DNA-FINGERPRINTING OF EIKENELLA-CORRODENS BY PULSED-FIELD GEL-ELECTROPHORESIS

In order to conduct molecular typing of Eikenella corrodens strains by macrorestriction fingerprinting, we evaluated different restriction e nzymes for digestion of genomic DNA and determined the optimal paramet ers for separating E. corrodens DNA by pulsed-field gel electrophoresi s. Ten E corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare-cutting restric tion endonucleases DraI, SmaI and XbaI usually used for pulsed-field g el electrophoresis analyses were not suitable for digestion of E. corr odens genomic DNA because they either did not digest the DNA or produc ed bands of similar molecular weights that could not be separated. Acc ordingly, among additional enzymes including BamHI, BglII, EcoRI and H ind III, we found BamHI and BglII to be the most suitable rare-cutting enzymes for pulsed-field gel electrophoresis analysis. They cleaved t he genomes of all the above strains into 15-20 fragment bands that wer e clearly separated by the following pulsed-field gel electrophoresis conditions: 140 V with a running time of 40 h, pulse times of 5 to 50 s with linear ramping and an electrical field angle of 120-degrees. Th ese conditions enabled us to distinguish 8 individual pulsed-field gel electrophoresis patterns from the 10 strains analyzed. However, only 4 identical outer membrane protein profiles were differentiated by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis. The data obtai ned in this analysis showed clonal divergence among members of the E c orrodens species, at the same time revealing this pulsed-field gel ele ctrophoresis as being a highly attractive procedure for epidemiologica l investigation of this organism, including its acquisition and transm ission.