Detection of Shiga Toxin-Producing Escherichia coli (STEC) in the Endocervix of Asymptomatic Pregnant Women. Can STEC Be a Risk Factor for Adverse Pregnancy Outcomes?

Abstract

The presence of Escherichia coli in the vaginal microbiome has been associated with pregnancy complications. In previous works, we demonstrated that Shiga toxin-producing Escherichia coli (STEC) can produce abortion and premature delivery in rats and that Shiga toxin type 2 (Stx2) can impair human trophoblast cell lines. The hypothesis of this work was that STEC may colonize the lower female reproductive tract and be responsible for adverse pregnancy outcomes. Thus, the aim of this work was to evaluate the presence and prevalence of virulence factor genes from STEC in the endocervix of asymptomatic pregnant women. For that purpose, endocervical swabs were collected from pregnant women during their prenatal examination. Swab samples were enriched in a differential medium to select Enterobacteria. Then, positive samples were analyzed by PCR to detect genes characteristic of Escherichia sp. (such as uidA and yaiO), genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfb_O157), and STEC virulence factor genes (such as stx1, stx2, eae, lpfA_O113, hcpA, iha, sab, subAB). The cytotoxic effects of stx2-positive supernatants from E. coli recovered from the endocervix were evaluated in Vero cells. Our results showed that 11.7% of the endocervical samples were positive for E. coli. Additionally, we found samples positive for stx2 and other virulence factors for STEC. The bacterial supernatant from an isolate identified as E. coli O113:NT, carrying the stx2 gene, exhibited cytotoxic activity in Vero, Swan 71 and Hela cells. Our results open a new perspective regarding the presence of STEC during pregnancy.

Keywords: Endocervical microbiota; STEC; Stx2; pregnancy; virulence factors.

Figure 1

Cytotoxicity of E. coli-positive endocervical samples carrying the stx2 gene on the viability of Vero cells. Vero cells were exposed to serial dilutions of bacterial supernatants (SN) from endocervical samples carrying the stx2 gene or different concentrations of purified Stx2 under growth-arrested conditions. Cell viability was determined by neutral red uptake after 72 h of incubation, and 100% represents cells incubated under identical conditions but without treatment. Data are shown as means ± S.D from at least three independent experiments performed in triplicate. **p < 0.01.

Figure 2

Neutralization of the Stx2 cytotoxicity of bacterial supernatants isolated from endocervical samples on the viability of Vero cells. The bacterial supernatant from the endocervical sample 14 (S14-SN, dilution 1:5000) was preincubated with the mouse monoclonal antibody 2E11 against the A-subunit of Stx2 (anti-Stx2 mAb, 25 µg/mL) at 37°C for 1 h. The mixture was then added to a 96-well culture plate containing Vero cells and incubated for 72 h. The CD₅₀ of purified Stx2 (1 ng/mL) incubated with anti-Stx2 mAb was used as control. Cell viability was analyzed by neutral red uptake. Data are shown as mean ± S.D from at least three independent experiments performed in triplicate. **p < 0.01, n=3.

Figure 3

Cytotoxic effects of STEC-positive endocervical samples on the viability of Swan 71 and HeLa cell lines. Cells were exposed to purified Stx2 or serial dilutions of the supernatant from endocervical sample S14 (S14-SN) for 72 h. Cell viability was analyzed by neutral red uptake. Data are shown as mean ± S.D from at least three independent experiments performed in triplicate. *p < 0.05, n=3.

Figure 4

Characterization of a STEC strain isolated from an endocervical sample by PCR assays. The STEC strain named 123/21 was subtyped for the stx2 gene. The amplicons were seeded on 1.5% agarose gel for 30 min at 80 Volts. A: STEC strain 123/21. C1: E. coli O157:H7 strain 125/99 (stx2a+ 349 bp; stx2 627 bp), C2: negative control of PCR; C3: E. coli O113:H21 (subAB; 556 bp); DNA size marker (MK).

Figure 5

Cytotoxic effects of the STEC isolate O113:NT obtained from an endocervical sample on the viability of Vero cells. Cells were exposed to purified Stx2 or serial dilutions of supernatants from STEC O113:NT or sample S14 for 72 h. Cell viability was analyzed by neutral red uptake. Data are shown as mean ± S.D from at least three independent experiments performed in triplicate. *p < 0.05.