Afssa, 23 avenue du Gnral de Gaulle, 94706 Maisons Alfort cedex, France) Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia, Canada

a r t i c l e

i n f o

a b s t r a c t
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 ndings 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 signicant 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 signicantly faster growth rates than others. Limiting diffusion in Fraser broth through the addition of agar signicantly 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 ndings support that the overgrowth phenomenon in most instances has a nutritional basis. 2009 Elsevier B.V. All rights reserved.

Article history: Received 22 May 2009 Received in revised form 16 October 2009 Accepted 24 October 2009 Keywords: Listeria monocytogenes Listeria innocua Listeria welshimeri Detection Competition Enrichment

1. Introduction Listeria monocytogenes is a Gram-positive non-spore forming short rod which has a ubiquitous distribution throughout the natural environment. Outbreaks of listeriosis result primarily from the post processing contamination of produce, dairy and meat products, which in the case of L. monocytogenes is of particular importance as this pathogen is able to grow and proliferate at refrigerator temperatures. Despite the low incidence, food borne listeriosis is characterised by a high rate of mortality in the elderly and immune-compromised and poses a serious risk for the foetus in pregnant women (Farber and Peterkin, 1991). Furthermore, the detection of L. monocytogenes in a food product has important economic consequences for the food

Corresponding author. Afssa LERQAP, 23 Avenue du Gnral de Gaulle, 94706 Maisons Alfort cedex, France. Tel.: +33 1 49 77 11 10; fax: +33 1 49 77 11 02. E-mail address: n.besse@afssa.fr (N. Gnanou Besse). 0168-1605/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.10.025

company including the recall/withdrawal of distributed products as well as other associated liabilities. The international standard method for the detection of L. monocytogenes in foods (ISO 11290-1; Scotter et al., 2001) consists of a double enrichment in Half Fraser and Fraser selective broths. The initial incubation in Half Fraser broth is carried out for 24 h at 30 C. The second step of the enrichment is carried out in Fraser broth for 48 h at a temperature of 37 C. Half Fraser broth contains half the concentration of nalidixic acid and acriavin as found in Fraser broth. The double enrichment can result in L. monocytogenes being overgrown by other Listeria species in samples where multiple species are present (Petran and Swanson, 1993; Curiale and Lewus, 1994; MacDonald and Sutherland, 1994; Cornu et al., 2002). The evolution of a Listeria population originating from a food sample during enrichment is dependent on a complex set of interactions. Factors which have been thought to impact on this evolution include the production of various inhibitors (Cornu et al., 2002), competition from the background ora (Dallas et al., 1991),

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differences in growth rates among the different Listeria species (Duh and Schaffner, 1993; Curiale and Lewus, 1994; MacDonald and Sutherland, 1994; Beumer et al., 1996; Cornu et al., 2002) and interactions with the food matrix (Santillan et al., 1997). From a practical perspective, the overgrowth by a non-pathogenic species of Listeria can mask the presence of low numbers of L. monocytogenes in the original food sample, and result in an increase in false negatives (Oravcova et al., 2008). In our previous work using naturally contaminated food samples, we demonstrated that there were no absolute correlations between individual species growth rates or inhibitory proles in terms of strain evolution during enrichment following the ISO 11290-1 procedure. Generally, strains which were predominant in the original sample, tended to remain the dominant strains at the end of the enrichment, although in some samples the relative proportion of any given strain could change signicantly over the enrichment process (Gnanou Besse et al., 2005). Here we have examined additional factors in the ISO 11290-1 isolation procedure and assessed their impact on listeria population evolution, including the physiological state of the cells, composition of the enrichment medium, temperature and nutritional competition. 2. Materials and methods 2.1. Bacterial strains and growth conditions Experiments were carried out using various isolates of L. monocytogenes, L. innocua and L. welshimeri (Table 1). All were originally isolated from food and have been previously characterized (Gnanou Besse et al., 2005). Stock cultures were maintained frozen at 80 C using cryobank (AES Laboratoires, Combourg, France). Cultures were revived by plating onto tryptone soy agar (Oxoid, Basingstoke, United Kingdom) with added yeast extract (6 g/L; TSAYE, Biokar Diagnostics, Beauvais, France). Listeria cultures grown in brain heart infusion broth (BHI, Oxoid, Basingstoke, United Kingdom) for 24 h at 37 C were used as inocula for all tests (stationary phase BHI cultures contain
Table 1 Listeria species, serotype and origin of strains utilized in this study. Species and strain code L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. monocytogenes 74 LM monocytogenes 77 LM monocytogenes 80 LM monocytogenes 86 LM monocytogenes 87 LM monocytogenes 88 LM monocytogenes 91 LM monocytogenes 92 LM monocytogenes AL75 monocytogenes AL78 monocytogenes SO49 monocytogenes IN2 monocytogenes 394 monocytogenes 66 LM innocua 2 LIN innocua 5 LIN innocua 6 LIN innocua 7 LIN innocua 12 LIN innocua 14 LIN innocua 20 LIN innocua 22 LIN innocua 23 LIN welshimeri 5 LW welshimeri 6 LW welshimeri 7 LW Serovar 1/2c 1/2c 1/2c 1/2a 1/2c 1/2c 4b 1/2a 1/2a 1/2c 1/2a 1/2a 4b 4c Food sample of origina 2 3 5 7 7 5 6 6

approximately 1 109 cfu/mL). To conrm the inoculum level, dilutions of the BHI cultures were enumerated by spreading on TSAYE. All serial dilutions were prepared using tryptone salt broth (TS; 8.5 g NaCl and 1 g peptone/L of deionised water, Oxoid, Basingstoke, United Kingdom). Stressed inoculums consisted of cells grown in BHI overnight, recovered by centrifugation, then re-suspended into sterile BHI containing 300 g NaCl/L at 25 C for 72 h. Salting is widely utilized in the food industry as natural preservation process, and exposure to high salt is known to induce stress in Listeria (Gnanou Besse et al., 2000). No decrease in viability was observed to result from this stress. 2.2. Microbiological growth kinetics Growth curves for individual isolates (Table 1) and combinations of isolates (L. monocytogenes 77 LM plus L. innocua 12 LIN or L. welshimeri 6 LW) was followed by direct plating, using the spiral plating system (Intersciences, Saint-Nom La Bretche, France). All serial dilutions were prepared using TS. Enumerations of pure cultures were carried by plating on TSAYE, whereas in the case of mixed cultures ALOA agar was utilized (AES Laboratoires, Combourg, France). Due to the production of a phospholipase, L. monocytogenes colonies growing on this selective chromogenic medium are surrounded by a halo making it possible to differentiate them from the colonies produced by L. innocua or L. welshimeri. For all experiments involving growth curves, two identical cultures for each individual isolate and each set of mixed cultures were inoculated separated by a 9 h interval. This was carried out in order to obtain data points covering the entire growth cycle. Samples from each broth culture were removed periodically to enumerate cells (cfu/mL). The resulting growth curves were tted to the Baranyi model (Baranyi and Roberts, 1994) using MicroFit software (Institute of Food Research, Norwich, United Kingdom). Curve tting was performed by minimizing the sum of squared errors between the data points. Growth parameters and their standard error were estimated by the model and compared.

Origin Cured dry sausage Raw pork/beef sausage Washed-rind soft cheese Raw sausage Raw sausage Washed-rind soft cheese Cold-smoked salmon Cold-smoked salmon Cheese Cheese Milk NR Cheese Food NR Cured dry sausage Cured dry sausage Cured dry sausage Raw pork/beef sausage Washed-rind soft cheese Raw sausage Cold-smoked salmon Cold-smoked salmon Cold-smoked salmon Raw pork/beef sausage Cured dry sausage

Particular properties

Low recovery on Oxford and Palcam agar Low recovery on Oxford and Palcam agar Hypovirulent Hypovirulent Slow growth in selective broth Fast growth in selective broth

2 2 2 3 5 7 6 6 6 3 2

Strains having the same food sample of origin were from food samples where the overgrowth of the enrichment broths by a single strain was previously described (Gnanou Besse et al., 2005). NR: not recorded. a Gnanou Besse et al. (2005).

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2.3. Overgrowth phenomenon in selective broths Growth experiments involving multiple species were carried out using L. monocytogenes 77 LM, L. innocua 12 LIN and L. welshimeri 6 LW, which were originally isolated from a naturally contaminated raw pork/beef sausage where the overgrowth phenomenon had previously been documented (Gnanou Besse et al., 2005). Application of the ISO 11290-1 detection method to this original food sample resulted in the loss of L. welshimeri, low numbers of L. monocytogenes (0.2% of nal cfu) and the overgrowth of the full Fraser enrichment broth by L. innocua. (Gnanou Besse et al., 2005). Growth curves for pure and mixed cultures were performed in parallel as described above, without agitation in Half Fraser broth at 30 C and in Fraser broth at 37 C for 48 h. For each species, broths were inoculated at an initial concentration of 102 cfu/mL. All experiments were performed in duplicate. 2.4. Impact of growth conditions on the overgrowth phenomena To evaluate the impact of both cell stress and enrichment medium constituents on strain evolution, we focused on the Half Fraser enrichment. The Half Fraser enrichment is the rst step in the ISO 11290-1 method and consequently would have the greatest impact on stressed or injured cells. The impact of each selective agent in Half Fraser medium and incubation temperature on the growth of L. monocytogenes 77 LM, L. innocua 12 LIN and L. welshimeri 6 LW was investigated using a Hadamard matrix (Plackett and Burman, 1943). Seven different parameters were assessed as follows: sodium chloride (Merck, Darmstadt, Germany) at 0 and 20 g/L; Fe3+ (ammonium iron citrate, VWR International, Leuven, Belgium) at 0 and 0.5 g/L; esculin (Merck, Darmstadt, Germany) at 0 and 1 g/L; lithium chloride (Merck, Darmstadt, Germany) at 0 and 3 g/L; nalidixic acid (Molekular, Shaftesbury, United Kingdom) at 0 and 0.01 g/L; acriavin (Molekular, Shaftesbury, United Kingdom) at 0 and 0.0125 g/L, and incubation temperatures of 30 and 37 C. For the Hadamard matrix, assays were performed using each species in pure culture. Separate assays were run using both nonstressed and salt-stressed inoculants. For each assay, individual species were grown in Half Fraser broth or the modied Half Fraser broths initially inoculated at levels ranging from 102 to103 cfu/mL. Complete growth curves were performed as described above in order to obtain all four growth parameters (lag phase, maximum growth rate, initial population level, maximal population attained). Results from the Hadamard matrix were modelled using multiple regression analysis. Signicant differences in regression coefcients were assessed using a Student t-test, and the global validity of the model conrmed using a Fisher test. In order to conrm the results we also performed an analysis using a general linear model (p = 0.05). In order to evaluate the impact of cell stress on species growth in enrichment medium, the growth parameters for all of the isolates listed in Table 1 were determined in Fraser broth using both nonstressed and stressed inoculums. Fraser broth was used for these experiments as the majority of the selective ingredients are present at twice the concentration as found in Half Fraser broth and it was reasoned that species associated growth effects might be more apparent than in Half Fraser broth. 2.5. Impact of limiting diffusion on the overgrowth in selective broth The impact of limiting diffusion by the inclusion of agar into Fraser broth on strain evolution in cultures containing multiple strains was assessed. Each pair of strains utilized was originally isolated from food samples where the overgrowth phenomenon had been previously observed (Gnanou Besse et al., 2005), and was listed in Table 3. Fraser medium containing agar (1.5% w/v) cooled to a temperature of 45 C was inoculated (102 cfu/mL), gently mixed, then poured into a sterile

Petri dish and allowed to solidify at room temperature. Following completion of the incubation half of the agar was aseptically removed from each plate, serially diluted and stomached in TS for 1 min. Following stomaching a 1.0 mL sample was removed, serially diluted in TS and the colony forming units determined as described above. All determinations were performed in duplicate following both 24 and 48 h incubation at 37 C. Experiments using Fraser broth and strains listed in Table 3 were run in parallel. 2.6. Substrate utilization The substrate utilization range of all Listeria isolates (Table 1) was determined using the Biolog GP2 plate for Gram positives (Biolog Inc., Hayward, California, U.S.A.). Individual plates were inoculated, incubated at 37 C and read following 48 h incubation according to the manufacturer's instructions. 3. Results 3.1. Overgrowth in pure and mixed cultures of Listeria spp. in Fraser broths Growth curves in Half Fraser (30 C) or Fraser broth (37 C) for pure cultures of L. monocytogenes 77 LM, L. innocua 12 LIN, L. welshimeri 6 LW, and for mixtures of each are shown in Fig. 1. In pure culture the growth rate of L. innocua 12 LIN was slightly more rapid than L. monocytogenes 77 LM in both Half Fraser and Fraser broth (Fig. 1, Panels A and C). In Fraser broth the growth rate of L. monocytogenes 77 LM was similar to that for L. welshimeri 6 LW (Fig. 1, Panel E), although the latter had a longer lag phase in Fraser broth (Fig. 1, Panel G). In Half Fraser broth at 30 C, all three isolates reached stationary phase following 30 h incubation. In Fraser broth, stationary phase was attained by all three strains following 24 h incubation. In each case using pure cultures, all three species attained similar nal numbers in stationary phase in both Fraser and Half Fraser broths (Fig. 1). In mixed cultures of Half Fraser and Fraser broths, when the faster growing L. innocua 12 LIN reached stationary phase, growth of the second species L. monocytogenes 77 LM also stopped (Fig. 1, Panels B and D). In contrast, in mixed cultures containing L. monocytogenes 77 LM and L. welshimeri 6 LW, the growth rate of L. welshimeri 6LW was slower (Fig. 1, Panels F and H) compared to that found in pure culture (Fig. 1, Panels E and G). Growth of both strains stopped simultaneously. For each mixed culture determination, marked differences in the relative proportion of each species over the enrichment process in each selective broth were also observed. In Half Fraser the nal proportion of the over grown strains was approximately half of the total number of the dominant strain, whereas in Fraser broth the nal proportion was less than 10% of the dominant strain (Fig. 1, Panels B, D, F and G). 3.2. Impact of temperature, medium composition and stress on growth in Fraser broths The impact of growth temperature, medium constituents and the physiological state of the inoculum on the observed differences in growth of L. monocytogenes 77 LM, L. innocua 12 LIN and L. welshimeri 6 LW in Half Fraser broth was determined. The time to attain a 5 log10 increase in the population of each of these three Listeria species was used as the determinative factor in the Hadamard matrix. For unstressed cells, the only signicant factor was NaCl and only on the growth of L. innocua 12 LIN (P = 0.027). A Hadamard matrix was also performed using stressed inoculum, although none of the seven factors could account for the differences in growth observed among these three strains in Half Fraser broth (results not shown). Global analysis of the complete data did however conrm signicant effects on growth between stressed and non-stressed cells and conrmed the obvious growth differences among these three strains (Table 2).

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Fig. 1. Growth curves of pure and mixed cultures of Listeria spp. in Half Fraser (48 h at 30 C) and Fraser broths (48 h at 37 C). Growth of pure cultures of L. monocytogenes 77 LM or L. innocua 12 LIN in Half Fraser broth (Panel A) and as a mixed culture in Half Fraser broth (Panel B). Growth of pure cultures of L. monocytogenes 77 LM or L. innocua 12 LIN in Fraser broth (Panel C), and as a mixed culture in Fraser broth (Panel D). Growth of pure cultures of L. monocytogenes 77 LM or L. welshimeri 6 LW in Half Fraser broth (Panel E), and as a mixed culture in Half Fraser broth (Panel F). Growth of pure cultures of L. monocytogenes 77 LM or L. welshimeri 6 LW in Fraser broth (Panel G), and as a mixed culture in Fraser broth (Panel H). Symbols are as follows: L. monocytogenes 77 LM (), L. innocua 12 LIN () and L. welshimeri 6 LW ().

N. Gnanou Besse et al. / International Journal of Food Microbiology 136 (2010) 345351 Table 2 Global analysis assessing the contribution of various factors on the observed differences in growth of L. monocytogenes 77 LM, L. innocua 12 LIN and L. welshimeri 6 LW in Half Fraser broths (P = 0.05). Parameters Species Physiological state Temperature NaCl Fe++ Esculin LiCl Nalidixic acid Acriavin Signicance (P-value) < 0.001 < 0.001 0.060 0.044 0.701 0.072 0.024 0.058 0.994

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Table 3 Final percentage of L. monocytogenes in mixed cultures in Fraser broth and Fraser agar, following 24 and 48 h incubation at 37 C. Pairs of strains tested Final percentage of L. monocytogenes Liquid 24 h L. innocua 12 LIN + L. monocytogenes 77 LM L. innocua 6 LIN + L. monocytogenes 74 LM L. monocytogenes 77 LM +L. welshimeri 6 LW L. innocua 14 LIN + L. monocytogenes 80 LM L. innocua 2 LIN + L. monocytogenes 66 LM 3.6 0.9 99.0 2.2 0.8 48 h 2.4 0.8 97.6 1.6 < 0.1 Solid 24 h 15.5 20.2 43.7 18.8 8.2 48 h 21.2 21.1 52.2 26.5 5.1

While salt concentration was also found to be a signicant factor, although not to the same degree as stress or strain growth differences (Table 2), repetition of the mixed culture growth experiments (see above) using Half Fraser broth containing no NaCl or LiCl had no impact on the growth kinetics or the nal proportion of each strain within each mixed culture (results not shown). In order to further examine the relationship between the physiological state of the inoculum, species and growth, the time to attain a 5 log10 increase in the population of each Listeria species listed in Table 1 using both stressed and non-stressed inoculums in Fraser broth was also determined (results not shown). Analysis of variance indicated that stress was the lone factor which signicantly impacted growth, (P = 0.001), and there was no signicant difference in the time to attain a 5 log10 increase between the species listed in Table 1 (P = 0.524) or a signicant species-treatment interaction (P = 0.352). 3.3. Impact of limiting diffusion on the over growth phenomenon Results from the Hadamard matrix failed to identify a single factor in Half Fraser broth which could contribute to the observed differences in the growth parameters among L. monocytogenes 77 LM, L. innocua 12 LIN and L. welshimeri 6 LW. However the growth kinetics in mixed cultures containing these strains did suggest that the overgrowth phenomenon could result from a competitive interaction. In order to further investigate, we attempted to reduce nutritional competition by reducing bulk transfer in the Fraser medium using 1.5% agar then re-assessing the impact on the overgrowth phenomenon. Culture evolution of mixed cultures was determined following 24 h and 48 h incubation at 37 C for these three model species as well as additional mixtures of Listeria isolated from food samples where the overgrowth phenomenon had previously been observed (Gnanou Besse et al., 2005). Table 3 shows a comparison of the percentage of L. monocytogenes in mixed cultures grown in Fraser medium (liquid and solid) following 24 and 48 h incubation. Under conditions where L. monocytogenes was over grown, the addition of agar to the medium increased the nal numbers over those found in Fraser broth. Similarly, where L. monocytogenes was the dominant strain in liquid culture, the addition of agar reduced its proportion of the total colony forming units by approximately half (Table 3). Cell numbers for each of the strains in each mixed culture exceeded 105 cfu/mL following 24 h incubation (results not shown), and exceeded 107 cfu/mL following 48 h. However, the overall proportion of each species (% of total) was similar at 24 h or 48 h incubation (Table 3). 3.4. Impact of additional nutritional factors Results from mixed cultures where agar was used to reduce bulk transfer through the medium supported that nutritional competition was an important factor in the nal evolution of a listeria population. We wondered if the ability of a particular strain to overgrowth other

strains during the enrichment process could be explained in terms of differences in the substrate utilization abilities. The substrate utilization abilities of all the strains listed in Table 1 were determined using the Biolog GP2 plates. Overall, these isolates utilized the same core substrates, although there were some minor differences in the ability of certain strains to utilize specic substrates (results not shown). However, substrate utilization capabilities did not explain why one strain could overgrow another strain during growth in Fraser broths. 4. Discussion The ISO standard method is based on a two-step enrichment using Half Fraser and Fraser broths. While the method is very effective, it is also quite involved and there are circumstances which can result in false negatives or cause difculties in isolating the L. monocytogenes strain responsible for a food borne outbreak from food samples containing multiple Listeria species. While it has been shown that the relative proportion of species and/or strains can change over the enrichment process, there is currently no data examining the growth kinetics of mixed Listeria cultures in Fraser broths over the entire enrichment, data which could aid our understanding of the overgrowth phenomena (Cornu et al., 2002). Previously, we demonstrated that the inhibitory activities produced by various Listeria isolates could impact culture evolution under conditions where initial contamination levels were high (107 cfu/mL; Cornu et al., 2002), although this was not an important factor when contamination levels were low as is typically found in contaminated foods (Gnanou Besse et al., 2005). While in some instances culture overgrowth could also be explained on the basis of differences in growth rates, we concluded that other factors were involved in the overgrowth phenomenon in the majority of instances (Gnanou Besse et al., 2005). Obviously a more complete perspective on this phenomenon might allow modications to the existing methodology which could address this problem. Initially we examined the growth kinetics of single and mixed cultures by plate counting over the entire course of the enrichment process. The three strains selected for these experiments were originally isolated from a food sample where the overgrowth of L. monocytogenes occurred (Gnanou Besse et al., 2005). While there were clear differences in growth parameters among all three isolates, the strains attained similar nal cell densities in selective broths when grown as monocultures. Following the complete growth curves in mixed cultures, rather than just the end points, provided further information concerning the overgrowth phenomenon. Firstly, the overgrowth of Fraser broths became very obvious in the late exponential and early stationary phase. Specically, we observed the simultaneous cessation of growth when the dominant species reached its stationary phase. This is referred to as a Jameson effect (Ross et al., 2000) and is generally attributed to a competition for a common nutritional resource, although it can also result from the accumulation of a toxic metabolite. Secondly, L. welshimeri 6 LW growth rates slowed in mixed culture compared to the rates observed in pure culture. Again, this nding supports that the

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inhibition of growth results from a nutritional competition, rather than from the accumulation of a toxic end product (Cornu et al., 2002). Furthermore, these ndings are consistent with our previous examination of the naturally contaminated food samples, where the dominant Listeria strain in the sample was more likely to remain the dominant strain throughout the enrichment process (Gnanou Besse et al., 2005). Half Fraser and Fraser broths contain various inhibitors added to suppress the growth of other species of bacteria associated with foods during the enrichment process. Previous work has suggested that Fraser media as well as certain constituents within this media can also affect the growth of Listeria (Beumer et al., 1996; Nexmann Jacobsen, 1999; Jasson et al., 2009). In addition factors such as the physiological state of the cells may also impact growth, as it is likely that cells contaminating foods would be subjected to a variety of stresses. Using a multi-factorial approach we assessed the impacts of these additional factors on the observed differences in growth among three different strains of Listeria originally isolated from a single food sample. While enrichment medium composition or incubation temperature turned out not to be signicant factors, cell stress was. In this case we used exposure to high salt to induce stress as it is widely used in the food industry as natural preservative. Pre-stressing was also found to signicantly impact the growth of a wider selection of strains in Fraser broth, although it affected each species to the same extent and provided no apparent growth advantage for any of the three species tested. While it is widely held that L. innocua has an advantage over L. monocytogenes during enrichment culturing due to faster growth rates (Duh and Schaffner, 1993; Curiale and Lewus, 1994; MacDonald and Sutherland, 1994; Beumer et al., 1996), we found no signicant differences in generation or lag times among various strains of these two species in Fraser broth, which is consistent with previous reports (Cornu et al., 2002). Cells immobilized in agar are subject to diffusion limits in terms of nutrients and metabolites, a situation quite different from dispersed growth in a liquid medium (Brocklehurst, 2003). We found that the overgrowth of one strain by another in mixed cultures could be signicantly reduced by decreasing bulk transfer through the medium by the addition of agar. Presumably, limiting diffusion inhibits the growth of the faster strain, allowing more time for the slower growing strain to increase in numbers. Again, this nding also supports that the overgrowth phenomenon has primarily a nutritional basis. We were however, unable to explain why one strain would overgrow another based on the range of substrates utilized among these strains. Furthermore, supplementation of Half Fraser broth with additional peptone or carbohydrates did not relieve the observed overgrowth in our model test system (Gnanou Besse and Kalmokoff, unpublished results). It is likely that the nutritional competition may be for multiple substrates, or reect differences in substrate uptake rates between individual strains or be a combination of many factors (KovarovaKovar and Egli, 1998). Results from this work indicate that nutritional competition can play an important role in the evolution of a Listeria population undergoing enrichment. A two-step enrichment procedure is likely to enhance overgrowth phenomenon. Recently, an isolation procedure utilizing a single enrichment in Half Fraser followed by plating onto ALOA medium was proposed and validated as an alternative to the ISO 11290-1 method (ALOA One Day method, Anonymous, 2008). While the majority of L. monocytogenes are detected following the primary enrichment in Half Fraser broth (Johansson et al., 2000; Scotter et al., 2001; Gnanou Besse et al., 2005; Oravcova et al., 2008; Loncarevic et al., 2008), the secondary enrichment in Fraser broth results in more L. monocytogenes strains, particularly when the original food sample contains low levels of L. monocytogenes or high levels of competing ora (Johansson et al., 2000; Scotter et al., 2001; Loncarevic et al., 2008). However, with a second enrichment some L. monocytogenes strains could be lost due to an overgrowth by other species of listeria (Johansson et al., 2000; Scotter et al., 2001; Loncarevic et al., 2008).

While we have demonstrated that limiting diffusion may reduce the extent of culture overgrowth in this situation, the use of agar does makes this somewhat impractical, particularly if large numbers of samples are being processed. The overgrowth of L. monocytogenes by other Listeria spp. during enrichment can result from a variety of factors including the effect of various inhibitors, differences in growth rates among individual isolates, and nutritional competition. It is unlikely that practical modications can be made to the existing method in order to overcome each of these factors. Previously, we suggested that 24 h incubation in Fraser broth was sufcient to attain the maximum population, in contrast to the current practice of 48 h (Gnanou Besse et al., 2005); a nding which we have conrmed here. However, this possibility would require the analysis and comparison of additional high number of naturally contaminated samples from various origins. This would reduce the total duration of the Standard detection method by 24 h, which represents a signicant improvement in its practicability. Acknowledgements This study was supported through Afssa as Community Reference Laboratories for Listeria monocytogenes, funded by the European Commission/General Directorate Health and Consumer Protection (DG SANCO) and through the Food Safety program of Agriculture and Agri-Food Canada. The authors would like to thank B. Heyd (AgroParisTech) and M. Laurentie (Afssa Fougres) for their helpful advices and statistical analysis, and A. Leclercq (Institut Pasteur, Paris) and M. Uyttendaele (Faculteit Bio-ingenieurswetenschappen, Ghent University) for providing some strains. References
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