Involvement of the Escherichia coli O157:H7(pO157) ecf operon and lipid A myristoyl transferase activity in bacterial survival in the bovine gastrointestinal tract and bacterial persistence in farm water troughs

Abstract

Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf (E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.

FIG. 1.

Defined deletion of the pO157 ecf operon and chromosomal lpxM. The partial diagram of E. coli O157:H7(pO157) has genomic nucleotide sequence numbers are from the GenBank database (accession no. AF080442 and AE005174). The arrows indicate the direction of translation.

FIG. 2.

Fatty acid analysis of the LPS purified from wild-type E. coli O157:H7, YH101 (Δecf), YH102 (ΔlpxM), and YH103 (Δecf ΔlpxM). LPS was isolated from E. coli cells grown at 37°C, subjected to methanolysis in 2 M methanolic HCl at 90°C for 18 h, extracted with hexane, and analyzed by GC/MS. C12:0, laurate; C14:0, myristate; C14:OH, 3′-hydroxymyristic acid.

FIG. 3.

Survival of wild-type E. coli O157:H7, YH101 (Δecf), YH102 (ΔlpxM), and YH103 (Δecf ΔlpxM) in synthetic bovine gastric juice. Individual E. coli strains grown at 37 or 24°C were exposed to synthetic bovine gastric juice (see Materials and Methods) for 15 min, and the surviving E. coli cells were counted by direct plating on LB agar. Data were obtained from at least three independent experiments.

FIG. 4.

Effect of bile on the growth of wild-type E. coli O157:H7, YH101 (Δecf), YH102 (ΔlpxM), and YH103 (Δecf ΔlpxM). Wild-type E. coli O157:H7 and mutant strains were grown either in LB broth at 37°C (A) or 24°C (B) or in LB broth containing 0.15% bile salts no. 3 at 37°C (C) or 24°C (D). All values represent the average optical density at 600 nm (OD₆₀₀) obtained from at least five independent experiments.

FIG. 5.

Colonization of the bovine RAJ by wild-type E. coli O157:H7 or YH103 (Δecf ΔlpxM). Three cattle per group were inoculated directly onto the RAJ mucosa with a single dose of 1.5 × 10¹⁰ CFU of wild-type E. coli O157LH7 (CH1, CH2, and CH3) or YH103 (Δecf ΔlpxM) (CH4, CH5, and CH6). The numbers of E. coli O157:H7 cells detected by RAMS culture are shown. ND, none detected; ER, RAMS sample culture positive for E. coli O157 after enrichment culture only and fewer than 30 CFU of E. coli O157 per swab.

FIG. 6.

Survival and persistence of wild-type E. coli O157:H7, YH101 (Δecf), YH102 (ΔlpxM), and YH103 (Δecf ΔlpxM) in farm water troughs. Spontaneously nalidixic acid-resistant wild-type and mutant strains were inoculated individually or as a mixture into microcosms simulating cattle water troughs with a single dose of 8 × 10⁹ CFU (see Materials and Methods). The number of surviving E. coli cells was determined by direct plating of water samples on LB agar containing 25 μg of nalidixic acid per ml. High and low daily air temperatures are shown. To simulate cattle drinking, one-half of the water volume was removed every day and replaced with fresh water. ND, none detected; ER, culture positive for E. coli O157 only after enrichment.

FIG. 7.

Motility of wild-type E. coli O157:H7 and mutant strains YH101 (Δecf), YH102 (ΔlpxM), and YH103 (Δecf ΔlpxM). Bacteria were stabbed into 0.3% soft agar and incubated at 37°C for 6 h or at 24°C for 12 h, and the diameter of the zone of bacterial growth (motility halo) was measured. A typical plate incubated at 24°C is shown on the left, and the data shown in the graph were obtained from three independent experiments.

FIG. 8.

EM analyses. (A) SEM analysis of wild-type E. coli O157:H7, YH101 (Δecf), YH102 (ΔlpxM), and YH103 (Δecf ΔlpxM). E. coli strains were grown at 37°C for 18 h, fixed with 2.5% glutaraldehyde, stained, and observed at a magnification of ×5,000. (B) TEM analysis of wild-type E. coli O157:H7 and YH103 (Δecf ΔlpxM). E. coli strains were grown at 37°C for 18 h, fixed with 2.5% glutaraldehyde, stained, and observed at magnifications of ×5,000 (left side) and ×50,000 (right side). As indicated by the arrows, about 10 to 20% of the YH103 cells had wrinkled or malformed membrane structures compared to the wild type and the two single-mutant strains.