Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.

The virulence potential of 51 Listeria monocytogenes isolates, including strains from cheese, cheese production environments and from human cases of listeriosis, was evaluated in this study. The isolates were used to infect HT-29 cell monolayers in an in vitro test of virulence, based on a plaque-forming assay (PFA). Fifteen selected isolates were used for subcutaneous footpad inoculation in mice and subsequent recovery of the bacterium from the spleen 3 days after inoculation. In the PFA, two isolates from milk (serovar 1/2a) were not significantly different (P,0.05) from the low-virulence strain (442) used as reference. Thirty-three isolates were not significantly different (P,0.05) from the virulent strain (EGDe) used as reference. Nine isolates were significantly more virulent (highly virulent) than the EGDe strain and seven isolates were significantly less virulent. The nine highly virulent isolates were either from humans (four), from cheese dairy environments (two isolates of a strain were found persistently in two dairies), from cheese (one), from milk (one) and the reference strain for serovar 1/2b (CECT 936). The two milk isolates with low virulence in the PFA were found to be virulent in mice. In conclusion, all the isolates from food and food-related environments were potentially virulent or highly virulent. These results stress the risk of listeriosis associated with the consumption of cheese contaminated with L. monocytogenes, and once more emphasize the importance of good manufacturing practices (GMPs) together with sanitation standard operating procedures (SSOPs) throughout the food chain.

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