title: Activity and expression of a virulence factor, gelatinase, in dairy enterococci.
abstract: In order to understand the virulence potential of dairy enterococci, 35 isolates from raw ewe's milk and traditionally fermented cheeses, identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus dispar and Enterococcus hirae, were screened for their capacity to produce gelatinase and for the presence of the genes gelE, sprE, fsrA, fsrB and fsrC. Studies correlating gelatinase production with maintenance and subculture of the isolates in the Laboratory environment, and growth in different media were performed. These studies were conducted with two dairy isolates identified as E. faecalis and E. durans, and one clinical isolate, E. faecalis OG1-10. RT-PCR was used for detection of transcripts of the above mentioned genes. Results demonstrated that the virulence factor gelatinase is disseminated among the genus Enterococcus and that dairy isolates are capable of producing gelatinase at comparable levels with clinical isolates, although this capacity is easily lost during conservation by freezing in the laboratory. Therefore, gelatinase production potential of dairy enterococci may be underestimated. The gene gelE was present in all studied isolates. The same was observed for the fsr operon, either complete or incomplete, revealing that the gelatinase genetic determinants, so far only described in E. faecalis, are a common trait in the genus. This work describes for the first time the detection of the complete Fsr-GelE operon in other species than E. faecalis, namely E. faecium and E. durans. The loss of expression of this virulence factor under laboratory culture conditions correlated with the loss of one or more genes of the regulatory fsr operon, although the gene gelE was maintained, demonstrating that a complete fsr operon is required for a positive GelE phenotype. Independent of the detection of any gelatinase activity, if both gelE and the complete fsr operon are present in the cell, all genes are transcribed, as revealed by RT-PCR, suggesting that regulation of gelatinase activity can also be post-transcriptional. The silent behavior of gelE was only observed in E. faecalis, but not in E. durans, suggesting different modulation mechanisms of gelatinase activity in these two species. Overall, these findings reopen the issue of food safety of enterococci and reinforce the need to further study the mechanisms responsible for triggering the virulence factor gelatinase in non-pathogenic enterococcal environmental isolates.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica (IBET)/Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Apartado 12, 2781-901 Oeiras, Portugal. flopes@itqb.unl.pt
authors.forename: Maria de Fátima Silva
authors.initials: Mde F
authors.lastname: Lopes
authors.forename: Ana Patrícia
authors.initials: AP
authors.lastname: Simões
authors.forename: Rogério
authors.initials: R
authors.lastname: Tenreiro
authors.forename: José Joaquim Figueiredo
authors.initials: JJ
authors.lastname: Marques
authors.forename: Maria Teresa Barreto
authors.initials: MT
authors.lastname: Crespo
mesh_terms: D000818:Animals; D001483:Base Sequence; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016983:Enterococcus / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D018093:Gelatinases / Q000235:genetics* / Q000378:metabolism*; D008892:Milk / Q000382:microbiology*; D008969:Molecular Sequence Data; D009876:Operon; D010802:Phylogeny; D012756:Sheep; D013045:Species Specificity; D014774:Virulence / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D018093:Gelatinases