Effects of azithromycin on shiga toxin production by Escherichia coli and subsequent host inflammatory response

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the human intestinal mucosa, produces Stx from phage, and causes the development of hemolytic-uremic syndrome via Stx-induced inflammatory cytokine production. Azithromycin exhibited strong in vitro activity against STEC without inducing Stx-converting phage, in marked contrast to norfloxacin. Azithromycin decreased the tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 production from Stx-treated human peripheral mononuclear cells or monocytes to a greater extent than did clarithromycin. In Stx-injected mice, azithromycin significantly suppressed Stx-induced TNF-alpha, IL-1beta, and IL-6 levels in serum and improved the outcome as assessed by survival rate. In the STEC oral infection experiment using immature mice immediately after weaning (weaned immature-mouse model), all mice died within 7 days postinfection. Azithromycin administration gave the mice 100% protection from killing, while ciprofloxacin administration gave them 67% protection. The data suggest that azithromycin (at least at higher concentrations) has a strong effect on Stx production by STEC and on the Stx-induced inflammatory host response and prevents death in mice. Azithromycin may have a beneficial effect on STEC-associated disease.

FIG. 1.

Stx2 phage induction (A) and Stx2 production (B) in Stx2-producing STEC incubated in the presence or absence of antimicrobial agents. STEC strain 1076 (producing Stx2) was treated with the indicated concentrations of antimicrobial agents for 30 min and then subjected to a 2-h incubation in drug-free medium. (A) Number of Stx2 phage induced. The antimicrobial agents used were norfloxacin (□) and azithromycin (▵). (B) Stx2 levels in the bacterial culture after treatment with antimicrobial agents at 0, 0.1, 1, or 10 μg/ml. The antimicrobial agents used were norfloxacin (open bars) and azithromycin (shaded bars).

FIG. 2.

Inhibition of cytokine production from Stx1-stimulated human peripheral blood mononuclear cells by azithromycin and clarithromycin. Human mononuclear cells were stimulated with Stx in the presence or absence of azithromycin (AZM) or clarithromycin (CAM). ∗, P < 0.05; ∗∗, P < 0.01, compared with Stx-stimulated mononuclear cells in the absence of antimicrobial agents. Similar inhibitory effects were observed even when human monocytes were used, although the cytokine levels were slightly lower compared with the data shown in this figure.

FIG. 3.

Dose-response effects of azithromycin on TNF-α (A), IL-1β (B), and IL-6 (C) levels in serum after Stx1 stimulation in mice. Groups of 10 mice were injected intraperitoneally with 55 ng of Stx1. Treatment (intraperitoneal injection) with various doses of azithromycin (AZM) or saline was carried out 5 min after the Stx1 injection. (A) TNF-α production 1.5 h after Stx1 stimulation in mice; (B) IL-1β production 3 h after Stx1 stimulation in mice; (C) IL-6 production 3 h after Stx1 stimulation in mice. Data are means ± standard deviations (error bars). ∗, P < 0.05; ∗∗, P < 0.01, compared with those of Stx-injected, azithromycin-untreated mice.

FIG. 4.

Lethality of Stx1 in mice with or without azithromycin treatment. Groups of 10 mice were injected intraperitoneally with 55 ng of Stx1. Treatment (intraperitoneal injection) with various doses of azithromycin (or saline) was carried out 5 min after the Stx1 injection. Mice injected only with saline (with no Stx injection) were used as controls (□). Doses of azithromycin were 0 mg/kg (⧫), 0.38 mg/kg (▪), 0.75 mg/kg (◊), 1.5 mg/kg (•) and 3 mg/kg (○). ∗, P < 0.05, compared with the Stx-injected, untreated controls (⧫).

FIG. 5.

Effect of antimicrobial agents on lethality after oral infection of mice with STEC. Three groups of immature mice (12 mice each) were infected orally with 10⁹ CFU of STEC strain 1076 (producing Stx2) via a tube on day 0 in this figure. In an additional group of 12 immature mice, the same volume of phosphate-buffered saline was given orally instead of STEC (□). At 1, 2, and 3 days after STEC inoculation, the two infected groups of mice were treated with azithromycin (10 mg/kg/day) (○) or ciprofloxacin (10 mg/kg/day) (•). The remaining infected group of mice (⧫) was not treated with antimicrobial agents. Survival of mice was monitored every 12 h after STEC inoculation. ∗, P < 0.05, compared with azithromycin-treated, STEC-infected mice.