Evaluation of pulsed-field gel electrophoresis as a tool for determining the degree of genetic relatedness between strains of Escherichia coli O157:H7

Abstract

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.

FIG. 1.

Correlation between Dice similarity indices for pairwise analysis of 62 isolates (r = 0.5318).

FIG. 2.

Effect of increasing numbers of enzymes on the correlation between Dice similarities. Triangles, actual data points; diamonds, extrapolations.

FIG. 3.

Isolate pairs AB, CD, CE, DE, FG, and HI were 100% similar after digestion with XbaI, PFGE by the standard protocol, and analysis in Bionumerics with a 1% position tolerance. Lanes λ contain lambda DNA concatemers. (a and c) PFGE gel image. (b and d) Nylon membrane with lambda probe hybridized to membrane. See the text for interpretation. The boxes mark occurrences of nonhomologous same-size bands.

FIG. 4.

Pulsed-field gels of isolate EDL933 after XbaI digestion. Shown are electrophoresis conditions according to the standard PulseNet protocol with switch times from 2.2 to 54.2 s over a 21-h run time (A) and 0.1 to 38 s over a 24-h run time (B).