Characterisation of Listeria monocytogenes to provide tools to predict biofilm formation during cheese making

- L. monocytogenes cheese dairy isolates demonstrate to have different susceptibilities to acid and salt. For example, L. monocytogenes A9 was the most acid susceptible strain but showed a high salt tolerance, whereas in contrast L. monocytogenes T8 is an innate acid tolerant strain but posses' a typical osmotolerance response. - Acid adaptation does not promote biofilm formation but acid sensitive strains may benefit. - Salt adaptation promotes biofilm formation. - When adherent to plastic L. monocytogenes cells became very resistant to sodium hypochlorite. - Planktonic L. monocytogenes cells are more susceptible than biofilm cells to disinfection. - A concentration of 250 ppm of sodium hypochlorite for 20 min completely inactivates L. monocytogenes biofilm cells attached to stainless steel.

The number of L. monocytogenes isolates was roughly equivalent for all dairies and, in both regions, it is evident that the incidence of this species is higher in the working rooms. Although there is a prevalence of L. monocytogenes and L. innocua in specific areas of the dairies, some sharing of ecological niches by these species was also observed. Regarding sampled locations, listeriae were recovered from many different types of surfaces including stainless steel, wood and a variety of plastics indicating that all materials are at potential risk of colonisation by listeriae and possible sources of cheese contamination. Since the incidence of L. monocytogenes diminished from working to ripening rooms in the dairies included in this study, the risk of cheese contaminated by L. monocytogenes reaching the consumer is expected to be concurrently lower for these cheeses. When cheese rind is not washed but scraped may also reduce the occurrence of this species in the final product. Even if the number of isolates obtained from each sampling campaign was, in some cases, very different, contamination of dairies with listeriae can occur at any time of the production cycle. L. monocytogenes, L. innocua and L. seeligeri isolates were obtained at almost all times of sampling. No association pattern, either qualitative or quantitative, could be established with the periods of the production cycle. More than one type could be simultaneously present in each dairy because each species and different strains occurred in the same location, even from different species. Listeria spp. in the artisanal dairies surveyed seems to be independent of dairy industrial level, i.e. production facilities, type of cheese produced or time of the year. Moreover, the pattern of types for a species within a dairy in the different sampling dates is not fixed, thus suggesting cycles of elimination and recontamination.

Gene lmo0435 was identified as coding for a surface protein that had similarities to a biofilm associated protein from staphylococci. An insertion mutation was made in gene lmo0435 of L. monocytogenes 10403s to create L. monocytogenes SJ78. Experiments with this mutant indicated that this putative extracellular protein is important for attachment to inanimate surfaces for strains that possess the gene. However, it is not an essential requirement for all L. monocytogenes strains because several lmo0435 -negative diary isolates demonstrated an ability to adhere to inanimate surfaces equivalent to that of lmo0435-positive strains.

Through sampling we have accumulated a comprehensive strain collection of Listeria species, particularly L. monocytogenes and L. innocua which were the two most prevalent species. These have been isolated from dairies in two distinct areas of Portugal. PCR serotyping was also performed for L. monocytogenes, showing the prevalence of serotype 4b (68% of isolates; 52% of genomic types), followed by serotypes 1/2b or 3b (23% of isolates; 24% of genomic types) and 1/2a or 3a (9% of isolates; 24% of genomic types).

A leaflet publicising the recommendations resulting from the project has been produced and widely circulated. This leaflet is available in English, French, Portuguese, Spanish and Italian. A poster publicising the recommendations resulting from the project has been produced for dairies to display prominently. This poster is available in English and Portuguese. The leaflet and poster were designed for use in artisanal cheese producing dairies. In order to cater for people with an interest in the data behind the recommendations, the project website has been expanded to include the background research and results. www.le.ac.uk/ii/eu/lmtooche An open workshop was organised by members of the LMTOOCHE consortium and was held on Friday 24th February 2006 at the Instituto de Ciencia Aplicada e Tecnologia, Lisbon. The workshop was entitled 'Occurrence of Listeria monocytogenes in artisanal and cheese making dairies'. Dairy industry experts from the three European countries involved in the project were invited to the workshop and it was well attended.

- Contamination of dairies with Listeria spp. can occur at any time of the year or any point in the production process. - There is no time of the year when the risk of contamination with Listeria spp. is highest. - All parts of the dairy are at risk of contamination. - Some surfaces have a higher incidence of Listeria monocytogenes than others. - Mixtures of Listeria monocytogenes and other species occur at any time suggesting multiple sources of contamination. - The presence of Listeria spp. other than Listeria monocytogenes neither positively nor negatively predicts the presence of Listeria monocytogenes. - The presence of Listeria spp. in the dairy is not a predictor of the presence of Listeria spp. in the cheese or vice versa. - A certain degree of heterogeneity and variability in the results shown for both types of cheese is characteristic of the fact that these cheeses are traditional products linked to a geographic origin and local know-how, contributing to the preservation of a natural diversity of flavours and to the variety of cheeses. - No conclusions could be withdrawn from the presence of L. monocytogenes in the cheeses, considering the limited number of positive samples found, and no correlation between the presence of L. monocytogenes and chemical composition of cheeses was established.

Our experiments used unique apparatus set-ups including modified radial flow chambers. They have provided quantitative information on the shear forces necessary to detach adherent cells of dairy isolates of L. monocytogenes. This information would be of particular value in, amongst other applications, the rational design of cleaning and decontamination processes. They may also be viewed as offering insights into the effects of cleaning and decontamination procedures involving the use of liquid shears to effect cell detachment. Whilst such procedures will be responsible for removing, organisms to an extent determined by the severity of the process, they will impose a selection pressure that will tend to favour cells of the phenotype typified by the strains we isolated from dynamic locations. The genetic basis for this in L. monocytogenes is currently not known. NaOH has often been used in studies of surface sanitisation of L. monocytogenes biofilms both under static conditions and under flow conditions. Our results show there to be no significant differences between decontamination under static and flow conditions. Moreover, it appears that surface roughness is not a significant parameter in determining biofilm removal. Our results do not show a correlation between the number of L. monocytogenes cells adhering to stainless steel and the force required to detach them. Moreover, the absence of the bap gene not only reduces the cells' ability to attach to a surface but also decreases the force necessary to remove cells already bound. However, bap-negative strains such as T8 and C897 require higher shear stresses for removal than the bap-positive 10403s.

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