An in vitro combined antibiotic-antibody treatment eliminates toxicity from Shiga toxin-producing Escherichia coli

Abstract

Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the "big six" serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.

FIG 1

Stx induction by MITO. (A) Stx1 sandwich ELISAs were conducted with cell-free supernatants from all Stx1-containing STEC cultures. The Stx1 sandwich ELISAs used MAb Stx1-3 (1 μg/ml) as a capture antibody and Stx1-1-biotin (0.5 μg/ml) as a detection antibody. All MITO-treated samples underwent the same treatment conditions and preparations, except that they were grown in 100 ng/ml MITO-containing medium. (B) Stx2 sandwich ELISAs were conducted with cell-free supernatants from all Stx2-containing STEC cultures. The Stx2 sandwich ELISAs used Stx2-1 (1 μg/ml) as a capture antibody and Stx2-5-biotin (0.5 μg/ml) as a detection antibody. All MITO-treated samples underwent the same treatment conditions and preparations, except that they were grown in 100 ng/ml MITO-containing medium.

FIG 2

Stx repression by TGC. (A) Stx1 sandwich ELISAs were conducted with TGC-treated and untreated cell-free supernatants from all Stx1-containing STEC cultures, using MAb Stx1-3 (1 μg/ml) as a capture antibody and Stx1-1-biotin (0.5 μg/ml) as a detection antibody. All TGC-treated samples were grown in 100 ng/ml TGC-containing medium. (B) Stx2 sandwich ELISAs were conducted with TGC-treated (100 ng/ml) and untreated cell-free supernatants from all Stx2-containing STEC cultures, using Stx2-1 (1 μg/ml) as a capture antibody and Stx2-5-biotin (0.5 μg/ml) as a detection antibody.

FIG 3

Cytotoxicity of Stxs from culture medium from bacteria incubated with antibiotics at subinhibitory concentrations. (A) Cell-free supernatants from isolates expressing Stx2 only (RM1913), Stx1 and Stx2 (RM2367), Stx1 only (RM13151), or no toxin (ATCC 25922), grown with 100 ng/ml TGC, 10 ng/ml CIP, or no antibiotic, were added to cell culture wells containing 10⁵ Vero cells, and plates were incubated for 24 h. All samples were added at 25 nl cell-free supernatant/well. (B) Cell-free supernatants from the experiment shown in panel A were added to Vero cell medium containing a mixture of anti-Stx antibodies (MAbs Stx1-1 and Stx2-5). This cell supernatant-antibody mixture was then added to cell culture wells containing 10⁵ Vero cells, and plates were incubated for 24 h. All samples were added at 25 nl cell-free supernatant/well. Antibodies were provided at 1 μg/ml each.

FIG 4

Effects of antibiotics at inhibitory concentrations on the viability of Vero cells cocultured with STEC. (A) STEC isolates expressing Stx2 only (RM1913), Stx1 and Stx2 (RM2367), Stx1 only (RM13151), or no toxin (ATCC 25922) were inoculated into Vero cell medium containing inhibitory concentrations of the indicated antibiotics (1 μg/ml each for CAZ and GEN, 2 μg/ml for TGC, and 0.2 μg/ml for CIP). (B) STEC isolates were combined with antibiotic, a mixture of neutralizing anti-Stx MAbs (Stx1-1 and Stx2-5 at 10 μg/ml each), and Vero cell medium and then were added immediately to Vero cells already present in cell culture wells. All Vero cell culture wells received 5 nl of stationary-phase bacteria (A₆₀₀ of 3) per well.

FIG 5

Effects of antibiotics and antibodies on the viability of Vero cells exposed to O104:H4 strains. (A) Cell-free medium from O104:H4 isolates (from the German outbreak in 2011) grown with 100 ng/ml TGC, 10 ng/ml CIP, or no antibiotic was added to cell culture wells containing 10⁵ Vero cells, and plates were incubated for 24 h. A subset of cell-free medium samples were also incubated with a MAb mixture (1 μg/ml each of Stx1-1 and Stx2-5). All samples were added at 25 nl cell-free supernatant/well. (B) O104:H4 isolates were inoculated into Vero cell medium containing inhibitory concentrations of the indicated antibiotics (1 μg/ml each for CAZ and GEN, 2 μg/ml for TGC, and 0.2 μg/ml for CIP). A subset of samples also received a MAb mixture (10 μg/ml each of Stx1-1 and Stx2-5). All Vero cell culture wells received 5 nl of stationary-phase bacteria per well.