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Antimicrobial Agents and Chemotherapy, August 2007, p. 2837-2841, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01397-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rifaximin Does Not Induce Toxin Production or Phage-Mediated Lysis of Shiga Toxin-Producing Escherichia coli

Theresa J. Ochoa,^1,2 Jane Chen,¹ Christopher M. Walker,¹ Elsa Gonzales,² and Thomas G. Cleary^1*

Center for Infectious Diseases, University of Texas Health Science Center, Houston, Texas,¹ Universidad Peruana Cayetano Heredia, Lima, Perú²

Received 8 November 2006/ Returned for modification 23 January 2007/ Accepted 14 May 2007

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Diarrhea in children is often caused by enteropathogen infections that might benefit from early empirical antibiotic therapy. However, when the definition of the pathogen requires sophisticated laboratory studies, the etiology of enteritis is not known early in illness. Empirical therapy may be dangerous if the child is infected with a Shiga toxin-producing Escherichia coli (STEC) strain because antimicrobials may increase Shiga toxin (Stx) release, resulting in increased risk of microangiopathic hemolytic anemia with acute renal failure (hemolytic-uremic syndrome [HUS]) and death. There is a need for antimicrobials that would be effective against multiple bacterial enteropathogens yet not induce Stx release or increase the risk of HUS. Rifaximin has been evaluated in adults for treatment of bacterial enteritis and has a good record for safety and efficacy, but it has not been evaluated extensively in children with gastroenteritis. We therefore evaluated rifaximin's potential for phage induction, drug-induced bacteriolysis, and toxin release in 57 STEC strains (26 O157 and 31 non-O157 strains). Growth in ciprofloxacin, a known Stx phage inducer, caused bacteriolysis and release of toxin in 25/26 (96%) O157 strains and 15/31 (48%) non-O157 strains. In contrast, rifaximin did not induce phage replication or lysis in any strain. Toxin release in the presence of rifaximin was not different from release in the absence of antibiotic. Rifaximin, unlike many antibiotics used to treat pediatric gastroenteritis, does not induce phage-mediated bacteriolysis and Stx release.

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Bacterial enteropathogens represent important pediatric pathogens worldwide. Many of the agents causing gastroenteritis are known or predicted to respond to antibiotic therapy (e.g., Shigella, Campylobacter, enteropathogenic Escherichia coli, enteroinvasive E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, etc.). Early empirical therapy for these agents is likely to result in more rapid resolution of illness. Unfortunately, diagnosis of some of these pathogens is not practical except in research labs. Thus, there is a need for low-cost safe oral agents which might be used for treatment of children with bacterial diarrhea.

An important issue with empirical therapy is that some episodes of bacterial diarrhea are due to Shiga toxin-producing E. coli (STEC; variously referred to in the literature as STEC, enterohemorrhagic E. coli, and verotoxin-producing E. coli). There are over 100 serotypes of STEC that cause disease in humans; important serotypes include E. coli O157:H7, O111:NM, and O26:H11. Strains may produce Shiga toxin 1 (Stx1), Stx2, variants of Stx1 or Stx2, or multiple toxins. Current in vitro data suggest that antibiotic exposure increases the risk of hemolytic-uremic syndrome (HUS) in children infected with STEC by inducing expression of Stx through replication of phages that carry stx genes (9, 24). However, clinical studies have given conflicting results (26, 32). Thus, this issue remains controversial.

STEC strains lack a specific secretory mechanism for release of toxins. The toxin genes are encoded on bacteriophages (20) and are released when bacteriophage-mediated bacteriolysis occurs. The Stx phages are closely related to phage , with similar promoters, repressors, terminators, antiterminators, lysis genes, and structural proteins. The stx genes are always located in the same region of these lambdoid phages. They are part of a late expressed module under the control of antiterminator Q (28) just upstream from the genes that code for breakdown of the bacterial cell membrane and peptidoglycan (S, R, and Rz). However, Stx phages show tremendous diversity (19, 22); they typically consist of modules derived from multiple different sources (15). DNA sequencing data demonstrate that STEC strains contain multiple phages (up to 13 -like phages or phage remnants) (7) and that these phages are defective due to deletions, insertions, and missense mutations (23).

There is a logic for evaluating rifaximin. The mechanism of action of rifamycins involves binding of antibiotic to the beta subunit of prokaryotic RNA polymerase to inhibit transcription. Sigma () factors of bacterial RNA polymerase mediate promoter recognition and opening in transcription initiation; ⁷⁰ is the primary factor of E. coli, and ³² is associated with transcription of most heat shock genes of E. coli (11, 31). The antiterminator Q of phage is central to the expression of stx genes; ⁷⁰ and Q alter RNA polymerase so that it reads through terminators (11) and stx genes are transcribed. However, rifampin inhibits ⁷⁰-dependent promoters more than ³²-dependent promoters (31). Rifaximin has not been evaluated for its effect on phage induction/Stx production. We therefore studied the effect of rifaximin on phage-mediated bacteriolysis, phage induction, and Stx release. Strains obtained from children with HUS as well as from those with milder illness were evaluated; both O157 and non-O157 strains were studied.

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Strains. Fifty-seven STEC strains, representing serogroup O157 (26 strains) and non-O157 serotypes (31 strains), were studied (Table 1). This collection of strains includes human STEC isolates from North and South America, Japan, and Great Britain. Strains were obtained from the STEC Center at Michigan State University; from M. Osterholm in Minnesota; from H. Lior at the Central Public Health Laboratory in Ottawa, Canada; from M. A. Karmali in Toronto, Canada; from the CDC; and from our patients in Argentina and Houston, TX. The prototype strains of Konowalchuk were obtained from the Central Public Health Laboratory in Ottawa, Canada (13). The O and H types listed reflect the designations from the primary labs, except for several O157 Houston strains that were typed using commercial antisera. Forty-eight strains were eae⁺ (84%), 13 strains had stx₁ only (23%), 13 strains had stx₂ only (23%), and 31 strains had stx₁ and stx₂ (54%). For 47 (83%) of the strains in this culture collection, we had clinical information; 27 strains were isolated from a diarrheal illness and 20 strains were from children with HUS. We did not have data regarding the presence of bloody versus nonbloody diarrhea.

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TABLE 1. Serotypes, strain designations, and genotypes of STEC strains studied, with associated clinical illnesses

Growth curve/bacteriolysis. The effects of serial twofold dilutions of ciprofloxacin or rifaximin (from 50 µg/ml to 0.049 µg/ml [final concentrations]) on growth and induction of phage-mediated bacteriolysis were determined spectrophotometrically over a 5-hour period, using an initial inoculum of 1.5 x 10⁵ CFU/ml. A bacteriolytic pattern was characterized by a growth curve at a subinhibitory concentration which showed a drop in the optical density after the initial 2 to 3 h of growth. Higher concentrations of drug that do not allow bacterial growth cannot be shown to induce lysis. Because Shiga toxin-producing lambdoid phages are often defective or cryptic (2, 14, 35), ciprofloxacin, a known Stx phage inducer, was used to characterize the inducibility of each strain.

Detection of Stx phage by real-time PCR. Each strain was confirmed to have stx₁ and/or stx₂ family genes by using previously described primers (1) on single colonies, whose DNAs were extracted by boiling for 5 min followed by centrifugation at 14,000 rpm for 10 min. The reaction mix included HF Phusion buffer with a final concentration of each deoxynucleoside triphosphate of 200 µM, MgCl₂ (4 mM), and Phusion polymerase (0.5 U) in a reaction volume of 25 µl. SYBR green was diluted as recommended by the manufacturer. The primers used were as follows: for stx₁, 5'-CTGGATTTAATGTCGCATAGTG and 5'-AGAACGCCCACTGAGATCATC; and for stx₂, 5'-GGCACTGTCTGAAACTGCTCC and 5-TCGCCAGTTATCTGACATTCTG. The eae primers were 5'-ATGCTTAGTGCTGGTTTAGG and 5'-GCCTTCATCATTTCGCTTTC. The amplification cycles consisted of incubation at 98°C for 50 s, 60°C for 20 s, 72°C for 30 s, and 75°C for 1 s; after 25 cycles, a melting curve using SYBR green fluorescence was determined with a ramp speed of 2.5°C/s between 73°C and 95°C, with a reading every 0.2°C. The resulting amplicons were confirmed to be 248 bp, 150 bp, and 255 bp by agarose gel electrophoresis and to have melting points of 83.4°C, 87°C, and 89°C for eae, stx₁, and stx₂, respectively. The number of free phage was determined for each STEC strain with filtered supernatants (0.45-µm filters; Fisher Chemical, Pittsburgh, PA) collected at the end of 5 hours of growth. Aliquots of supernatants of each strain grown in ciprofloxacin, rifaximin, or buffer control were serially diluted 10-fold and evaluated by real-time PCR for the presence of free stx genes to indicate the presence of free phage DNA. The presence of eae was used to indicate that samples had been diluted adequately so that chromosomal DNA was no longer detected. Based on observations of dilutions at which phage could be demonstrated with eae⁺ strains, a positive PCR at a dilution of >1:100 was used as a criterion for released phage for the eae-negative strains.

Extracellular Shiga toxin measurement. The 5-hour growth curve filtered supernatants used for phage induction studies were also evaluated by enzyme immunoassay (Meridian Diagnostics, Cincinnati, OH) for the presence of free Shiga toxins. The assay was performed according to the manufacturer's instructions. This assay detects both Stx1 and Stx2 but does not differentiate between them. Standard curves were prepared using pure Shiga toxin (Sigma, St. Louis, MO).

Data analysis. Data are presented as frequencies and percentages for categorical variables and as medians and interquartile ranges (IQR) for nonparametric continuous variables. The chi-square test was used for comparison of proportions. The Wilcoxon signed rank test and Wilcoxon rank sum test were used to assess differences between independent or related groups of nonparametric continuous variables. Data were stored in Microsoft Excel and analyzed with SPSS 12.0 for Windows (SPSS Inc.).

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Induction of bacteriolysis. Bacterial lysis growth curves for a typical strain (STEC 1370) are shown in Fig. 1. None of the strains grown with inhibitory or subinhibitory concentrations of rifaximin demonstrated bacteriolysis (0/57 strains [0%]). However, STEC strains usually (40/57 samples [70%]) had a growth pattern demonstrating ciprofloxacin-induced bacterial lysis at subinhibitory drug concentrations. Thus, the machinery for induction of vegetative growth was often intact. Interestingly, there was a significant difference in frequency of the lytic growth pattern induced by ciprofloxacin between O157 and non-O157 strains (25/26 samples [96%] versus 15/31 samples [48%]; P < 0.001).

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FIG. 1. Induction of bacteriolysis. Growth curves for E. coli O26:H11 strain 1370 in the presence of serial twofold dilutions of antibiotics. (Top) Ciprofloxacin at 0.049 µg/ml () and no-antibiotic control (); (bottom) rifaximin dilutions at 50 µg/ml (•), 12.5 µg/ml (), and 0.049 µg/ml () and no-antibiotic control (). Growth was determined spectrophotometrically (optical density [OD] at 620 nm) over a 5-hour period. The curves show that no concentration of rifaximin was associated with a drop in OD after the initial period of growth; in contrast, the subinhibitory concentration of ciprofloxacin allowed initial growth similar to that of the no-drug control, followed by lysis of organisms, with the OD approaching baseline over the next several hours.

Detection of extracellular phage. At the end of a 5-hour growth period, real-time PCR assays demonstrated that strains grown in ciprofloxacin usually had detectable extracellular free phage (34/57 samples [60%]), in contrast to those grown in rifaximin, which did not have detectable extracellular phage (0/57 samples [0%]). Strains that were grown in subinhibitory concentrations of ciprofloxacin were more likely to have detectable phage if they were serogroup O157 strains than if they were strains of other serogroups (25/26 samples [96%] versus 15/31 samples [48%]; P < 0.001). Strains that had a ciprofloxacin-triggered lytic growth pattern usually also had detectable phage, while strains that did not have a lytic growth pattern did not have detectable phage (34/40 samples [85%] versus 0/17 samples [0%]; P < 0.001). There were six strains that demonstrated bacteriolysis with ciprofloxacin but had no detectable Stx phage, presumably reflecting the fact that ciprofloxacin induces lambdoid phages whether they contain stx genes or not.

Extracellular Shiga toxins. The amount of free Shiga toxin differed between STEC strains grown in ciprofloxacin (median, 359 ng/well; IQR, 22 to 1,573 ng/well) and those grown in rifaximin (median, 3 ng/well; IQR, 0 to 29 ng/well [P < 0.001]) or a medium control (median, 6 ng/well; IQR, 0 to 36 ng/well [P < 0.001]) (Table 2). The data are dispersed, reflecting the inherent strain variability related to basal production and competence of phages. Strains that were O157 had higher levels of toxin induced by ciprofloxacin than did strains that were non-O157 (P < 0.01). Strains that had a lytic growth pattern induced by ciprofloxacin had higher levels of toxin than did strains without a lytic grown pattern (P < 0.001). Two strains (O103:NM [TW07697] and O111:NM [TW08607]) had large increases in toxin when grown in rifaximin and in the control without antibiotic compared to mean levels. However, these strains did not have evidence of rifaximin-induced bacteriolysis or release of phage, suggesting that some strains may release toxin without induction of phage replication.

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TABLE 2. Extracellular Shiga toxin induced by ciprofloxacin and rifaximin

Clinical correlation. There was a correlation between ciprofloxacin-induced toxin levels and clinical illness, with higher levels of toxin found in strains from children with HUS than in diarrhea-associated strains. HUS-related strains were more likely than diarrhea-associated strains to demonstrate ciprofloxacin-induced bacteriolysis and phage induction, especially with stx₂ (Table 3). There were no significant differences for O157 and non-O157 strains, analyzed separately, for each of the examined parameters (bacteriolysis, phage induction, and Shiga toxin release) between diarrhea-associated and HUS strains. There was one strain (O91:H21 [TW01393]) isolated from a child with HUS that had no detectable lysis, phage induction, or toxin production. STEC strains have been reported to mutate or variably express phages during culture (8, 17), and strains from a single outbreak may differ significantly (27). We suspect that this strain may have had critical mutations occur during subculture.

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TABLE 3. Clinical correlation between HUS-associated and diarrhea-associated strains and bacteriolysis, phage induction, and amount of Shiga toxin released

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It has been hypothesized that release of Stx-producing phages leads to infection of nonpathogenic E. coli and amplification of toxin production (4, 5). Animal model data suggest that these in vitro data are likely to be relevant. When intestinal E. coli strains from mice are resistant to Stx phages, toxin can rarely be detected in feces, while toxin is commonly detected if STEC-infected mice are colonized with phage-susceptible E. coli (3). Agents that induce Stx phages (e.g., ciprofloxacin) have been shown to increase toxin levels in feces (3) and to cause death in STEC-infected mice (21, 36).

As expected (16, 25), we found that many STEC strains are defective in the ability to produce lytic Stx phages. However, it is unclear whether defective prophages may be complemented in trans by other lambdoid phages so that their late genes (including stx₁, stx₂, and cell lysis genes) might be upregulated. For example, the Q antiterminator protein from one phage might induce late genes of another Q-defective strain. Although Q is typically specific for its cognate qut sites, Q from phage 933W can induce Stx2 production in E. coli O157 strain Thai-12 and other strains that have 21 phage-like qut elements (12). Similar findings have been reported for two coli lambdoid phages, namely, H19B and 933W (19).

This study confirms the previously reported dramatically different amounts of toxin produced by different O157 strains carrying phages (30). We suspect that O157 strains are infected with phages that can be induced to make more Stx than that made by phages that infect non-O157 strains. O157 strains isolated from humans with HUS can be induced to make more Stx2 than O157 bovine strains (25).

The literature is currently filled with contradictions about which drugs may pose a risk of increasing toxin production. Typically, small numbers of strains have been studied, so the tremendous variability that is typical of these strains has been ignored. For example, the amount of Stx1 was found to be increased in strain E. coli O157:H7 by minocycline, gentamicin, cefazolin, doxycycline, and fosfomycin, while the Stx2 level was decreased by all of these drugs. In contrast, nine other strains showed an increase in Stx1 by fosfomycin, with the Stx2 level usually unchanged (34). Studies using subinhibitory concentrations of 13 antibiotics (including trimethoprim-sulfamethoxazole, azithromycin, gentamicin, penicillins, streptomycin, ciprofloxacin, fosfomycin, and expanded-spectrum cephalosporins) found that all antibiotics increased the amount of toxin in at least one of three strains studied (6). Others have suggested that macrolides do not stimulate toxin production (18, 21, 33). DNA gyrase inhibitors (quinolones), folate metabolism inhibitors (trimethoprim-sulfamethoxazole), and to a lesser extent, cell envelope-active agents (penicillins and cephalosporins) have all been described as inducing toxin gene expression, while drugs that interfere with translation or transcription do not induce expression (10). A major concern regarding the prior literature is that many studies depended on the demonstration of toxin production without a concomitant demonstration that phage had been induced and bacteriolysis had occurred. Free toxin alone may not be a reliable indicator of antibiotic-induced phage production and bacterial lysis. Antibiotics that act on the cell wall may cause a leak of toxin (along with release of endotoxin) (29), and this may be interpreted as phage induction if additional studies have not been done.

We propose that before drugs are used in children with possible STEC enteritis, the drugs should be evaluated as described herein as part of preclinical safety evaluation. Rifaximin, unlike many antibiotics used for treating bacterial enteritis, does not induce Stx production and phage-mediated STEC lysis. The limitation of these data is that this is an in vitro study. Conclusions should not be made regarding the effectiveness and safety of rifaximin in treating diarrhea due to STEC in the absence of data from clinical trials.

 
This work was supported in part by Salix Corporation. T. J. Ochoa was supported by the Baylor-Cayetano Heredia Training Program in Global Infectious Diseases (D43TW006569).

We thank T. Whittam and the STEC Center (Michigan State University) for providing some of the strains used in this study. We also thank M. Karmali, H. Lior, and M. Osterholm for the strains they provided.

 
* Corresponding author. Mailing address: University of Texas School of Public Health, Center for Infectious Diseases, P.O. Box 20186, Houston, TX 77225. Phone: (713) 500-5714. Fax: (713) 500-5688. E-mail: Thomas.G.Cleary{at}uth.tmc.edu

Published ahead of print on 25 May 2007.

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Antimicrobial Agents and Chemotherapy, August 2007, p. 2837-2841, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01397-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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