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In order to understand the causes of the low virulence of Listeria monocytogenes field strains, five low-virulence strains were analysed. These five strains showed changes in relation to invasion, phosphatidyl-inositol phospholipase C (PI-PLC) activity, plaque formation and in vivo virulence. Molecular analyses revealed the same mutations in the picA, inlA and inlB genes in all five strains. The Thr262Ala substitution in the PI-PLC protein was responsible for the absence of PI-PLC activity. This residue, conserved in certain L. monocytogenes species, is located at the outer rim of the active site pocket and could impair the cleavage activity of the enzyme. The low invasion rate of these strains was due to a nonsense codon leading to a lack of InlA protein synthesis, and to an Ala117Thr substitution in the leucine-rich repeat of InlB, which altered the interaction with the Met receptor. Single trans complementation with the [inlA.sub.EGDe], [inlB.sub.EGDe] or [plcA.sub.EGDe] genes restored the capacity of low-virulence strains either to enter epithelial and fibroblastic cells or to express PI-PLC activity. Complementation by allelic exchange of the [plcA.sub.EGDe] gene on the chromosome and trans complementation with either the [inlA.sub.EGDe] or the [inlB.sub.EGDe] gene restored the ability to form plaques, but only partly restored the in vivo virulence, suggesting that there were other gene mutation(s) with consequences that could mainly be observed in vivo. These results indicate that the low virulence of L. monocytogenes strains can be explained by point mutations in a number of virulence genes; these could therefore be important for detecting low-virulence strains. Moreover, the fact that all the strains had the same substitutions suggests that they have a common evolutionary pathway.

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGD[DELTA]prfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix [alpha]H. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.

Twenty-six field Listeria monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in subcutaneous inoculation test on mice by assigning four groups (I, II, III, IV). The results demonstrates a relationship between the phenotypic classification and the genotypic modifications for at least group I and III and suggest a common evaluation of these strains within the group.

Aims: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. Methods and Results: A total of 60 isolates of L.  monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/ AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque-forming assay and by mouse virulence assay. Conclusions: Our findings suggest that L. monocytogenes strains found in non-sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. Significance and Impact of the study: This is the first study dealing with the characterization of L. monocytogenes isolates from non-sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land. [ABSTRACT FROM AUTHOR]

Abstract: We have previously shown a relationship between the virulence level of Listeria monocytogenes strains and their detection on PALCAM medium. To account for the fact that only 40% of low-virulence field strains of L. monocytogenes were detected on PALCAM medium compared to 92% on ALOA medium, the detection of virulent and low-virulence strains on decomposed selective ALOA and PALCAM media was compared. This showed that better detection of the strains was not explained by the growth factors added to the ALOA medium. On the other hand, the presence of acriflavine in the PALCAM medium partly explained the delay in detection of the low-virulence strains, while the presence of ceftazidime was related to growth inhibition. However, the effect of these two components was modified when they were combined in the PALCAM medium. As some of these low-virulence strains had an inactive PrfA (the transcriptional activator of the main virulence genes of L. monocytogenes), its role in the poor detection of these low-virulence strains was investigated. However, complementing these strains with the wild-type prfA gene or deleting the prfA gene from a virulent strain suggested that this poor detection was unrelated to PrfA, but was related to their higher susceptibility to the antimicrobial components in the selective media. [Copyright &y& Elsevier]