Potentiation of Antibiotics by a Novel Antimicrobial Peptide against Shiga Toxin Producing E. coli O157:H7

Abstract

Infection with Shiga toxin-producing Escherichia coli (STEC) results in hemorrhagic colitis and can lead to life-threatening sequelae including hemolytic uremic syndrome (HUS). Conventional treatment is intravenous fluid volume expansion. Antibiotic treatment is contraindicated, due in part to the elevated risk of HUS related to increased Shiga toxin (Stx) release associated with some antibiotics. Given the lack of effective strategies and the increasing number of STEC outbreaks, new treatment approaches are critically needed. In this study, we used an antimicrobial peptide wrwycr, previously shown to enhance STEC killing without increasing Stx production, in combination with antibiotic treatments. Checkerboard and time-kill assays were used to assess peptide wrwycr-antibiotic combinations for synergistic STEC killing. Cytotoxicity and real-time PCR were used to evaluate Stx production and stx expression, respectively, associated with these combinations. The synergistic combinations that showed rapid killing, no growth recovery and minimal Stx production were peptide wrwycr-kanamycin/gentamicin. Transmission electron microscopy revealed striking differences in bacterial cell morphology associated with various treatments. This study provides proof of principle for the design of an antibiotic-peptide wrwycr combination effective in killing STEC without enhancing release of Shiga toxins. It also offers a strategy for the repurposing of antibiotics for treatment of STEC infection.

Figure 1

STEC survival over the course of a 24 hour killing assay as determined by serial plate count assay and expressed as viable counts per mL of culture. Each graph provides survival curves for STEC treatment with the subMIC concentrations of each of antibiotic or peptide or antibiotic-peptide combination relative to growth of untreated STEC (bacterial control). In all cases, the peptide dose was 32 µg/mL and the FICI value for peptide-antibiotic combinations was 0.38. Time kill curves for peptide alone or in combination with (A) Gentamicin (0.5 µg/mL) (B) Kanamycin (2.0 µg/mL) (C) Chloramphenicol (1.0 µg/mL) (D) Ciprofloxacin (0.008 µg/mL) (E) Meropenem (0.008 µg/mL). All assays were carried out in triplicate and on at least two different occasions.

Figure 2

Vero cell cytotoxicity levels with secreted (SE) and seriplasmic (PE) lysates from STEC 86–24 following treatment with peptide or antibiotic after (A) 6 hr and (B) 15 hr. Antimicrobial doses included both MIC and subMIC doses: peptide (128 µg/mL and 64 µg/mL), gentamicin (4 µg/mL and 2 µg/mL), kanamycin (16 µg/mL and 8 µg/mL), chloramphenicol (8 µg/mL and 4 µg/mL), ciprofloxacin (0.063 µg/mL and 0.031 µg/mL) and meropenem (0.063 µg/mL and 0.031 µg/mL). All assays were carried out in triplicate and on at least two different occasions. Data bars represent means ± standard deviation. *Significantly different from subMIC treatment; P < 0.05; Unpaired student’s test with Welch’s correction.

Figure 3

Vero cell cytotoxicity levels with secreted and periplasmic lysates from STEC 86–24 following treatment with peptide or antibiotic or peptide-antibiotic combinations after 6 hr and 15 hr. (A) with subMIC peptide-gentamicin combinations with specified FICI values (B) with subMIC peptide-kanamycin combinations with specified FICI values. All assays were carried out in triplicate and on at least two occasions. Data bars represent means ± standard deviation. *Significantly different from both individual treatments (peptide or antibiotic); ^†Significant different from antibiotic treatment. ^‡Significantly different from peptide treatment; P < 0.05; One-way ANOVA with post hoc Tukey comparisons.

Figure 4

TEM micrographs of resin-embedded thin sections of STEC 86–24 following treatments with (A) no treatment (control) (B) peptide (128 μg/mL) (C) kanamycin (16 μg/mL) (D) gentamicin (4 μg/mL) (E) kanamycin (4 μg/mL) and peptide (8 μg/mL) (F) kanamycin (4 μg/mL) and peptide (4 μg/mL) (G) gentamicin (0.25 μg/mL) and peptide (32 μg/mL) (H) gentamicin (1 μg/mL) and peptide (16 μg/mL). Scale = 1 μm. These images are representative of images collected on two different occasions.