STEC strain O104:H4 caused the large outbreak of STEC in spring 2011 in Germany. Antibiotic treatment of STEC infected patients is generally not recommended, because enhanced release of STX from STEC O157:H7 has been reported associated with the fear of enhancing the frequency of HUS and fatalities (reviewed in [2
]). This report characterizes the response of the German outbreak STEC strain O104:H4 in comparison to the prototypic STEC O157:H7. The results of this study should help to illuminate present and future medical practice.
The mechanisms of the antibiotic-induced production and release of STX by STEC have extensively been characterized in vitro
for the most frequent STEC strain, O157:H7. Our study confirms previous reports showing enhanced STX production and release by O157:H7 in the presence of diverse antibiotics. In stark contrast, the German outbreak STEC strain O104:H4 responded to several antibiotics differently with either no release of STX or even reduced STX-titers. These data further confirm and extend previous reports that the release of STX by STEC in response to antibiotics is highly dependent on the strain of STEC and the concentration of the antibiotic [3
For this study, two randomly picked different isolates, P5711 and P5765, of E. coli O104:H4 were used that were isolated from two independent patients at the Medical Center of Cologne University during the German outbreak of STEC O104:H4 in spring 2011. It should be noted that these isolates responded highly concordant to antibiotic treatment as it should be expected due to the assumed clonal origin of pathogenic microorganisms during a defined outbreak.
The impact of antibiotics on STX release merits further consideration. Despite an early induction of STX2-transcripts, meropenem does not enhance the release of STX from STEC O104:H4. The 4x MIC of meropenem even decreases STX titers and activity in supernatants of O104:H4. Since after i.v. application of meropenem peak concentrations in the relevant tissues are reached within about 1
], the observed moderate induction of STX2-transcripts should not be clinically relevant. Indeed, our data suggest that meropenem is safe for the treatment of STEC O104:H4. Similarly, ciprofloxacin at concentrations equal to or beyond 4x MIC reduces the release of STX2 by STEC O104:H4 below that of untreated controls and therefore should be a safe therapeutic option against this STEC strain. These conclusions are of clinical relevance because with standard doses of either meropenem [13
] or ciprofloxacin [12
] concentrations far beyond the 4x MIC are achieved in humans within 1
The antibiotics fosfomycin, gentamicin and chloramphenicol also appear to be suited to treat patients infected with STEC O104:H4 without increasing the release of STX. This means that there are several well-established antibiotics at hand for the treatment of infections with STEC O104:H4. Since inhibitory concentrations of these antibiotics can be achieved in patients rapidly, treatment with these substances would greatly diminish the number of, if not eradicate the bacteria and thereby prevent the sustained production and release of STX.
Previous recommendations to refrain from antibiotic treatment of STEC were not only deduced from in vitro
]. They were also drawn from clinical observations of more frequent and more severe symptoms of STEC infection up to increased frequencies of fatalities after treatment with antibiotics (reviewed in [2
]). However, those in vitro
as well as in vivo
studies have to be interpreted cautiously with regard to the specific experimental conditions or to the particular STEC outbreaks. Some in vitro
studies addressed the response of STEC only to subinhibitory concentrations of antibiotics [3
]. A rationale for this may have been the consideration that in the beginning of antibiotic therapy, the STEC may be exposed to such low concentrations of antibiotics. However, after application of standard antibiotic doses to humans, rapid achievement of high tissue concentrations within 1
h has been reported e.g. for ciprofloxacin [12
] or for meropenem [13
] more than 20 or 10
years ago, respectively.
Published clinical studies are mostly retrospective studies rather than well-controlled, blinded studies which is due to the unexpected outbreaks of STEC. As a consequence, they allow only correlative conclusions rather than revealing causative mechanisms. One carefully designed prospective study [14
] suffered from its small sample size as reported in a recent metaanalysis [15
]. Other clinical studies have individual limitations depending on the specific conditions of the respective outbreaks. For example, Dundas et al. report about the central Scotland outbreak of STEC O157:H7 in 1996 [8
]. These authors state that coincidental treatment of STEC-infected patients with antibiotics for other diseases is a risk factor for HUS and fatalities. However, such a coincidental, non-targeted antibiotic treatment cannot replace a validated, high-dose treatment specifically targeted against a defined STEC strain. Similarly, in a Japanese outbreak of STEC O157:H7 among school children, fosfomycin was used as the “most commonly prescribed antimicrobial agent in Japan” but not because it was validated as effective and safe in the treatment of this STEC strain [9
Other clinical studies [16
] as well as a metaanalysis [15
] did not reveal a correlation between the use of antibiotics and the frequencies of the development of HUS. Consequently, in medical practice antibiotic treatment of patients infected with STEC is avoided. However, it seems unjustified to forfeit generally the antibiotic eradication of STEC and resort only to symptomatic treatment of STEC patients.
Animal studies have revealed that treatment with various antibiotics on days 1 to 3 after infection with STEC O157:H7 reduced in mice the STX levels in the blood and stool, shortened the duration of excretion of the bacteria, and all antibiotic-treated mice survived the otherwise lethal infection [19
]. Similarly, mice infected with STEC O157:H7 showed enhanced survival after treatment with rifampicin alone [20
] or after a sequential therapy with low dose rifampicin followed by high dose gentamicin [21
During the final preparation of this report, Karch´s group published similar data of their concurrent study of the effects of subinhibitory concentrations of antibiotics on the German outbreak strain STEC O104:H4 with regard to the induction and release of STX [22
]. In both studies, almost identical responses of STEC O104:H4 to the antibiotics meropenem, fosfomycin, gentamicin, rifampicin, and chloramphenicol were observed. At the first glance, the responses of both the outbreak strain O104:H4 and the reference strain O157:H7 seemingly differs somewhat between both reports. However, these differences are apparently due to differences in the experimental conditions applied by each group. Among these are (i) different bacterial densities at the start of antibiotic treatment (OD600
of 0.5 in Bielaszewska´s study versus 1x108
cells/ml (corresponding to an OD600
of 0.1 in our hands)), (ii) analysis of induction of STX2-transcripts after 15
h versus 2
h of antibiotic treatment, (iii) or incubating Vero cells in cytotoxicity assays for 72
h versus 48
h with STX2-containing supernatants. Altogether, both reports with slightly different concepts and approaches confirm each other and therefore clearly show the potential for future controlled clinical studies using antibiotic treatment of patients infected with specific STEC strains. Newly emerging outbreak strains of STEC can be rapidly tested for the release of STX in response to relevant antibiotics. STX release can be assessed by EIA, which takes only about 2
h. Thus, the results of these assays can be available already one day after the isolation of the suspected causative STEC. Our data show that the results of the EIA and of the cytotoxicity assay on Vero cells are highly concordant. Lack of STX release in response to a specific antibiotic should provide a rationale to conduct clinical studies with the required statistically significant power that provide definitive answers to burning questions as to the potential of antibiotics to eradicate STEC, to diminish the length of carrier status, and to attenuate the development of HUS.