The hypothesis that antimicrobial therapy of STEC infections will increase the risk of developing HUS has been widely discussed (11
). Since the production of Stx seems to be regulated by an induction of the integrated bacteriophage that encodes the toxins, it is suggested that the regulation of Stx-encoding phages play a direct role in STEC pathogenesis (19
The majority of strains investigated carried the gene encoding intimin (eae). One strain producing Stx1 (C118-05) did not have eae; however, its response was similar to the responses of the other strains carrying stx1, indicating that this virulence gene did not contribute to cytotoxicity. Furthermore, the control strains, enteropathogenic E. coli strain C135-03 and E. coli K-12 strain D2103, were included in the study to demonstrate that no other bacterial factors affected cytotoxicity. These observations confirmed that the cytotoxicity measured is a function of the amount of Stx released.
Interestingly, relative average decreases in toxin release were seen for all strains producing seven Stx2 toxin variants incubated with protein synthesis inhibitors at MICs. The 25 strains producing seven Stx2 variants exhibited relative average decreases ranging from 94.7% (Stx2d-O8-C466-01B) to 14.0% (Stx2-O157-EDL933). In contrast, the strain producing Stx1 generated an average increase (4.6%) in toxin release, which may occur due to induction of the stx
-carrying bacteriophage. The stx
genes are located directly downstream of the late phage promoter (pR′
), and transcription from pR′
due to phage induction is crucial for the production of Stx2 but is unnecessary for high-level Stx1 production (18
). These findings may confirm that protein synthesis inhibitors are able to suppress Stx2 production but not Stx1 production. The pR′
promoter is the primary regulatory element in Stx2 production (19
). Different decreases in the amounts of Stx2 released from the seven strains studied that produced Stx2 variants may therefore be due to variations in the pR′
regions present in the individual strains.
The decreases in the amounts of Stx2 released upon exposure to protein synthesis inhibitors found in the present in vitro study are of clinical interest, since the most severe clinical complications of STEC infections are associated with strains producing subtype Stx2 and subtype Stx2c (6
). In contrast, the use of protein synthesis inhibitors is not obvious in drug therapy of infections due to Stx1-producing strains.
Significant differences in average suppression of toxin release were identified for toxin variants of the same Stx2 subtype. The toxin variants of subtype Stx2 (Stx2-O157-EDL933 and Stx2-O48-94C) showed average suppressions of 14.0% and 48.5%, respectively; furthermore, the toxin variants of subtype Stx2d (Stx2d3-O157-7278, Stx2d-O73-C165-02, and Stx2d-O8-C466-01B) were suppressed 33.9%, 76.5%, and 94.7%. These variations within subtypes indicate that the toxin variant can be a marker of the toxin release following application of protein synthesis inhibitors.
STEC strains belonging to O-serogroup O157 have traditionally been considered to cause more severe clinical outcome than strains of other O serogroups (13
). However, epidemiological studies indicate that the development of HUS is attributed to other risk factors, such as the presence of the stx2
genes but is not independently associated with the O-serogroup O157 of the infecting STEC (5
). Nevertheless, the present data show that it is significantly more difficult to suppress toxin release of Stx1 and toxin variant Stx2-O157-EDL933 from O157 strains compared to non-O157 strains when the strains were exposed to protein synthesis inhibitors. The promoter strength of pR′
might be stronger for the strain producing Stx2-O157-EDL933 than for strains producing other Stx2 variants, and in association with a presently unknown factor that covaries with the O157 strains producing Stx2-O157-EDL933, this could explain the lower decrease in the relative toxin release for this O serogroup.
To avoid introducing bias when comparing toxin variants due to a higher ratio of strains representing O-serogroup O157, strains included in this study were selected to achieve an equal number of O157 strains and non-O157 strains producing each toxin variant. This was attained for Stx1, Stx2-O157-EDL933, and Stx2c-O157-FLY16. Toxin variant Stx2-O48-94C is represented by only one strain of O-serogroup O157 out of four. However, Stx2-O48-94C was still found to be significantly different from Stx2-O157-EDL933 when all O157 strains producing the two toxin variants were omitted from the statistical test or if all non-O157 strains were excluded (P < 0.001 in both cases). Furthermore, the observed difference between the strains producing toxin variants of Stx2d cannot be due to bias that arose from the ratio of O157 strains, as no strains of this subtype are O157.
Used at MICs, the protein synthesis inhibitors significantly suppressed the toxin release from all strains producing Stx2, except from two strains producing toxin variant Stx2-O157-EDL933 (C532-03 and C528-03). Thus, the use of protein synthesis inhibitors for treatment of infections with most STEC strains carrying stx2 appears promising. However, treating infections caused by Stx2-O157-EDL933-producing strains of O-serogroup O157 is not feasible, since both strains that caused an increase in toxin release were of serotype O157:H7. Consequently, these data suggest that it should be mandatory to determine the O serogroup of Stx2-O157-EDL933-producing strains prior to antimicrobial treatment with protein synthesis inhibitors.
In a Danish STEC cohort (255 strains), isolated in the period from January 1997 to October 2003, the Stx2 subtype/variant distribution was determined and associated with clinical data, including HUS. All 20 HUS cases were associated with subtype Stx2 and/or Stx2c. Nineteen HUS cases involved toxin variant Stx2-O157-EDL933 or Stx2-O48-94C either alone (11 cases) or in combination with variant Stx2c-O157-FLY16 (8 cases). Finally, one strain causing HUS produced the Stx2c-O157-FLY16 variant (15
). Of the 11 strains producing Stx2-O157-EDL933 and the 8 strains producing Stx2-O48-94C, 8 strains and 1 strain, respectively, belonged to O-serogroup O157. In this cohort, 12 HUS cases might have benefitted from antimicrobial treatment with protein synthesis inhibitors prohibiting fulminant STEC pathogenesis, including HUS.
Ciprofloxacin applied at MIC caused an increase in toxin release from approximately half of the strains tested, whereas only two strains producing Stx2-O157-EDL933 increased the amount of toxin released as a result of incubation with protein synthesis inhibitors. Ciprofloxacin was observed to suppress the toxin release from a strain producing Stx2d-O8-C466-01B by 92.2%, which could indicate that this toxin variant is independent of phage induction, which was observed for an activatable Stx2d strain (O91:H21, B2F1) by Teel et al. (17
The subinhibitory concentration is clinically relevant only until the concentration of antimicrobial drug reaches the therapeutic level of the target, because the administered doses are recommended to be well above the MIC. Hence, it will be of importance that the antimicrobial reaches the inhibitory concentration quickly when therapy is initiated and maintained well above the MIC until the infection is cured in order to avoid increase of toxin release of the STEC. Increase in toxin release is expected to enlarge the risk of systemic damage to the renal endothelial cells (22
) and is therefore to be avoided. In this study, incubation with protein synthesis inhibitors at sub-MICs displayed highly variable responses, including increases in toxin release.
The level of decrease in toxin release for the different toxin variants shows much variation. In a clinical perspective, it might be more beneficial to treat infections due to STEC producing toxin variants that are more easily suppressed in vitro. Therefore, it seems important to determine the toxin variant of the infecting STEC before antimicrobial therapy is initiated. The Stx2 subtype and variant determination should be referred to national reference laboratories of clinical microbiology if not feasible in local clinical laboratories.
In conclusion, the significant difference in response to protein synthesis inhibitors between Stx2 toxin variants presented in this study emphasizes the relevance of determining not only the Stx2 subtype but also the toxin variant before antimicrobial treatment is considered. Further clinical studies are required to confirm the possible beneficial effect of protein synthesis inhibitors in management of STEC infections. In general, antimicrobial treatment of STEC increases the risk of HUS because of toxin liberation from lysed bacterial cells. Therefore, clinical studies of antimicrobial treatment of STEC raise serious ethical considerations. Our in vitro study suggests that introductory clinical studies should focus on protein synthesis inhibitors.