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Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle - PubMed

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Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle

Ji Youn Lim et al. Appl Environ Microbiol. 2007 Apr.

Abstract

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic DeltapO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the DeltapO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the DeltapO157 mutant. The DeltapO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the DeltapO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the DeltapO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed approximately 50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.

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Figures

FIG. 1.
FIG. 1.

Comparison of WT and ΔpO157 mutant genomic DNA. (A) PFGE of XbaI-digested genomic DNA from the WT or the ΔpO157 mutant. (B) Southern blot hybridization of A with a pO157-specific ecf1 probe. The size fragments shown at the left were estimated using the λ ladder CHEF DNA size standard (Bio-Rad). Lane 1, WT; lane 2, the ΔpO157 mutant. Arrows indicate the location of the 92-kb pO157.

FIG. 2.
FIG. 2.

Comparison of the WT, the ΔpO157 mutant, and its complement for growth, competition, salt tolerance, and heat tolerance. Three strains were grown with aeration in LB medium or M9 minimal medium at 37°C (A). Growth was measured every 30 min spectrophotometrically using a Bio-Tek Power XS reader (optical density at 600 nm). Competitive growth in mixed culture was determined by incubating 2.0 × 108 CFU of both the WT and the ΔpO157 mutant in LB medium for 48 h at 25°C or 24 h at 37°C. After incubation, cell numbers were determined by plate counting on LB agar, and WT and ΔpO157 mutant colonies were distinguished by PCR differentiation. Representative ratios of cell types after competitive growth are shown (B). Heat tolerance (C) and salt tolerance (D) were assessed by exposure to prewarmed TSB (55°C) for 2 h and exposure to TSB with 2.5 M NaCl (15%) at 25°C for 2 weeks. The percent survival was calculated by direct plate counting on LB agar before and after exposure.

FIG. 3.
FIG. 3.

Effect of pO157 on survival of E. coli O157:H7 in synthetic bovine gastric fluid, bile, or low-pH medium. The WT or the ΔpO157 mutant was exposed to each medium for 1 h at 37°C. The number of E. coli cells was determined by direct plate counting on LB agar before and after exposure, and the percent survival was calculated. SGF 3.0, synthetic bovine gastric fluid adjusted to pH 3.0; SGF 2.2, synthetic bovine gastric fluid adjusted to pH 2.2; bile, TSB with 0.15% bovine bile (final concentration); 2.2, TSB adjusted to pH 2.2; bile 2.2, TSB with 0.15% bovine bile (final concentration) adjusted to pH 2.2.

FIG. 4.
FIG. 4.

Analysis of proteins from the WT and the ΔpO157 mutant. After overnight incubation at 37°C, either cells were collected by centrifugation, divided, washed in buffer, and lysed (whole-cell lysate) (A) or the membrane proteins (C) were extracted using the Sigma membrane extraction kit. Cell supernatants (B) were collected by centrifugation and concentrated 10× by centrifugal filtration. These protein preparations were separated by SDS-PAGE and visualized by Coomassie blue staining (A and C) or Silver staining (B). The thick arrows indicate the unique proteins (∼50 kDa) present in ΔpO157 mutant preparations. The thin arrow indicates the lack of enterohemolysin because the strain used to create the complement was Δehx. Lane M (A and B), PageRuler protein ladder (Fermentas Life Sciences); lane M (C), Bench Marker protein ladder (Invitrogen); lanes 1, 4, and 6, WT; lanes 2, 5, and 7, the ΔpO157 mutant; lane 3, complement strain.

FIG. 5.
FIG. 5.

Whole-cell lysate protein profiles of WT (A and C) and the ΔpO157 mutant (B and D) using two-dimensional gel electrophoresis. Whole-cell lysates from the WT and the ΔpO157 mutant were prepared using a French press and divided into two fractions by centrifugation; the insoluble proteins (A and B) and the soluble proteins (C and D) are shown. Proteins were separated by 2-DE using pH 3 to 11 IPG and 12.5% SDS-PAGE. Proteins were visualized by Coomassie blue staining. The pH gradient of the first-dimension electrophoresis is shown at the bottom of the gel, and the migration of molecular mass markers for SDS-PAGE in the second dimension is shown at the left in kDa. Protein spots with intensity differences of more than threefold between the WT and the ΔpO157 mutant are indicated by arrows (thick arrow, increased expression; thin arrow, decreased expression).

FIG. 6.
FIG. 6.

Protein profiles of the WT (A) and the ΔpO157 mutant (B) in the membrane fraction using two-dimensional gel electrophoresis. Cell membrane fractions from the WT and the ΔpO157 mutant were extracted using the Sigma membrane extraction kit and separated by 2-DE using pH 3 to 11 IPG and 12.5% SDS-PAGE. Proteins were visualized with Coomassie blue staining. The pH gradient of the first-dimension electrophoresis is shown at the top of the gel, and the migration of molecular mass markers for SDS-PAGE in the second dimension is shown on the left in kDa. Protein spots with intensity differences of more than threefold between the WT and the ΔpO157 mutant are indicated by arrows. The boxed panels (C and D) show enlargement (×2) of the 50-kDa area in A and B, respectively, and in this box, arrows indicate protein spots with intensity differences of more than twofold between the WT and the ΔpO157 mutant (thick arrow, increased expression; thin arrow, decreased expression).

FIG. 7.
FIG. 7.

Effect of pO157 on survival of E. coli O157:H7 in cattle following oral administration of bacteria. Four steers (steers 1 to 4) were given a single oral dose of 1.0 ×1010 CFU containing both the WT and the ΔpO157 mutant. RAMS samples were cultured by direct plating onto SMAC-CTMV, and sorbitol-negative MUG-negative colonies were confirmed to be the O157 serotype by latex agglutination. At least 15 isolates from each sample were subcultured and differentiated as the WT or the ΔpO157 mutant by PCR. Bar heights represent to total number of E. coli O157:H7 isolates recovered from the steer expressed as log CFU/swab. The proportion of isolates that were either the WT (open white bars) or the ΔpO157 mutant (dotted bars) is shown. Asterisks indicate significant differences between the WT and the ΔpO157 mutant (day 1, P < 0.001; day 3, P < 0.05).

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