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 enzymes for digestion of genomic DNA and determined the optimal parameters for separating E. corrodens DNA by pulsed-field gel electrophoresis. Ten E. corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare-cutting restriction endonucleases DraI, SmaI and XbaI usually used for pulsed-field gel electrophoresis analyses were not suitable for digestion of E. corrodens genomic DNA because they either did not digest the DNA or produced bands of similar molecular weights that could not be separated. Accordingly, among additional enzymes including BamHI, BglII, EcoRI and Hind III, we found BamHI and BglII to be the most suitable rare-cutting enzymes for pulsed-field gel electrophoresis analysis. They cleaved the genomes of all the above strains into 15-20 fragment bands that were 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. These 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data obtained in this analysis showed clonal divergence among members of the E. corrodens species, at the same time revealing this pulsed-field gel electrophoresis as being a highly attractive procedure for epidemiological investigation of this organism, including its acquisition and transmission.
Steffens
L
L
Institut für Hygiene und Mikrobiologie der Universität Würzburg, Germany.
Franke
S
S
Nickel
S
S
Schwarzkopf
A
A
Flemmig
T F
TF
Karch
H
H
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001426:Bacterial Proteins; D015373:Bacterial Typing Techniques; D016172:DNA Fingerprinting / Q000379:methods*; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D016124:Eikenella corrodens / Q000145:classification / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D015245:Deoxyribonuclease BamHI; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific