AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohara, T.
Right arrow Articles by Yamamoto, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohara, T.
Right arrow Articles by Yamamoto, T.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, November 2002, p. 3478-3483, Vol. 46, No. 11
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.11.3478-3483.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Effects of Azithromycin on Shiga Toxin Production by Escherichia coli and Subsequent Host Inflammatory Response

Tatsuki Ohara,1,2 Seiichi Kojio,1 Ikue Taneike,1 Saori Nakagawa,1 Fumio Gondaira,1 Yukiko Tamura,1 Fumitake Gejyo,2 Hui-Min Zhang,1 and Tatsuo Yamamoto1*

Division of Bacteriology, Department of Infectious Disease Control and International Medicine,1 Division of Respiratory Medicine, Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan2

Received 27 March 2002/ Returned for modification 16 May 2002/ Accepted 31 July 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-1ß (IL-1ß), 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-1ß, 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Shiga toxin (Stx)-producing Escherichia coli (STEC), alternatively called enterohemorrhagic E. coli, was identified as an emerging bacterial pathogen of food-borne infections in the United States in 1982 (27). In Japan during 1996, explosive outbreaks of STEC infection occurred, with 17,877 people infected and 12 fatalities (5).

STEC colonizes the intestinal mucosa and elaborates Stx (14, 21). Stx is divided into two subtypes, Stx1 and Stx2 (19), both of which are encoded by prophages lysogenized in STEC strains (17, 23, 24, 35). Stx prophages are induced by agents that damage DNA or inhibit DNA replication. At this stage, accumulated small DNA fragments (such as the Okazaki fragment) bind to and activate the RecA protein (with protease activity), resulting in cleavage of the phage repressor as well as LexA protein (repressor for the recA gene) and prophage induction (15, 18, 26). Stx prophage induction is associated with increased Stx gene copy numbers and increased Stx production (18, 20).

The Stx production is followed by the development of abdominal symptoms and by serious systemic disorders such as hemolytic-uremic syndrome (HUS), especially in young children (1, 4, 25).

Stx is a major factor responsible for damaging human endothelial cells and leads to HUS via overproduction of inflammatory cytokines (11, 33, 36). Cytokines such as tumor necrosis factor alpha (TNF-{alpha}) and interleukin-1ß (IL-1ß) regulate the expression on the endothelial cell membrane of a glycolipid globotriaosylceramide (Gb3), which serves as the Stx receptor (8, 30, 32, 33).

Treatment regimens that down-regulate such inflammatory cytokines are one option for management of STEC infection. Macrolides including erythromycin, clarithromycin, and azithromycin exhibit immunomodulatory activities, such as inhibition of neutrophil chemotaxis and oxidative burst and the release of proinflammatory cytokines from monocytes (7, 13, 28, 29). However, whether macrolides can modulate Stx-induced inflammatory-cytokine production has not been investigated.

Newer fluoroquinolones such as norfloxacin and ciprofloxacin exhibit strong in vitro activity against STEC. However, they do induce Stx-converting phage (18, 39), resulting in higher levels of Stx production. In this study, we investigated whether azithromycin induces Stx-converting phage and whether azithromycin down-regulates inflammatory cytokine production and prevents death in mice exposed to Stx or challenged with STEC.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. The STEC strains used in this study were 208 clinical isolates and included strains derived from outbreaks in Japan in 1996 and from sporadic cases occurring from 1996 to 2001. There were 183 strains of serotype O157:H7, 2 strains of serotype O157:H-, 9 strains of serotype O26:H11, 6 strains of serotype O111:H-, 5 strains of serotype O145:H-, 1 strain each of serotypes O128:H2 and O128:H12, and 1 strain of serotype O86:H- (34). All strains were stored at -80°C in 3% skim milk (Difco Laboratories, Detroit, Mich.) supplemented with 5% glucose (Difco).

Media and bacterial growth. For bacterial growth, nutrient agar (Eiken Chemical, Tokyo), Mueller-Hinton agar (Difco), and Luria-Bertani (LB) agar (Difco) were used as solid media. LB broth (Difco) was used as the liquid medium, which was inoculated and incubated at 37°C for 12 to 18 h with agitation.

Antimicrobial agents. The antimicrobial agents (pure substances not including a preservative) were gifts from the manufacturer. They included azithromycin (Pfizer Pharmaceuticals Inc., Tokyo, Japan), clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), erythromycin (Shionogi & Co., Osaka, Japan), rokitamycin (Asahi Kasei Corp., Tokyo, Japan), roxithromycin (Aventis Pharma, Tokyo, Japan), oleandomycin (Pfizer Pharmaceuticals Inc., Tokyo, Japan), kitasamysin (Asahi Kasei Corp., Tokyo, Japan), midecamycin (Meiji Seika, Tokyo, Japan), and josamycin (Yamanouchi Pharmaceutical Co., Tokyo, Japan) for macrolides, and norfloxacin (Daiichi Pharmaceutical Co., Tokyo, Japan) and ciprofloxacin (Bayer Yakuhin, Osaka, Japan) for newer fluoroquinolones.

Susceptibility testing. Susceptibility testing of bacterial strains was performed by the agar dilution method with Mueller-Hinton agar by using standard procedures (10).

Preparation of Stx. Stx1 produced from E. coli strain 87-27 (carrying the cloned Stx1 gene) and Stx2 produced from E. coli strain Tp8 (carrying the cloned Stx2 gene) were purified to homogeneity as described previously (22, 37). Purified Stx1 and Stx2 (0.2 mg of protein per ml) were stored at 4°C in phosphate-buffered saline. The Stx1 and Stx2 preparations contained no detectable endotoxin contamination (less than 0.05 endotoxin unit per ml) as determined by a Limulus amebocyte lysate assay.

Stx-converting phage induction. In this experiment, an Stx2-producing STEC strain (serotype O86:H-) isolated from a patient with HUS (strain 1076 [34]) was grown to log phase (ca. 5.0 x 108 CFU/ml) at 37°C in LB broth containing 10 mM CaCl2 and incubated for 30 min in the presence or absence of various concentrations of antimicrobial agents. The bacterial cells were washed and resuspended in the same volume of fresh LB broth and incubated for 2 h. The resultant bacterial culture was divided into two parts.

One part was centrifuged, the supernatant was filtered through a membrane with a pore size of 0.45 µm, and the filtrate was subjected to phage titer determination. For this, 10-fold serial dilutions of the supernatant were made, phages in each dilution were adsorbed onto indicator E. coli cells (strain C600), and the cells were incubated overnight at 37°C in LB soft agar containing 10 mM CaCl2. The numbers of phage plaques that developed in the LB soft agar plates (see Fig. 1) were determined. The plaques (at least 20 plaques in each plate) were examined for the Stx2 gene by the PCR assay (21); the data suggested that the plaques were all from the Stx2 phage.



View larger version (20K):
[in this window]
[in a new window]
  		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 ({square}) and azithromycin ({triangleup}). (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).

 
The remaining part of the bacterial culture was subjected to sonification and centrifugation, and the amount of Stx in the supernatant (which was filtered through a membrane with a pore size of 0.45 µm) was determined by the passive latex agglutination test using a VT detection kit (Denka Seiken Co., Tokyo, Japan). The Stx titers (the levels of Stx production) represented the highest dilution (after twofold serial dilutions) to yield positive results.

Preparation of human peripheral blood mononuclear cells and monocytes. Human mononuclear cells were prepared from peripheral blood of healthy volunteers by using Ficoll-Hypaque (Gibco BRL, Grand Island, N.Y.) gradient centrifugation. Adherent monocytes were obtained after incubation in 24-well culture plates (A/S Nunc, Roskilde, Denmark) for 60 min at 37°C in a CO2 incubator with subsequent washing under high pressure (2).

Cytokine induction by Stx-stimulated human peripheral blood mononuclear cells and monocytes. The human mononuclear cells (1 x 106 to 1.5 x 106/ml) or monocytes (1 x 105/ml), prepared as above, were stimulated with Stx (5 ng/ml) in the presence or absence of antimicrobial agents for 20 h at 37°C in a CO2 incubator. After incubation, TNF-{alpha}, IL-1ß, and IL-6 in the supernatants were assayed with TNF-{alpha}, IL-1ß, and IL-6 kits (Genzyme, Cambridge, Mass.) as specified by the manufacturer.

Stx challenge experiment in mice. Specific-pathogen-free male C57BL/6 mice aged 5 to 6 weeks old and weighing 15 to 20 g were used. They were purchased from Charles River Japan Inc. (Yokohama, Japan) and fed a standard diet and water. The animals were randomized into six groups (10 mice each), five Stx-injected groups and one saline-injected group. The five Stx-injected groups of mice were injected intraperitoneally with 2.8 µg of Stx1 per kg (approximately 55 ng/mouse). This Stx1 dose was chosen because it was shown previously to result in a mortality of >50% between 3 and 4 days after injection. Of the five Stx-injected groups of mice, four were treated with various doses of azithromycin 5 mins after the Stx1 injection. Azithromycin was initially dissolved in ethanol at 12 mg/ml and then diluted 100-fold or more with sterile saline to make a solution of 120 µg/ml (or less). Volumes of approximately 0.5 ml of the azithromycin solutions were administered to the mice to achieve doses of 0.38, 0.75, 1.5, and 3 mg/kg. The Stx-injected, azithromycin-untreated mice received approximately 0.5 ml of sterile saline. Administration of 0.5 ml of sterile saline containing 1% ethenol had no effects on mouse mortality. Survival of the mice was monitored every 24 h after Stx injection. Cumulative mortality was recorded over 7 days after Stx1 injection.

In parallel experiments (as above), whole-blood samples were obtained from the blood vessels of the eyeground by inhalation at the indicated time points (0, 1.5, 3, 6, and 24 h) after Stx injection. Serum samples were preserved at -30°C until used for measurement of cytokinese levels. Concentrations of TNF-{alpha}, IL-1ß, and IL-6 in serum were assessed using enzyme-linked immunosorbent assay kits (Genzyme-THCHNE, Minneapolis, Minn.) with a sensitivity of 5.1, 3.0, and 3.1 pg/ml, respectively.

STEC challenge experiment in mice. In mouse infection experiments (designated the weaned immature-mouse model), immature mice (body weight, approximately 9.4 g) immediately after weaning were used. They were specific-pathogen-free male and female C57BL/6 mice and were purchased from CLEA Japan Inc., Tokyo, Japan. The immature mice were divided into four experimental groups (12 animals each): non-STEC infection group (control), STEC infection group, STEC infection and subsequent ciprofloxacin treatment group, and STEC infection and subsequent azithromycin treatment group. STEC (serotype O86 strain 1076) was grown on a nutrient agar plate at 37°C, and the STEC colonies were suspended in PBS at 1010 CFU/ml and used for inoculation. In the infection groups, 0.1 ml of the bacterial suspension (109 CFU) was orally administered to immature mice via a tube. Ciprofloxacin was dissolved in water and adjusted to 1 mg/ml, and 0.1 ml of the solution was administered; the dose was 10 mg/kg. Azithromycin was prepared with ethanol and adjusted to 100 mg/ml. This ethanol solution was 100-fold diluted with water to 1 mg/ml, and approximately 0.1 ml of this solution was administered; the dose was 10 mg/kg. To antimicrobial agent treatment groups, the agents were orally administered via a tube 24, 48, and 72 h after STEC inoculation. Survival of the immature mice was monitored every 12 h after STEC inoculation.

Statistical analysis. Data concerning cytokine levels were evaluated by Mann-Whitney's U test. Data concerning survival rate in mice were evaluated by Fisher's exact probability test. The level of significance was defined as a P value of <0.05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro activity against STEC. The MICs of the antimicrobial agents against the STEC strains are summarized in Table 1. Among the macrolides tested, azithromycin showed the greatest activity (MICs, <=8 µg/ml). The MICs of the newer fluoroquinolones, norfloxacin and ciprofloxacin, were low (<=0.5 µg/ml).


View this table:
[in this window]
[in a new window]
  		TABLE 1. MICs of macrolides, norfloxacin, and ciprofloxacin for 208 clinical isolates of Stx-producing E. coli strains belonging to serogroups O157, O26, O86, O111, O128, and O145

 
Effect on Stx phage induction. Next, the effect of azithromycin on Stx-converting phage induction was examined. Norfloxacin, whose MIC for strain 1076 was 0.06 µg/ml, markedly induced Stx phage when used at 0.1 µg/ml or more (Fig. 1A). The number of PFU was 103- to 104-fold greater than for spontaneous phage induction. In accordance with phage induction, marked Stx production was also observed at a norfloxacin concentration of 0.1 µg/ml (Fig. 1B); the Stx titer was increased 512-fold compared with that observed in the absence of antimicrobial agents. In marked contrast, azithromycin did not induce Stx phage (Fig. 1A) and did not stimulate Stx production (Fig. 1B). Similar results were obtained with Stx1 phage induction and Stx1 production in experiments using an Stx1-producing STEC strain (data not shown).

Inhibitory effect on cytokine induction by Stx-stimulated human peripheral blood mononuclear cells and monocytes. Stx stimulates the production of TNF-{alpha} and IL-1ß from human peripheral blood monocytes. The effects of azithromycin on modulating such Stx-induced inflammatory cytokine production were then examined.

Data for Stx1 stimulation of human mononuclear cells are shown in Fig. 2. Azithromycin inhibited the Stx1-stimulated cytokine production in a dose-dependent manner. The percent inhibition at an azithromycin concentration of 10 µg/ml was 61.7% for TNF-{alpha}, 60.4% for IL-1ß, and 47.7% for IL-6. With clarithromycin, much less or no significant inhibition was observed at 10 µg/ml (P < 0.01); the inhibition was 4.7% for TNF-{alpha}, 14.8% for IL-1ß, and 1.3% for IL-6.



View larger version (21K):
[in this window]
[in a new window]
  		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.

 
Similar inhibitory effects of azithromycin were obtained even when human monocytes were used or when Stx2 stimulation was used instead of Stx1 stimulation; inhibition of TNF-{alpha} induction by azithromycin (10 µg/ml) in human monocytes was 64.5% for Stx1 and 66.6% for Stx2, and inhibition of Stx2 stimulation of IL-1ß and IL-6 was 91.5 and 80.6%, respectively.

Inhibitory effect on serum cytokine levels. The effect of azithromycin on serum cytokine levels after Stx1 stimulation was examined in mice. First, we determined the induction of cytokines in mouse sera by Stx1. The TNF-{alpha} level generally peaked around 1.5 h and IL-1ß and IL-6 usually appeared around 3 h after Stx1 injection (data not shown). Therefore, we evaluated the effects of azithromycin on Stx1-induced TNF-{alpha}, IL-1ß, and IL-6 production at 1.5, 3, and 3 h after injection, respectively.

The effects of azithromycin on serum cytokine levels after Stx1 stimulation in mice are shown in Fig. 3. Treatment with various doses of azithromycin resulted in a marked decrease in Stx1-induced TNF-{alpha} production (Fig. 3A). Compared with the TNF-{alpha} levels in saline-treated mice, azithromycin at doses as low as 0.38 mg/kg significantly inhibited Stx1-induced TNF-{alpha} production (P < 0.05).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. Dose-response effects of azithromycin on TNF-{alpha} (A), IL-1ß (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-{alpha} production 1.5 h after Stx1 stimulation in mice; (B) IL-1ß 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.

 
The degree of enhancement of IL-1ß production in mice injected with Stx1 was low (Fig. 3B). We determined the concentration of IL-1ß in serum 3 h after treatment with various doses of azithromycin. Azithromycin at 0.38 mg/kg significantly reduced the peak concentration of IL-1ß in serum compared with that in saline-treated mice (P < 0.05).

Stx1 showed a greater enhancement of IL-6 than of TNF-{alpha} and IL-1ß production in mouse sera (Fig. 3C). Increased concentrations of IL-6 were observed 1.5 h after injection of Stx1 and reached a peak after 3 h. We found that azithromycin at 3 mg/kg also significantly inhibited Stx1-induced IL-6 production (P < 0.05). However, at doses of less than 3 mg/kg, this agent had no significant inhibitory effect (Fig. 3C). In these experiments, the administration of azithromycin alone did not produce increases in the concentration of TNF-{alpha}, IL-1ß, or IL-6 (data not shown).

Protective effect against Stx challenge. The protective effect of azithromycin against Stx1 challenge was examined in mice. We compared the lethality of Stx1 by using C57BL/6 mice with or without azithromycin treatment (Fig. 4). Stx1 injection (2.8 µg/kg) resulted in 100% mortality in saline-treated mice (0% survival). In contrast, treatment with azithromycin at 3 mg/kg resulted in a significantly higher rate of survival (40%) than that in the Stx-injected, saline-treated controls (P < 0.05). However, lower doses of azithromycin (0.38, 0.75, and 1.5 mg/kg) resulted in a survival of 20%, which was not significantly different from that of the Stx-injected, saline-treated controls (P > 0.05).



View larger version (25K):
[in this window]
[in a new window]
  		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 ({square}). Doses of azithromycin were 0 mg/kg ({blacklozenge}), 0.38 mg/kg ({blacksquare}), 0.75 mg/kg ({lozenge}), 1.5 mg/kg (•) and 3 mg/kg ({circ}). *, P < 0.05, compared with the Stx-injected, untreated controls ({blacklozenge}).

 
Protective effect against STEC challenge. The STEC oral-infection experiment was performed using immature mice immediately after weaning. Survival of the immature mice was monitored every 12 h after STEC inoculation (Fig. 5). In the STEC infection group, the immature mice began to die 48 h after inoculation and all mice died within 5 days after inoculation. In contrast, in the ciprofloxacin treatment group, death was observed 60 h after inoculation but the mortality rate was only 33% (survival rate, 67%) 1 week after inoculation; this was the case even 2 weeks after inoculation. In the azithromycin treatment group, no deaths were observed and all animals were alive even 2 weeks after inoculation.



View larger version (21K):
[in this window]
[in a new window]
  		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 ({square}). At 1, 2, and 3 days after STEC inoculation, the two infected groups of mice were treated with azithromycin (10 mg/kg/day) ({circ}) or ciprofloxacin (10 mg/kg/day) (•). The remaining infected group of mice ({blacklozenge}) 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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inflammatory cytokines up-regulate the expression of the receptor for Stx on endothelial cells and allow the endothelial cells to be more sensitive to the toxic effect of Stx (32, 33). Elevated concentrations of TNF-{alpha} and IL-6 in plasma have been reported in HUS patients, and the degree of HUS is closely related to the levels of inflammatory cytokines (12, 16). Therefore, inflammatory cytokines are thought to be important in modifying the disease caused by STEC infection.

The roles of antimicrobial agents in the prevention and amelioration of HUS remain controversial, and an optimal treatment regimen for STEC infection has not been established (9, 31). In this study, we demonstrated that azithromycin has a strong effect on Stx production by STEC. Azithromycin inhibited the in vitro growth of STEC strains and did not induce Stx-converting phage or stimulate the production of Stx at a wide range of concentrations in vitro. This was in sharp contrast to newer fluoroquinolones such as norfloxacin and ciprofloxacin (18, 39), which markedly induced Stx-converting phage and stimulated the production of Stx.

In addition to the merits of azithromycin reported above, the results of the present study suggested that azithromycin is capable of inhibiting the Stx1- or Stx2-induced production of inflammatory cytokines (TNF-{alpha}, IL-1ß, and IL-6), which are considered to be essential for the development of HUS. Preliminary data suggested that azithromycin also inhibited Stx1- or Stx2-induced production of IL-8 from human monocytes by 67 to 78%. Azithromycin treatment at 3 mg/kg resulted in the greatest reduction in cytokine levels and improved outcome as assessed by monitoring the survival rate. Although further studies are necessary to investigate the mechanism of this effect, we speculated that improvement of the survival of mice may in part be due to the anti-inflammatory effect of azithromycin.

In healthy volunteers, azithromycin achieved maximum concentrations in serum of 0.09 µg/ml following a single oral dose of 125 mg (approximately 2.2 mg/kg), 0.24 µg/ml following a single oral dose of 250 mg (approximately 3.6 mg/kg), 0.58 µg/ml following a single oral dose of 500 mg (approximately 8.3 mg/kg), and 0.74 µg/ml following a single oral dose of 1,000 mg (approximately 15.4 mg/kg) (3). However, concentrations of azithromycin in tissue are much higher than are concentrations in serum. For instance, after two 250-mg (approximately 3.5-mg/kg) oral doses given 12 h apart, peak azithromycin concentrations exceeded 3 mg/kg in the prostate, tonsils, and many other tissues (6). Therefore, the dose of 3 mg/kg used in this study for mice seems to be relevant to human dosing levels.

We previously reported that a Chinese medicine, anisodamine (a vasoactive alkaloid drug extracted from a Chinese herb), inhibits TNF-{alpha}, IL-1ß, and IL-8 production by Stx1-stimulated human peripheral blood monocytes and increases the survival of Stx1-treated mice (38). The protective effect of azithromycin, observed in this study, was at a level similar to or slightly lower than that of anisodamine. Anisodamine, however, had no effects on the in vitro growth of STEC strains (MICs, >=256 µg/ml).

In this study, we also performed the infection experiment using immature mice immediately after weaning (designated the weaned immature-mouse model). Immature mice immediately after weaning were susceptible to STEC. The animals began to die 2 days after oral inoculation, and all animals died within 5 days after inoculation. Investigation of the pathology of STEC infection and the cause of death in weaned immature mice is in progress. When ciprofloxacin was administered in this experimental system, the mortality after 1 week (or even after 2 weeks) was 33% and the survival rate was 67%. The infection-preventing effect of azithromycin was higher than that of ciprofloxacin (P < 0.05), and the survival rate was 100% even 2 weeks after inoculation. Ciprofloxacin administration induced Stx phages, which may have been the cause of the lower infection-preventing effect of ciprofloxacin than that of azithromycin.

In conclusion, the present study demonstrated that azithromycin (at least at higher concentrations) has a strong effect on Stx production by STEC and the Stx-induced inflammatory host response and prevents death in mice exposed to Stx or STEC. Azithromycin may have a beneficial effect on STEC-associated disease. Further studies to investigate the potential usefulness of azithromycin are necessary.


   ACKNOWLEDGMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and a grant (97-1) from the Organization for Pharmaceutical Safety and Research (OPSR), Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 757 Ichibanchou, Asahimachidori, Niigata, Japan. Phone: 81-25-227-2050. Fax: 81-25-227-0762. E-mail: tatsuoy{at}med.niigata-u.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Gastroenterological Association. 1995. Consensus conference statement: Escherichia coli O157:H7 infections—an emerging national health crisis, July 11-13, 1994. Gastroenterology 108:1923-1934.[CrossRef][Medline]
  2. Bhakdi, S., M. Muhly, S. Korom, and G. Schmidt. 1990. Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release. J. Clin. Investig. 85:1746-1753.
  3. Cyong, J., K. Kodama, A. Yafune, M. Takebe, and H. Takayanagi. 1995. Phase I study of azithromycin—single dose and multiple dose for 3 days. Jpn. J. Chemother. 43(Suppl. 6):139-163.
  4. Fong, J. S., J. P. de Chadarevian, and B. S. Kaplan. 1982. Hemolytic-uremic syndrome. Current concepts and management. Pediatr. Clin. North Am. 29:835-856.[Medline]
  5. Food Sanitation Division Environmental Health Bureau Ministry of Health and Welfare, Japan. 1997. Report on enterohemorrhagic Escherichia coli O157 infections. Ministry of Health and Welfare, Tokyo, Japan.
  6. Foulds, G., R. M. Shepard, and R. B. Johnson. 1990. The pharmacokinetics of azithromycin in human serum and tissues. J. Antimicrob. Chemother. 25(Suppl. A):73-82.[Abstract/Free Full Text]
  7. Fujii, T., J. Kadota, T. Morikawa, Y. Matsubara, K. Kawakami, K. Iida, R. Shirai, H. Taniguchi, M. Kaseda, S. Kawamoto, and S. Kohno. 1996. Inhibitory effect of erythromycin on interleukin 8 production by 1{alpha},25-dihydroxyvitamin D 3-stimulated THP-1 cells. Antimicrob. Agents Chemother. 40:1548-1551.[Abstract]
  8. Harel, Y., M. Silva, B. Giroir, A. Weinberg, T. B. Cleary, and B. Beutler. 1993. A reporter transgene indicates renal-specific induction of tumor necrosis facter (TNF) by Shiga-like toxin. Possible involvement of TNF in hemolytic uremic syndrome. J. Clin. Investig. 92:2110-2116.
  9. Izumikawa, K., Y. Hirakata, T. Yamaguchi, H. Takemura, S. Maesaki, K. Tomono, S. Igimi, M. Kaku, Y. Yamada, S. Kohno, and S. Kamihira. 1998. Escherichia coli O157 interaction with human intestinal Caco-2 cells and the influence of fosfomycin. J. Antimicrob. Chemother. 42:341-347.[Abstract/Free Full Text]
  10. Japan Society of Chemotherapy. 1981. Committee report. Chemotherapy (Tokyo) 29:76-79.
  11. Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151:775-782.[Medline]
  12. Karpman, D., A. Andreasson, H. Thysell, B. S. Kaplan, and C. Svanborg. 1995. Cytokines in childhood hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Pediatr. Nephrol. 9:694-699.[CrossRef][Medline]
  13. Khan, A. A., T. R. Slifer, F. G. Araujo, and J. S. Remington. 1999. Effect of clarithromycin and azithromycin on production of cytokines by human monocytes. Int. J. Antimicrob. Agents 11:121-132.[CrossRef][Medline]
  14. Kresse, A. U., C. A. Guzman, and F. Ebel. 2001. Modulation of host cell signalling by enteropathogenic and Shiga toxin-producing Escherichia coli. Int. J. Med. Microbiol. 291:277-285.[CrossRef][Medline]
  15. Kuzminov, A. 1999. Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63:751-813.[Abstract/Free Full Text]
  16. Lopez, E. L., M. M. Contrini, S. Devoto, M. F. de Rosa, M. G. Grana, M. H. Genero, C. Canepa, H. F. Gomez, and T. G. Cleary. 1995. Tumor necrosis factor concentrations in hemolytic uremic syndrome patients and children with bloody diarrhea in Argentina. Pediatr. Infect. Dis. J. 14:594-598.[Medline]
  17. Makino, K., K. Yokoyama, Y. Kubota, C. H. Yutsudo, S. Kimura, K. Kurokawa, K. Ishii, M. Hattori, I. Tatsuno, H. Abe, T. Iida, K. Yamamoto, M. Onishi, T. Hayashi, T. Yasunaga, T. Honda, C. Sasakawa, and H. Shinagawa. 1999. Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak. Genes Genet. Syst. 74:227-239.[CrossRef][Medline]
  18. Matsushiro, A., K. Sato, H. Miyamoto, T. Yamamura, and T. Honda. 1999. Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin. J. Bacteriol. 181:2257-2260.[Abstract/Free Full Text]
  19. Melton-Celsa, A. R., and A. D. O'Brien. 1998. Structure, biology, and relative toxicity of Shiga toxin family members for cells and animals, p. 121-128. In J. B. Kaper and A. D. O'Brien (ed.), Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains. American Society for Microbiology, Washington, D.C.
  20. Mühldorfer, I., J. Hacker, G. T. Keusch, D. W. Acheson, H. Tschape, A. V. Kane, A. Ritter, T. Olschlager, and A. Donohue-Rolfe. 1996. Regulation of the Shiga-like toxin II operon in Escherichia coli. Infect. Immun. 64:495-502.[Abstract]
  21. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.[Abstract/Free Full Text]
  22. Noda, M., Yutsudo, T., Nakabayashi, N., Hirayama, T. and Takeda, Y. 1987. Purification and some properties of Shiga-like toxin from Escherichia coli O157:H7 that is immunologically identical to Shiga toxin. Microb. Pathog. 2:339-349.[CrossRef][Medline]
  23. O'Brien, A. D., L. R. Marques, C. F. Kerry, J. W. Newland, and R. K. Holmes. 1989. Shiga-like toxin converting phage of enterohemorrhagic Escherichia coli strain 933. Microb. Pathog. 6:381-390.[CrossRef][Medline]
  24. O'Brien, A. D., J. W. Newland, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal. 1984. Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 9:694-696.
  25. Pickering, L. K., T. G. Obrig, and F. B. Stapleton. 1994. Hemolytic-uremic syndrome and enterohemorrhagic Escherichia coli. Pediatr. Infect. Dis. J. 13:459-475.[Medline]
  26. Plunkett, G., III., D. J. Rose, T. J. Durfee, and F. R. Blattner. 1999. Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181:1767-1778.[Abstract/Free Full Text]
  27. Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308:681-685.[Abstract]
  28. Rubin, B. K., H. Druce, O. E. Ramirez, and R. Palmer. 1997. Effect of clarithromycin on nasal mucus properties in healthy subjects and in patients with purulent rhinitis. Am. J. Respir. Crit. Care Med. 155:2018-2023.[Abstract]
  29. Rubin, B. K., and J. Tamaoki. 2000. Macrolide antibiotics as biological response modifiers. Curr. Opin. Investig. Drugs 1:169-172.[Medline]
  30. Tesh, V. L., J. E. Samuel, L. P. Perera, J. B. Sharefkin, and A. D. O'Brien. 1991. Evaluation of the role of Shiga and Shiga-like toxins in mediating direct damage to human vascular endothelial cells. J. Infect. Dis. 164:344-352.[Medline]
  31. Trachtman, H., and E. Christen. 1999. Pathogenesis, treatment, and therapeutic trials in hemolytic uremic syndrome. Curr. Opin. Pediatr. 11:162-168.[CrossRef][Medline]
  32. van de Kar, N. C., T. Kooistra, M. Vermeer, W. Lesslauer, L. A. Monnens, and V. M. van Hinsbergh. 1995. Tumor necrosis factor alpha induces endothelial galactosyl transferase activity and verocytotoxin receptors. Role of specific tumor necrosis factor receptors and protein kinase C. Blood 85:734-743.
  33. van Setten, P. A., L. A. Monnens, R. G. Verstraten, L. P. van den Heuvel, and V. W. van Hinsbergh. 1996. Effects of verocytotoxin-1 on nonadherent human monocytes: binding characteristics, protein synthesis, and induction of cytokine release. Blood 88:174-183.[Abstract/Free Full Text]
  34. Yamamoto, T., I. Taneike, Y. Tamura, J. Nishi, and K. Kawakami. 2000. A novel hemagglutinin of enterohemorrhagic Escherichia coli O86:NM associated with hemolytic uremic syndrome in a child. Acta Med. Biol. 48:147-152.
  35. Yokoyama, K., K. Makino, Y. Kubota, M. Watanabe, S. Kimura, C. H. Yutsudo, K. Kurokawa, K. Ishii, M. Hattori, I. Tatsuno, H. Abe, M. Yoh, T. Iida, M. Ohnishi, T. Hayashi, T. Yasunaga, T. Honda, C. Sasakawa, and H. Shinagawa. 2000. Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak. Gene 258:127-139.[CrossRef][Medline]
  36. Yoshimura, A., Y. Hara, T. Kaneko, and I. Kato. 1997. Secretion of IL-1ß, TNF-{alpha}, IL-8 and IL-1ra by human polymorphonuclear leukocytes in response to lipopolysaccharides from periodontopathic bacteria. J. Periodontal Res. 32:279-286.[CrossRef][Medline]
  37. Yutsudo, T., N. Nakabayashi, T. Hirayama, and Y. Takeda. 1987. Purification and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin. Microb. Pathog. 3:21-30.[CrossRef][Medline]
  38. Zhang, H. M., Z. L. Ou, F. Gondaira, M. Ohmura, S. Kojio, and T. Yamamoto. 2001. Protective effect of anisodamine against Shiga toxin-1: inhibition of cytokine production and increase in the survival of mice. J. Lab. Clin. Med. 137:93-100.[CrossRef][Medline]
  39. Zhang, X., A. D. McDaniel, L. E. Wolf, G. T. Keusch, M. K. Waldor, and D. W. K. Acheson. 2000. Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J. Infect. Dis. 181:664-670.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, November 2002, p. 3478-3483, Vol. 46, No. 11
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.11.3478-3483.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohara, T.
Right arrow Articles by Yamamoto, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohara, T.
Right arrow Articles by Yamamoto, T.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS