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Jill Roberts

Advisor: Barbara Murray, MD

Expression of the Enterococcus faecalis virulence-related protein gelatinase requires the fsr two-component regulation system. Mutation of fsr genes or gelE, the gene encoding gelatinase, leads to attenuation of the organism in animal models of infection. We investigated the production of gelatinase by 194 clinical isolates from infected patients and by 21 fecal isolates from healthy individuals. A gelatin plate test assay and hybridization results confirmed that gelatinase activity is not essential for E. faecalis to cause infection and also showed that it is not produced more commonly in clinical isolates as previously suggested. Furthermore, the fsr locus, which positively regulates the expression of gelatinase through the production of an activating pheromone, is absent in a number of clinical isolates. Previous studies on urine isolates from a single hospital reported a 23.9-kb deletion in the fsr locus leading to loss of gelatinase activity. In the current projects, sixty isolates (28% of the total number of isolates tested) were found to contain this deletion as confirmed by positive gelE hybridization results, negative fsrB hybridization results, and amplification of a 1.8-kb fragment using primers located outside of the deleted region. The deletion was present in 17.5% of endocarditis isolates, 15.6% of blood isolates, 44% of urine isolates, and 23.8% of our healthy community fecal isolates. The deletion results in a joining of the 5′-end of an open reading frame designated EF1841 in the TIGR V583 E. faecalis genome database, and the 3′-end of the fsrC gene. The junctions of 10 isolates (four endocarditis, two blood, two urine, and two community fecal) were sequenced and were found to be identical. Thirty-five deletion isolates from geographically distinct locations collected over a 27-year period were analyzed using pulsed-field gel electrophoresis. Based on banding patterns we determined that many of these isolates represent at least 22 distinct strains. These data show that the fsr genes, and the gelatinase enzyme regulated by fsr, are not more common in clinical versus healthy isolates and are not required for E. faecalis pathogenesis.

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Research Info

Molecular Epidemiology of the fsr Locus of Enterococcus faecalis