Role for flagella but not intimin in the persistent infection of the gastrointestinal tissues of specific-pathogen-free chicks by shiga toxin-negative Escherichia coli O157:H7

Abstract

Shiga toxin (Stx)-positive Escherichia coli O157:H7 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative E. coli O157:H7 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157 E. coli in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in E. coli O157:H7 isolate NCTC12900. These mutants were used to inoculate (10(5) CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intimin-aflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.

FIG. 1.

Southern blot hybridization of E. coli O157:H7 (NCTC12900) and isogenic intimin- and flagellum-deficient mutants to demonstrate insertional inactivation of target genes. (A) Lanes: 1, 3, 5, 7, and 9, NCTC12900; 2, 4, 6, 8, and 10, DM3 (eae::Cam^r) digested with PvuII (lanes 1 and 2), EcoRI (lanes 3 and 4), SalI (lanes 5 and 6), EcoRV (lanes 7 and 8), and AvaI (lanes 9 and 10). (B) Lanes: 11, 13, 15, 17, and 19, NCTC12900; lanes 12, 14, 16, 18, and 20, DM4 (fliC::Str^r) digested with ClaI (lanes 11 and 12), MfeI (lanes 13 and 14), EcoRI (lanes 15 and 16), EcoRV (lanes 17 and 18), and BglI (lanes 19 and 20). DM5 (eae::Cam^r, fliC::Str^r) gave a pattern that was a combination of both DM3 and DM4. Purified probes were created using the primers listed for the amplification of fliC and eae genes in the construction of mutants section of Materials and Methods.

FIG. 2.

Adhesion and invasion of HEp-2 cells by Stx-negative E. coli O157:H7 NCTC12900 wild-type, intimin, and aflagellar mutants. DM3, isogenic intimin-deficient mutant; DM4, isogenic flagellum-deficient mutant; DM5, isogenic intimin-flagellum-deficient mutant. Mean log 10 = CFU/ml.

FIG. 3.

Observation of E. coli O157 adherence patterns for intimin- and flagellum-deficient mutants as demonstrated by SEM (A through D) and FAS (E through H). Wild-type NCTC12900 (A and E), isogenic intimin-deficient mutant DM3 (C and F), isogenic flagellum-deficient mutant DM4 (B and G), and isogenic intimin-flagellum-deficient mutant DM5 (D and H) are shown. Bar = 20 μm. FAS magnification, ×1000.

FIG. 4.

Colonization and invasion of SPF chicks by Stx-negative E. coli O157:H7 NCTC12900 wild-type and intimin- and flagellum-deficient mutants. Post mortem examinations were performed on five birds from each group on days 1, 2, and 5; on two birds from each group on days 57 and 92; and on three birds from each group on day 211. DM3, isogenic intimin-deficient mutant; DM4, isogenic flagellum-deficient mutant; DM5, isogenic intimin-flagellum-deficient mutant. *, P < 0.05. Statistical comparisons were not applicable when the number of birds in each group was less than five.

FIG. 5.

The distribution of bacterial shedding over time by SPF chicks of wild-type NCTC12900 (A), DM3 (isogenic intimin-deficient mutant) (B), DM4 (isogenic flagellum-deficient mutant) (C), and DM5 (intimin-flagellum-deficient mutant) (D). Recovery of E. coli O157 strains was scored as follows: high, confluent growth; medium, >200 colonies; low, <200 colonies; positive after enrichment; and clear, no colonies.

FIG. 6.

Association of bacteria specifically stained with somatic anti-E. coli O157 sera during the first 5 days of experimental infection. Magnification, ×1,000. Cecal tissue from SPF chicks dosed orally by gavage with wild-type NCTC12900 (A), isogenic intimin-deficient mutant DM3 (B), isogenic flagellum-deficient DM4 (C), and isogenic intimin-flagellum-deficient mutant DM5 (D) are shown.