Evaluation of pulsed-field gel electrophoresis as a tool for determining the degree of genetic relatedness between strains of Escherichia coli O157:H7.
Pulsed-field gel electrophoresis (PFGE) has been used extensively to investigate the epidemiology of Escherichia coli O157:H7, although it has not been evaluated as a tool for establishing genetic relationships. This is a critical issue when molecular genetic data are used to make inferences about pathogen dissemination. To evaluate this further, genomic DNAs from 62 isolates of E. coli O157:H7 from different cattle herds were digested with XbaI and BlnI and subjected to PFGE. The correlation between the similarity coefficients for these two enzymes was only 0.53. Four additional restriction enzymes (NheI, PacI, SfiI, and SpeI) were used with DNAs from a subset of 14 isolates. The average correlations between similarity coefficients using sets of one, two, and three enzymes were 0.405, 0.568, and 0.648, respectively. Probing with lambda DNA demonstrated that some DNA fragments migrated equal distances in the gel but were composed of nonhomologous genetic material. Genome sequence data from EDL933 indicated that 40 PFGE fragments would be expected from complete XbaI digestion, yet only 19 distinguishable fragments were visible. Two reasons that similarity coefficients from single-enzyme PFGE are poor measures of relatedness (and hence are poorly correlated with other enzymes) are evident from this study: (i) matching bands do not always represent homologous genetic material and (ii) there are limitations to the power of PFGE to resolve bands of nearly identical size. The findings of the present study indicate that if genetic relationships must be inferred in the absence of epidemiologic data, six or more restriction enzymes would be needed to provide a reasonable estimate using PFGE.
Davis
Margaret A
MA
Field Disease Investigation Unit, Department of Veterinary Clinical Sciences, Washington State University, Pullman, Washington 99164, USA. madavis@uidaho.edu
Hancock
Dale D
DD
Besser
Thomas E
TE
Call
Douglas R
DR
D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods / Q000706:statistics & numerical data; D002417:Cattle; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods* / Q000706:statistics & numerical data; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D013045:Species Specificity
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; C025580:endodeoxyribonuclease XBAI; C064053:CCTAGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific