A novel antimicrobial peptide significantly enhances acid-induced killing of Shiga toxin-producing Escherichia coli O157 and non-O157 serotypes

Abstract

Shiga toxin-producing Escherichia coli (STEC) colonizes the human intestine, causing haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Treatment options are limited to intravenous fluids in part because sublethal doses of some antibiotics have been shown to stimulate increased toxin release and enhance the risk of progression to HUS. Preventative antimicrobial agents, especially those that build on the natural antimicrobial action of the host defence, may provide a better option. In order to survive the acid stress of gastric passage, STEC is equipped with numerous acid resistance and DNA repair mechanisms. Inhibition of acid-induced DNA repair may offer a strategy to target survival of ingested STEC. We report here that brief pretreatment with a novel antimicrobial peptide, which was previously shown to inhibit bacterial DNA repair, significantly and profoundly reduces survival of acid-stressed O157 : H7 and non-O157 : H7 STEC seropathotypes that are highly associated with HUS. Reduction in survival rates of STEC range from 3 to 5 log. We also show that peptide/acid treatment results in little or no increase in toxin production, thereby reducing the risk of progression to HUS. This study identifies the peptide wrwycr as a potential new candidate for a preventative antimicrobial for STEC infection.

Fig. 1.

Survival after acid stress. Survival of strains 86-24 (a), CL106 (b), N99-4390 (c) and EC2-211 (d) after incubation in LB at 37 °C at either pH 7.0 (black bars) or pH 2.5 (grey bars). Each experiment includes two biological replicates and three technical replicates. Asterisks indicate a statistically significant difference relative to the corresponding control, confidence interval 95 %. Error bars indicate sd.

Fig. 2.

Peptide wrwycr/acid treatment. The impact of temperature, time and peptide concentration on survival of STEC strain 86-24 after 5 min incubation with PBS (black bars), 25 µM wrwycr (grey bars), 50 µM wrwycr (dark-grey bars, c only) or 75 µM wrwycr (white bars, c only) followed by timed acid stress exposure. Both wrwycr and acid stress incubations were conducted at room temperature (a), 30 °C (b) or 37 °C (c). Asterisks indicate a peptide treatment with a statistically significant difference relative to untreated control, confidence interval 95 %. U, Unstressed. Error bars indicate sd.

Fig. 3.

Comparison of survival of O157 and non-O157 strains after peptide/acid treatment. Survival after incubation with wrwycr for 5 min at room temperature followed by timed 37 °C acid stress exposure. Survival of control [incubation in PBS followed by pH 2.5 treatment (black bars)] or sample [incubation in 25 µM wrwycr in PBS followed by pH 2.5 treatment (grey bars)] of strain 86-24 (seropathotype A) (a), strain CL106 (seropathotype B) (b), strain N99-4390 (seropathotype C) (c), strain EC2-211 (seropathotype E) (d) or MG1655 (e). Each experiment includes two biological replicates and three technical replicates. Asterisks indicate a statistically significant difference relative to the corresponding control, confidence interval 95 %. U, Unstressed. Error bars indicate sd.

Fig. 4.

Effect of wrwycr/acid stress treatment on periplasmic STx production as measured by the Vero cell cytotoxicity assay for STEC strains 86-24 (a), CL106 (b) and N99-4390 (c). Following the indicated treatment, periplasmic extracts were prepared from each strain and diluted serially on Vero cells. After 72 h, adherent, viable cells were stained with crystal violet and absorbance was read at 570 nm. Results are expressed as the mean absorbance±sd versus the dilution of extract and are representative of three independent experiments. ▴, Control (no STx); ⧫, pH 2.5; ▪, pH 2.5 + 25 µM wrwycr. Standard curves for STx1 (▴) and STx2 (⧫) are shown in (d). Error bars indicate sd.