Characterization of urinary tract infection-associated Shiga toxin-producing Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.

FIG 1

Localized adherence pattern of EHEC isolates from hospitalized patients with UTIs. The adherence patterns of the urine isolates were investigated on HEp-2 cells (A) and on T24 urinary bladder epithelial cells (B). (a) EAEC strain 042 (positive control); (b) E. coli K-12 strain C600 (negative control); (c) EPEC O157:H45 strain 1083/97 (bfp positive); (d) EPEC O157:H45 strain 904/90 (bfp negative); (e) EHEC O157:H7 strain EDL933; (f) UR131; (g) UR5703/202; (h) UR5779/201. Bars = 10 μm.

FIG 2

Stx2-mediated detection of Gb3Cer and Gb4Cer in neutral GSL preparations from Vero and T24 cells. (A to C) GSLs in amounts equivalent to those from 5 × 10⁵ cells were applied (lanes b, Vero cells; lanes c, T24 cells). GSL reference mixture 1, composed of Gb4Cer as the predominant GSL and Gb3Cer in a lower abundance (lanes a), and reference mixture 2, containing equimolar amounts of Gb3Cer and Gb4Cer (lanes d), were present at 2 μg and 1 μg, respectively. Supernatants of EHEC isolates UR5703/202 (Stx2a) (A), UR5779/201 (Stx2a) (B), and UR131 (Stx2b) (C) were investigated.

FIG 3

Positive-ion-mode ESI–Q-TOF mass spectra of antibody-detected Gb3Cer species from T24 cells. (A) Overview of the MS¹ spectrum obtained from the silica gel extract of the TLC overlay assay (shown in the inset) performed with an aliquot of GSL at an amount corresponding to the amount from 1 × 10⁶ cells. The area comprising the immunostained double band, from which the silica gel was scraped off, is indicated by a dotted rectangle. (B) Low-energy CID MS² spectrum of monosodiated Gb3Cer (d18:1, C_24:0) with m/z 1,158.80. (C) Molecular structure and explanatory fragmentation scheme of Gb3Cer (d18:1, C_24:0). arb., arbitrary.

FIG 4

Positive-ion-mode ESI–Q-TOF mass spectra of antibody-detected Gb4Cer species from T24 cells. (A) Overview of the MS¹ spectrum obtained from the silica gel extract of the TLC overlay assay (shown in the inset) performed with an aliquot of GSL at an amount corresponding to the amount from 6 × 10⁵ cells. The area comprising the immunostained double band, from which the silica gel was scraped off, is indicated by a dotted rectangle. (B) Low-energy CID MS² spectrum of monosodiated Gb4Cer (d18:1, C_16:0) with m/z 1,249.75. Fragment ions generated by internal cleavages are marked by asterisks. (C) Molecular structure and explanatory fragmentation scheme of Gb4Cer (d18:1, C_16:0).

FIG 5

Real time-PCR-based quantification of transcript levels of different stx alleles in EHEC O157:H7 isolate EDL933 and urine isolates UR5703/202, UR5779/201, and UR131. Data represent the normalized fold expression levels of the different stx alleles upon growth in pooled human urine, LB, RPMI 1640, or SCEM after 2 h, 4 h, and 12 h of growth. Error bars show the standard errors of the means of three independent experiments and technical replicates. The individual stx transcript levels were normalized to the transcript level of the housekeeping gene gapA and compared with the level at the starting point (2 h). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Urovirulence of EHEC urine isolates. Bladder (A) and kidney (B) colonization levels were determined 72 h after transurethral inoculation of female C57BL/6 mice. The mice were independently challenged with UPEC strain 536 (positive control), EHEC urine isolate UR131, UR5703/202, or UR5779/201, or nonpathogenic E. coli K-12 strain MG1655 (negative control). Horizontal bars represent the mean number of CFU/g tissue for each population; the whiskers display the respective standard errors of the means. Significant differences in the bacterial organ load compared to that for the negative controls are indicated: n.s., no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.