Article pp.415-429 du Vol.28 n°6 (2008)

(1)

L’ALIMENTATION ET LA VIE FOOD AND LIFE

Evaluation of an alternative method for Salmonella detection in food products using Flow cytometry

W. Yao

^1,3

, C. Simonnet

²

*, A. Boubetra

¹

, A.-P. Le Foll

¹

, M. Bouix

³

1. ISHA : Institut scientifique d’Hygiène et d’Analyse.

2. AES Chemunex.

3. AgroParisTech.

* Correspondance : c.simonnet@aeschemunex.com RÉSUMÉ

Évaluation d’une méthode alternative pour la détection de Salmonella spp dans les aliments par cytométrie en flux.

La cytométrie en flux est une technologie éprouvée dans le domaine de la microbiologie. L’originalité de ce travail est de comparer une méthode basée sur le principe de la cytométrie (méthode alternative) pour la détection des salmonelles à une méthode de référence (ISO 6579) selon le référentiel ISO 16140. Les essais ont porté sur la sélectivité et la justesse de la méthode alternative par rapport à la méthode de référence.

L’ensemble des résultats montre une bonne spécificité de la méthode alternative vis-à-vis des souches cibles et des souches non-cibles. Les données de l’exacti- tude relative, de sensibilité relative et de la spécificité relative montrent que les deux méthodes sont équivalentes.

La praticabilité de la méthode alternative a été évaluée et on constate que le délai de rendu des résultats est raccourci pour les échantillons négatifs.

Mots clés

Salmonella, cytométrie en flux, microbiologie alimentaire, ISO 16140, méthode de référence, méthode alternative.

Dans chaque numéro, cette rubrique met en avant un article traitant d’un des aspects de la nutrition, du rôle des technologies agroalimentaires sur la qualité des aliments jusqu’à la

« cuisine », en passant par les problèmes nutritionnels, la toxicologie alimentaire, et plus générale- ment les conséquences sur la santé des pratiques alimentaires. Les articles retenus sont soit des travaux de synthèse de haut niveau faisant le point sur une question, soit des publications origina- les rendant compte de travaux de recherche appliquée récents apportant un regard nouveau.

La Société scientifique d’hygiène alimentaire (SSHA), société savante créée en 1904 pour contri- buer à la diffusion des connaissances en nutrition et sécurité sanitaire, est aujourd’hui formée de deux départements : l’Institut supérieur de l’alimentation (ISA) développe des actions de formation, d’information et de conseil ; l’Institut scientifique d’hygiène et d’analyse (ISHA) propose un catalogue complet d’analyses (composants nutritionnels, contaminants, analyse sensorielle, microbiologie…).

Les propositions d’articles, remarques et suggestions peuvent être envoyées à : Claude Bourgeois

SSHA

Rue du Chemin-Blanc, BP 138, Champlan F-91163 Longjumeau cedex Tél. : + 33 (0)1 69 79 31 50

Fax : + 33 (0)1 64 48 82 49 http://www.ssha.asso.fr cbourgeois@ssha.asso.fr

(2)

SUMMARY

The flow cytometry is a technology used in microbiology. The originality of this study is to compare a method based on the principle of the cytometry (alternative method) for the detection of Salmonella against a reference method (ISO 6579) according to ISO standard 16140. The essays concerned the selectivity and the accuracy of the alternative method comparing at the reference method.

All the results show a good specificity of the alternative method for the target and non-target strains. The data of Relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) shows that the two methods are equivalent.

The results show that flow cytometry method is very fast compared to the reference method for negative samples.

Keywords

Salmonella, flow cytometry, food testing, ISO 16140, reference method, alterna- tive method.

1 – INTRODUCTION

Salmonella is the most prevalence foodborne pathogen in the world. Salmonella can cause serious and sometimes fatal infections to weak immune systems (e.g:

young children, frail, elderly people). Healthy persons experience fever, diarrhea (which may be bloody), nausea, vomiting, and abdominal pain. In rare circumstan- ces, the infection can lead to septicemia resulting in more severe illness.

The outbreak of Salmonella is increasing, a total of 111 non-typhoid Salmonella bacteraemia patients were studied in Denmark from 1994 to 2003. The incidence rate (mean 2.3/100 000 person-years) increased steadily from 1.9/100 000 person per year in the 40–49-year age group to 14.6/100 000 person per year. Twelve (11%) and 24 (22%) patients died within 30 and 180 days, respectively (GRADEL et al.

2006).

Several epidemiological studies have described outbreaks caused by Salmo- nella. The latest outbreak was found in U.S., for which we had a peak in November 2008 and January 2009. Centers for Disease Control and Prevention reported a total of 116 patients hospitalised, from September 1, 2008, to January 16, 2009. During this period the infection might have contributed to eight deaths. Epidemiologist and laboratory research indicate that peanut butter and peanut paste are the source of the outbreak. These products are also the main ingredient in food process manufacturing (CENTERS FOR DISEASE CONTROL AND PREVENTION (CDC) 2009).

Different types of food could be contaminated and then transfer Salmonella, such as meat from pork, chicken, beef, fish, eggs, milk and its derivates. It was reported that major food safety issue like Salmonella contamination occurs in pork meat. Slaughterhouse and food process manufacturing appeared to be the most cri- tical stages in the supply chain to occur the prevalence of Salmonella contaminating carcasses (VAN DER GAAG et al. 2005). Therefore, rapid detection of Salmonella contamination is important for an efficient and prompt investigation.

The reference method (NF EN ISO 6579) for detection and identification of Sal- monella includes pre-enrichment and selective enrichment in a liquid culture media.

(3)

Negative results are obtained in three days and positive result in six to seven days so as biochemical and serological methods. However a confirmation of the colonies grown on agar plates is required. These techniques are widely accepted neverthe- less they present some limits regarding the time required for identification. This limit could have serious economical consequences and public health issues. That’s why rapid detection and identification methods are required for analysis of the contaminated food.

The development of rapid detection methods has a different approach nowadays based on Flow Cytometry. This technique is potentially a valuable analytical method for microbiology providing the ability to rapidly analyse large numbers of individual microorganisms by several parameters. Flow cytometry is widely implemented in food industries (LAPLACE-BHUILE et al. 1993, BOUIX and LEVEAU 2001) like dairy (DUMAIN et al. 1990), wine (BRUESTSCHY et al. 1994, BOUIX et al. 1997), fruit juice (PETTIPHER et al. 1991, BOUIX et al. 2003), vegetables (SILVESTRI et al. 1997) and meat (LETEILLIER et al. 2003). This technology can detect the total viable bacteria or yeast and mould contaminants in a food product. More recently this technique has been used for specific application, like enterobacteria in vegetable products (SIL- VESTRI et al. 1997).

Flow cytometry combined with fluorescently labelled polyclonal antibodies offers advantages of speed and sensitivity for detection of specific pathogenic bacteria in foods.

The aim of this work is to evaluate the performance of a flow cytometry method for detection of Salmonella spp. compared to the ISO reference method according to the ISO 16140 standard.

The following characteristics are studied:

– selectivity of the flow cytometry method;

– relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP);

– practicability of the flow cytometry method.

2 – MATERIALS AND METHODS

2.1 Origin of the bacterial strains

The bacterial strains used in this study are listed in Table 1A (column 1 and 2) for Salmonella species and Table 2 (column 1 and 2) for non-Salmonella species. We have chosen some ATCC and CIP strains from our collection as reference test. The strains used were mostly isolated from naturally contaminated food products analysed by the ISHA laboratory (Institut Scientifique d’Hygiène & Analyse). The identification of the strains was performed by AFSSA (Agence Française de Sécurité Sanitaire des Aliments) with serotyping technique.

2.2 Cells cultures

The strains were stored at – 20°C on beads and then cultivated in tryptone soy broth (TSB, AES Chemunex, France) at 37°C for 6-8 hours.

(4)

The cell suspensions were then subcultured in 20 ml of TSB at 37°C for 18 +/– 2 hours, centrifuged at 3000 rpm, 20°C during 15 min. The supernatant was discarded and the pellet was resuspended in 20 ml of physiological saline solution.

The cell suspension was used for further artificial spiking of food products. In order to control our spiking level, we did plate the different cell suspension dilution onto TSA agar plates (AES Chemunex, France), which were incubated at 37°C for 24 +/– 2 hours.

2.3 Preparation of stressed cells

For sample spiking, strains were stressed using two types of treatment:

1) Heating at 50°C during 20 min;

2) Heating at 50°C during 20 min then storage at 4°C for 20 days.

The stress intensity was evaluated by CFU count differences between TSYE (non selective media) and Hektoën (selective media). If the difference was over 0.5 Log, calibrated suspensions were prepared in order to contaminate with less than 30 cells in 25 g of product.

In this study, Salmonella detection was performed with “Fluorassure Detection Kit Salmonella spp” associated to flow cytometer (AES Chemunex, France). Briefly, this kit contains: ChemBoost S, ChemIdS, ChemSol B30 and CS30.

2.4 Selectivity (inclusivity-exclusivity)

A concentration of 10 to 10² Salmonella cells was inoculated into 250 ml of Buf- fered Peptone Water (BPW) and 1 ml of Chemboost S (selective supplement used to inhibit the growth of Gram positive bacteria) was added.

For non-Salmonella strains, BPW was inoculated with 10⁵Cells/ml without addi- tion of Chemboost S. After inoculation, each preparation was incubated at 37°C for 18 +/– 2 hours prior to flow cytometry analysis.

Contamination levels were confirmed by plate counts on TSA incubated at 37°C for 24 +/– 2 hours. The negative control was performed by incubating BPW with Chemboost S at 37°C for 18 +/– 2 hours. In order to determine the reproducibility of our results, we have done 3 replicates.

2.5 Sample preparation

In a filter bag 25 g of food product was homogenised and mixed with 225 ml of BPW and 1 ml of ChemBoost S. Then the sample was incubated at 37°C for 18 +/– 2 hours prior to flow cytometry analysis. The experimental protocol is detailed in figure 1.

Two types of samples are prepared:

a) non-contaminated food products

Food products were selected among dairy (20 products), meat (10 products), fish (10 products) and egg products (10 products) (table 3, column 1). Each sample was tested for the absence of Salmonella spp. in 25 g of product. The samples were prepared as described above in the section “sample preparation”. Each non-contaminated sample was analysed by both methods.

(5)

b) Artificial contamination of food products

For the artificial contamination we have selected 30 different food products (10 of meat and 20 of dairy). The samples were prepared as described previously and the artificial spiking was performed by inoculation of less than 30 stressed cells/

25g of product.

Reference method Food

dt

Incubation (37°C, 18 h ± 2 h) Add 1 ml ChemBoost S (0.4% V/V)

Flow cytometry analysis (250 µl sample)

Positive

Weight 25 g food product

Add 225 ml buffered peptone water in filter bag

Negative

0.1 ml in 10 ml RVS**

Incubation (41.5°C, 24 h ± 2 h)

ASAP*

Pos

XLD*

Neg Pos Neg

Incubation (37 °C, 24 h)

1 ml in 10 ml MKTT**

0.1 ml in 10 ml RVS**

XLD*

Positive

Biochimical Confirmati Weight 25 g food product

Add 225 mL buffered peptone water in filter bag

Incubation (37°C, 18 h)

Positive Negative

Incubation (37°C, 24 h) Hektoen* XLD* Hektoen*

41.5°C, 24 h 37°C, 24 h

Nutrient agar Negative

37°C, 24 h

37 °C, 24-48 h

Nutrient agar (4 Separated colonies)

37°C, 24 h

Negative

* ASAP, XLD and Hektoen: selective agar for Salmonella.

** RSV and MKTT: selective broth for Salmonella.

Positive Fluorassure Detection kit

D 0

D 1

D 2

D 3

D 1

D 2

D 3

D 4

D 5

D6

Figure 1

Different steps of analysis for a sample with the reference method and the flow cytometry method.

(6)

In table 4, column 1 and 2, the type of each product and the parameters for the artificial contamination (strain used, number of cells added and type of stress) are reported.

As for non-contaminated sample, each contaminated sample was analysed firstly by the “Fluorassure Detection kit Salmonella spp” with flow cytometry analysis and secondly by the reference method.

Inoculums levels were confirmed by plate counts on TSA incubated at 37°C for 24 hours, and 10 replicates were performed in order to determine the reproducibility.

2.6 Procedure for Flow cytometric analysis

After 18 hours incubation, 250 µl of sample was transferred into a 20 ml tube fit- ted with a ChemFilter25 (AES Chemunex, France), 2 ml of ChemSol B30 was added, and then centrifuged (3200 rpm for 8 min). The supernatant was discarded and the pellet was labelled with 50 µl of Chemld S, a FITC-antibody solution for Salmonella spp, and then incubated at 40˚C for 1 hour. After incubation, 100 µl of CS30 was added to minimise the background fluorescence. After 1 min contact time 1 ml of ChemSol B30 was added into the 20 ml tube. Twenty five µl of the labelled sample was transferred into a 3 ml tube to which we added 600 µl of ChemSol B30. Finally, the tube was placed onto the flow cytometer apparatus (BactiFlow, AES Chemu- nex., France) for automatic analysis. The microorganisms were aligned and focused into a laminar fluid stream.

The labelled cells are fluorescent and passing through the flow cell onto which the laser beam is focused. Each cell is individually detected and counted by sensi- tive photo-multipliers.

Results were displayed on the flow cytometer as microbial count/ml of labelled sample. The result is positive if the microbial count is superior to the threshold value set. Levels for pass or negative (green) and action or positive (red) can be pre-set to enable easy visual display of results.

3 – RESULTS AND DISCUSSION

3.1 Selectivity (inclusivity-exclusivity)

The selectivity is a measure of inclusivity and exclusivity:

– Inclusivity means the ability of the alternative method to detect a wide range of Salmonella serotypes.

– Exclusivity means the ability of the alternative method not to detect a relevant range of non-target microorganisms.

The selectivity test was also used to establish the optimal condition of Salmo- nella detection by flow cytometry method.

The objective of this study is to ensure that all the strains of Salmonella are detected by flow cytometry method and that no cross reactions with non- Salmo- nella species occur.

The results are detailed in table 1A for Salmonella serotypes and in table 2 for non-Salmonella species.

(7)

Table 1A

Results of analysis of different Salmonella serotypes (47 strains) with flow cytometry method.

Salmonella species Origin Flow cytometry

results Initial level of contamination (CFU/250mL)

S. Typhimurium CIP 104115 + 8

S. Typhimurium pork + 3

S. Typhimurium steak + 5

S. Typhimurium Pork meat + 7

S. Virchow CIP 105 355 + 8

S. Virchow 6838 (lact +) + 6

S. spp + 5

S. Schwarzengrund beef powder + 8

S. Kaneshie vegetable + 5

S. Bredeney chicken + 5

S. Bredeney turkey hen + 100

S. Montevideo beef + 10

S. Montevideo AES Sal 17.8 + 36

S. Brandenburg duck + 10

S. Brandenburg cooked meat + 97

S. Orion duck + 13

S. Derby pork + 6

S. Derby pork + 92

S. Paratyphi B chicken + 12

S. Agona milk + 91

S. Enteritidis egg product + 87

S. Gallinarum CIP A 255 + 97

S. Heidelberg poultry meat + 41

S. Indiana beef + 27

S. Javiana mushroom + 37

S. Paratyphi B chicken + 12

S. Paratyphi B CIP 54 100 + 42

S. Paratyphi B AES Sal 19.1 + 36

S. Paratyphi B AES Sal 19.2 + 102

S. Saintpaul turkey hen + 24

S. Schwarzengrund pork + 46

S. Senftenberg CIP 105 343 + 38

S. Paratyphi A CIP A 220 + 13

S. Enteritidis chicken + 5

S. Infantis Meat powder + 6

S. Anatum Dry sausage + 34

S. Anatum Peeled sesame + 25

S. London sea snail + 19

S. Dugbe* Vegetable - 5

S. Paratyphi A* CIP 55 41 - 4

S. Kottbus* vegetable - 8

S. Virchow* 11337 (intox) - 4

S. Typhi* CIP 54 136 - 6

S. Typhimurium* meat - 9

S. Paratyphi C* CIP 55 108 - 8

* Strains detected positive in presence of matrix (Table 1B)

(8)

In table 1 A, 47 strains of Salmonella were tested with flow cytometry method. 39 strains were detected (positive results) and 8 strains were not detected (negative results). For these 8 strains of Salmonella we observed no growth in the broth (BPW with ChemBoost S). The hypothesis was that these strains of Salmonella are not able to multiply in absence of matrix.

To confirm this hypothesis, bacon cubes were contaminated with these 8 strains of Salmonella. The results are shown in table 1B. In the presence of food products all results are positive and all 8 strains of Salmonella are detected.

These results show that the inclusivity is good and the method with flow cytometry allows the detection of all tested Salmonella strains.

Table 1B

Results of bacon cubes analysis, artificially contaminated with 8 strains of Salmonella.

In table 2, 30 strains of non-Salmonella were tested with flow cytometry after enrichment in buffered peptone water without ChemBoost S, in order to evaluate the specificity of the labelling solution. The initial contamination added in the broth for all strains is around 10⁵ CFU/ ml. After determination of the final concentration, the results show that all strains grew from 2 to 4 Log.

These results are in accordance with what we expected as all non–target strains were detected negative. It means that the exclusivity of flow cytometry analysis is favourable.

All the test with Salmonella strains were detected positive and all the others test with non Salmonella strains were detected negative. We can conclude that we had no cross reaction with the selected antibody and that the labelling solution used (ChemId S) is specific for Salmonella species.

3.2 Applicability to a wide range of food products

For this test, 50 food products were randomly chosen in a supermarket. The objective of this study is to evaluate the performance of flow cytometry and reference method on contaminated and non-contaminated samples. This experiment allows also to verify whether there was any inhibition of the labelling of Salmonella cells due to the food matrix.

Results of both methods are shown in table 3.

Salmonella species Origin Flow cytometry

results Initial level of contamination (CFU/250mL)

S. Kottbus vegetable + 15

S. Kottbus vegetable + 20

S. Virchow 11337 (intox) + 16

S. Typhi CIP 54 136 + 24

S. Typhimurium Meat + 15

S. Paratyphi C CIP 55 108 + 21

S. Dugbe vegetable + 15

S. Paratyphi A CIP 55 41 + 23

(9)

Table 2

Results of analysis of different non-Salmonella strains with flow cytometric method.

Non-Salmonella species Origin

Flow cytometry

results

Initial level of contamination N₀ (CFU/mL)

Concentration after enrichment

N_t=18h (CFU/mL)

Bacillus cereus CIP 549 – 3.3 _✕10⁵ 4.7 _✕10⁷

Bacillus cereus milk – 2.1 _✕10⁵ 1.9 _✕10⁷

Bacillus circulans milk – 9.6 ✕ 10⁵ 2.7 ✕ 10⁸

Bacillus subtilis cream dessert – 4.1 _✕10⁵ 4.4 _✕10⁸

Enterococcus hirae CIP 5855 – 1.4 _✕10⁵ 4.6 _✕10⁸

Staphylococcus epidermis environment – 4.9 ✕ 10⁵ 2.4 ✕ 10⁷

Staphylococcus aureus ATCC 6538 – 8.3 ✕ 10⁵ 8.0 ✕ 10⁸

Escherichia coli milk – 3.1 _✕10⁵ 1.5 _✕10⁹

Escherichia coli carrot – 1.9 ✕ 10⁵ 4.7 ✕ 10⁹

Escherichia coli ATCC 8739 – 2.8 ✕ 10⁵ 1.9 ✕ 10⁹

Escherichia coli CIP 54127 – 3.1 _✕10⁵ 5.0 _✕10⁷

Enterobacter aerogenes milk – 3.0 _✕10⁵ 6.2 _✕10⁸

Enterobacter aerogenes CIP 6086T – 2.4 ✕ 10⁵ 2.0 ✕ 10⁹

Enterobacter cloacae – 3.7 _✕10⁵ 1.7 _✕10⁹

Enterobacter cloacae CIP 6085 – 2.1 _✕10⁵ 3.3 _✕10⁸

Hafnia alvei tabouli – 4.1 ✕ 10⁵ 2.1 ✕ 10⁹

Klebsiella pneumoniae pastry – 2.7 ✕ 10⁵ 1.4 ✕ 10⁹

Klebsiella oxytoca soja – 2.8 _✕10⁵ 2.9 _✕10⁸

Klebsiella pneumoniae CIP 8291 – 1.3 ✕ 10⁵ 7.1 ✕ 10⁸ Pseudomonas aeruginosa CIP 100720 – 2.0 ✕ 10⁵ 1.6 ✕ 10⁹ Pseudomonas aeruginosa ATCC 19429 – 3.5 _✕10⁵ 4.2 _✕10⁸ Pseudomonas fluorescens CIP 69.13T – 2.0 _✕10⁵ 1.8 _✕10⁸ Pseudomonas fluorescens CIP 102127 – 3.3 ✕ 10⁵ 5.6 ✕ 10⁷

Citrobacter freundii ATCC 8090 – 4.4 _✕10⁵ 1.2 _✕10⁹

Citrobacter koserii CIP 7211 – 2.1 _✕10⁵ 1.1 _✕10⁸

Citrobacter freundii CIP 5362 – 4.2 ✕ 10⁵ 1.5 ✕ 10⁹

Candida albicans ATCC 10231 – 6.0 ✕ 10⁵ 2.0 ✕ 10⁶

Acinetobacter baumanii turkey hen – 1.8 _✕10⁵ 2.6 _✕10⁸

Shigella flexneri CIP 82.48T – 2.9 ✕ 10⁵ 1.0 ✕ 10⁹

Shigella sonnei ATCC 9290 – 1.7 ✕ 10⁵ 1.1 ✕ 10⁹

(10)

Table 3

Analysis of non-inoculated food products with flow cytometry and reference method.

Product Flow cytometry

results

Confirmation of Flow cytometry results

Reference method

Raw pig leg – / –

Raw turkey hen cuelet – / –

Raw lamp leg – / –

Cooked white fowl + – –

Minced steak – / –

Meat gruel – / –

Whole fat liver – / –

Duck’s breast – / –

Ham – / –

Dry sausage – / –

Young turbot and salmons’ meat – / –

Smoked salmon – / –

Prawn – / –

French beans – / –

Cucumber nature – / –

Hoki filet – / –

Prawn, shrimp – / –

Raw young mackerel – / –

Raw sea bass – / –

Raw back – / –

Whole egg – / –

Egg white + – –

Egg product – / –

Mayonnaise – / –

Yolk – / –

Mayonnaise of hardboiled egg – / –

Omelette – / –

Nature hard-boiled egg – / –

Gruyere – / –

Emmental + – –

Raw milk – / –

Comté – / –

Gruyere – / –

Cantal – / –

Fresh cheese – / –

(11)

Only five products gave positive results with flow cytometry method. These positive results were not confirmed by biochemical tests. All other products gave negative results with both flow cytometry and reference method.

As we had no positive results, we decided to perform artificial spiking of food products with Salmonella. Dairy and meat products were chosen for the test. We used stressed strains of Salmonella. The results are given in table 4 and a synthesis of all products results is detailed in table 5.

All products gave identical results for both reference and flow cytometry method except 2 products (Roquefort and Cantal). The analysis of Roquefort gave a negative result with flow cytometry method and a positive result with the reference method.

The analysis of Cantal gave a positive result with flow cytometry method and a negative result with the reference method. The Roquefort and the Cantal contain a high concentration of non Salmonella flora which can slow the growth of Salmonella.

Since the results are obtained from two separate contaminated aliquots and it is a low level inoculum, an heterogeneity of growth can be expected between the alternative and the reference method. In the case of the other food products the non Sal- monella flora is reduced leading to an absence of interference.

3.3 Relative accuracy, relative sensitivity and relative specificity

The relative accuracy is the degree of correspondence between the response obtained by the reference method and the response obtained by the flow cytometry method on identical samples.

The relative sensitivity is the ability of the flow cytometry method to detect the microorganism when it is detected by the reference method.

The relative specificity is the ability of the flow cytometry method to not detect the target microorganism when it is not detected by the reference method.

Relative accuracy, relative sensitivity and relative specificity of flow cytometry method are detailed in table 6.

The Percentage of non-confirmed presumptive positive results has been calcula- ted and the result is 7.5 %.

The results show that the values of relative accuracy, relative sensitivity and relative specificity for the cytometry method are acceptable.

Half skimmed pasteurized milk – / –

Comté – / –

Gruyere – / –

Comté – / –

Milk powder – / –

Cream + – –

Fresh milk – / –

Roquefort + – –

Fresh cheese – / –

Infantile milk powder – / –

Yoghurt – / –

Product Flow cytometry

results

Confirmation of Flow cytometry results

Reference method

(12)

Table 4

Results of artificially contaminated samples.

Products

Parameters of artificial contamination Flow cytometry results and confirmation

Reference method Strain CFU Stress Log reduction

Sausage

S. Agona 25

50°C 20 min

0.6

+ +

Roast chicken + +

Lamb + +

Raw rumsteak + +

Fish + +

Sauted turkey hen

S. Indiana 35 0.5

+ +

Raw beef thigh + +

Sausage with much

spice + +

Sauted turkey hen + +

Blanquette veal + +

Pasteurized Milk

S. Agona 28

20 min at 50°C + 20 days at 4°C

0.5

– –

Gruyere + +

Laguiole + +

Raw milk + +

Cantal

S. Dublin 6

25 min at 50°C

0.7

+ –

Raw milk – –

Light cheese spread – –

Half skimmed pasteurized milk

S. Enteritidis 22 0.7

+ +

Comté + +

Fresh cheese + +

Gruyère

S. Agona 24 20 min at 50°C + 20 days at 4°C

0.5

+ +

Roquefort – +

Fresh cheese + +

Comté

S. Dublin 20

25 min at 50°C

0.7

+ +

Milk powder + +

Cream + +

Fresh milk + +

Cantal

S. Montevideo 7 1.2

+ +

Comté + +

* flow cytometry results confirmed by biochemical tests

(13)

Table 5

Results synthesis of all food products categories.

Table 6

Calculation of the relative accuracy, the relative sensitivity and the relative specificity.

3.4 Practicability of the flow cytometric method

The comparison of required time for each method is detailed in table 7. This comparison was made for negative and positive samples.

The results show that flow cytometry method is much fast than reference method for negative sample. For positive samples no difference is shown.

Table 7

Time to results for positive and negative samples analyzed with both methods

4 – CONCLUSION

Response Reference method positive (R+)

Reference method

negative (R–) Total

Flow cytometry analysis

positive (A+) PA = 25 PD = 1 26

Flow cytometry analysis

negative (A–) ND = 1 NA = 53

PPNA = 5* 54

Total 26 54 80

PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive deviation, PP: presumed positive before confirmation.

A+: confirmed positive.

A–: immediatly negative or negative after confirmation when presumed positive.

*with 5 non confirmed presumptive positive results by flow cytometry.

PA NA ND PD Total

N Relative accuracy AC (%) [100 x (PA+NA)]/ N

N+

PA+ND Relative Sensitivity SE (%) (100 x PA) / N+

N–

NA+PD Relative Specificity SP (%) (100xNA)/N–

Total 25 53 1 1 80 98 26 96 54 89

Negative sample Positive sample

Method Flow cytometry Reference Flow cytometry Reference

Pre-enrichment J0 J0 J0 J0

BactiFlow analysis J1 – J1 –

Enrichment – J1 J1 J1

Plating out on selective agars – J2 J2 J2

Reading – J3 J3 J3

Confirmation tests (purification, biochemical and serological tests)

– – J4 to J7* J4 to J7*

*: identification of the first colony at J5, and after 48 hours after for the others.

(14)

All results show that the flow cytometry method (alternative method) is equivalent to the ISO method (6579) for the food samples tested. Different kinds of products were analysed, meat, dairy, egg products and sea produts.

Selectivity data shows the ability of the alternative method to detect different serotypes. 50 strains of Salmonella spp were tested. For the non-target strains no cross-reaction was observed with the 30 strains tested.

Concerning the relative accuracy, relative sensitivity and relative specificity, the data shows acceptable results for the 80 food samples analysed except 2 discord- ant results (1 PD and 1 ND). This conclusion is in accordance with the ISO 16140.

The advantage of the alternative method is to give negative results in 24 hours, this can be very interesting in the screening analysis.

Moreover automation of the alternative method can be an advantage by increasing productivity.

REFERENCES

BOUIX M., GRABOWSKI A., CHARPENTIER M., LEVEAU JY., DUTEURTRE B. 1999.

Évaluation rapide des niveaux de contamination des moûts de champagne par cytométrie en flux, J. Int. Sci. Vigne. Vins, 33, n° 1, 25-32.

BOUIX M., LEVEAU J.Y., 2001. Les applica- tions de la cytométrie en flux en microbiologie. Bulletin de la Société Française de Microbiologie, 16, 210-218.

BOUIX M., LEVEAU JY., CHARPENTIER M., MANGEOT G. 2003- La cytométrie en flux : un outil pour la maîtrise de la qualité organoleptique et de la qualité microbiolo- gique des produits par la conduite des procédés, Ind. Agric. Alim., cahier scientifique, septembre, p. 8-12

BRUESTSCHY A., LAURENT M., JACQUET R., 1994. Use of flow cytometry in oeno- logy to analyse yeast. Letters in Appl.

Micro. 18, 343-345.

CENTERS FOR DISEASE CONTROL AND PREVENTION (C.D.C.), 2009. Multistate outbreak of Salmonella infections associated with peanut butter and peanut butter- containing products--United States, 2008- 2009. MMWR. Morb. Mortal. Wkly. Rep.

58, 85-90.

DUMAIN P.P., DESNOUVEAUX R., BLOCH L., LECOMTE C., FURHMANN B., DECO- LOMBEL E., PLESSIS M.C., VALERY S., 1990. Use of flow cytometry for yeast and mould detection in process control of fer-

mented milk product – a factory study, Biotech. Forum Europe. 3, 224-22.

GRADEL K.O., SCHONHEYDER H.C., PEDERSEN L., THOMSEN R.W., M. NOR- GAARD M., NIELSEN H., 2006. Incidence and prognosis of nontyphoid Salmonella bacteraemia in Denmark: a 10-year county-based follow-up study. European Journal of Clinical Microbiology & Infec- tious Diseases. 25, 151-158.

LAPLACE-BUILHE C., HANE K., HUNGER W., TIRILLY Y., DROCOURT J.L., 1993. Appli- cation of flow cytometry to rapid microbial analysis in food and drinks industries. Bio.

cell. 73, 123-128.

LETEILLIER C., BOUIX M., DROCOURT J.L., 2003. Dénombrement de la flore totale du jambon par cytométrie en flux. IAA, novembre 2003.

NF EN ISO 6579, 2002. Reference method for Salmonella spp: Microbiology of food and animal feeding stuffs. Horizontal standard for the detection of Salmonella. NF EN ISO 6579 (December, 2002).

NF EN ISO 16140, 2003. Reference method for Validation of alternative methods:

Microbiology of food and animal feeding stuffs: Protocol for the validation of alternative methods. NF EN ISO 16140, 2003 (October, 2003).

PETTIPHER G.L. 1991. Preliminary evaluation of flow cytometry for the detection of

(15)

yeast in soft drinks. Letters in Appl. Micro- biol. 12, 109-112.

SILVESTRI A.C., BOUIX M., LEVEAU J.Y., DROCOURT J.L. 1997. Extraction of Ente- robacteriacecae from foods by immuno- magnetic separation before flow cytometric detection. Sci. Aliments, 17, 361-370.

VAN DER GAAG M.A., H.W. SAATKAMP H.W., VOS F., VAN BOVEN M., P. VAN BEEK P., HUIME R.B.M., 2005. Simulation of the epidemiology of Salmonella in the pork supply chain. Operations Research Proceedings. 8, 263-270.

(16)