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Dr. Devleeschouwer, ULB - Biocontaminants et Microbiologie et Hygiène, Inst. de Pharmacie - CP 205/2
Boulevard du Triomphe, 1050 Bruxelles
Prof J. Dony, ULB - Unité des Biocontaminants et risques physico-chimiques du milieu
Boulevard du Triomphe, 1050 Bruxelles
To reach this goals several approaches were considered:
(a) A study of cheese contamination by Listeria spp. This study performed on 1902 samples allowed us to compare three detection methods for Listeria. The aims of this part were:
Sample analysis and strain identification were performed by ULB. IHE confirmed the identifications and performed serotyping.
(b) A study of the characteristics of Belgian Listeria monocytogenes strains isolated from various foods and from sporadic episodes of human listeriosis. This part required from the IHE to develop a novel typing method based on esterase polymorphism. This study concerned 832 strains. A comparison of serotyping and esterase typing was performed on two strain populations (clinical and dairy strains) and discrimination indexes were calculated. The study showed that the population of Listeria monocytogenes isolated from cheese was different of that isolated from clinical cases. This could not be explained by differences of secretion of virulence marker PI-PLC neither by differences of pathogenicity in a murine model of these strains.
The esterase typing was for the first time used by the IHE to incriminate a contaminated food in a sporadic case of listeriosis.
Furthermore, ULB characterised 54 strains of Listeria monocytogenes, 27 human strains given by IHE and 27 cheese strains, by a study of hybridation polymorphism using ARNr 16s and 23s probes on restriction profiles generated by two endonucleases. This study was performed in collaboration of the University of Liege.
(c) A study aiming to reduce or eliminate the contamination by means of technical improvements. Inhibitory strains of Listeria monocytogenes were isolated from Listeria-free cheese. The strains were identified and the factors influencing the production of the inhibitor were studied more extensively for two inhibitory strains. Some properties of the inhibitory molecules were compared to those of nisin.
The study has been divided in three parts. We first evaluated the contamination of Belgian and foreign cheeses by Listeria monocytogenes. The second part aimed to adapt to L. monocytogenes a ribotyping method used for Clostridium perfringens. The third part of the work was dedicated to the isolation of bacterial strains able to inhibit the growth of L. monocytogenes. We then characterized the antagonistic fractions secreted by these bacteria.
To reach to goals of the first part of the study, we adapted and compared three detection methods of Listeria: the direct method, the method by twofold enrichment and the cold enrichment method. Serology and confirmation of the identification of the strains of Listeria monocytogenes have been made at the National Reference Centre for Listeria, Institute of Hygiene and Epidemiology (Doctor P. ANDRE). In the second part of the work, we studied the restriction profiles of some Listeria strains, profiles obtained with the EcoR1 and HindIII endonucleases. Using those profiles, we have done hybridization using either a rRNA 16S probe from Bacillus subtilis or a rRNA 23S probe from Clostridium perfringens. These experiments were done at the microbiology laboratory of the faculty of veterinary medicine of the "Université de Liège" (Professor KAECKENBEECK and Doctor G. DAUBE). In the third part of the work, we have isolated, from cheeses not contaminated by Listeria sp., strains that inhibit Listeria sp. We further studied the antagonistic properties of strains whose the inhibition was not due to the production of acid metabolites or hydrogen peroxide.
The first step of the study involved, during three successive periods, the analysis of 1902 cheese samples (1520 of Belgian origin and 382 imported ones). Listeria sp. have been isolated from 446 samples (23.4%) and Listeria monocytogenes from 159 samples (8.4%). We have recorded a diminution of the contamination rate of L. monocytogenes during the study. Indeed, this contamination reached 11% (period June 1988 to September 1990), then 6.9% (period March 1991 to February 1992) and finally 6.4% (period April 1992 to February 1993). In 1995, 52 new samples have been analysed. This last sampling suggests that the contamination persist for one producer. The two most frequently isolated species were L. innocua and L. monocytogenes. Serotype 1/2a represented 77.9% of the strains of L. monocytogenes and serotype 4b 12.6%.
The soft cheeses are the most often contaminated. The contamination is located mainly in the surface layer or in the whole cheese. A significant relation could be found between the contamination by Listeria and the presence of faecal coliforms. The numbers of L. monocytogenes isolated per gram of cheese range from less than 100 to one million.
The restriction profiles obtained with the two endonucleases show too many strips to be easily used. Nevertheless, the ribotypings, that associate an EcoR1 restriction and a hybridization with a rRNA 23S probe from C. perfringens, or a HindIII restriction with a hybridization with a rRNA 16S probe from B. subtilis, gave the best discrimination of the strains, particularly in association with serotyping. The combination of serotyping and ribotyping has allowed us to show that two different cheeses from the same producer were relatively homogeneously contaminated, while there was a heterogeneous contamination of a third cheese. Ribotyping is thus a useful technique when studying contamination of dairy products.
In the third part of our work, we hold, on a total of 1320 tested strains, 25 inhibitory strains, namely, 22 Enterococcus faecalis strains, 2 Lactococcus lactis and one Pediococcus pentosaceus strain. An Enterococcus and the Pediococcus strains have been selected to further study the properties of their antagonistic fraction. Several parameters have been studied: production conditions of the antagonist, its sensitivity to proteolytic enzymes and to heat, and the influence of the pH and of casein on the inhibitory action. These experiences showed that these strains secreted molecules with the properties of bacteriocins. Like nisin, they are bactericidal and thermostable. Unlike nisin, they have a narrow bactericidal spectrum. The production of the antagonist fraction (enterocin B57) is plasmid dependent. Attempts to transfer this plasmid to lactic bacteria did however not succeed.
We recommend a regular control of the dairy products for L. monocytogenes. Soft cheeses in particular should be regularly controlled and cheeses should no longer be made with raw milk but made only with pasteurized milk. Improvement of manufacturing hygiene could be reached by implementation of the HACCP (hazard analysis critical control point) system and by using normalised fast detection methods, adapted to each specific pathogen and each specific product.
The cheese protection could be ensured by using pure bacteriocins added before ripening instead of using the antagonist strains themselves. On the other hand, the natural lactic bacteria, into which the plasmids producing the bacteriocins are transferred, could be used for the production of safer cheeses.
Listeria monocytogenes strains recovered in Belgium, fom different foodstuffs and from human sporadic cases of listeriosis, were analysed. The distribution of serovars differed in each of these populations. The bacteria isolated from cheeses and from human cases of listeriosis were further studied by a newly developed typing method. We have analysed L. monocytogenes esterases by starch gel electrophoresis. Five esterases, numbered EST 1 to EST5 in order of decreasing anodal migration, were identified. The EST1, EST 3, EST 4, and EST 5 set was most active toward a-naphtyl propionate, while EST2 was most active toward a-naphtyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed and used to developed a new typing method (esterase typing). Twenty esterase patterns were defined in the population of strains originating from cheeses and from human sporadic cases of listeriosis. These 20 esterase patterns were not equally distributed in these two populations. The secretion of the virulence determinant phosphatidylinositol-specific phospholipase C, and the pathogenicity level of strains in immunocompromised mice couid not explain the different distribution of esterase types. The discrimination of esterase typing (Dl = 0.868) was compared with that of serotyping (Dl = 0.666) and with that of the two combined methods (Dl = 0.899).
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