Verocytotoxigenic Escherichia coli (VTEC), also referred to as Shiga toxin-producing E. coli (STEC), are zoonotic pathogens associated with a high variety of clinical outcomes such as diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Human infections are in most cases acquired through water or food directly or indirectly contaminated with cattle feces. The key virulence factors are the verocytotoxins. There are two main types, namely VT1 and VT2, which can be further divided into subtypes based on their sequence analysis. The nomenclature is not definite and new variants are constantly being described. These VT-encoding genes (vtx) are generally encoded by a heterogenous group of temperate lambdoid bacteriophages and are expressed when the lytic cycle is activated [1,2].

In a cross-sectional survey, both vtx-negative and vtx-positive strains belonging to the same serogroup were detected on two cattle herds [3]. The absence of vtx genes in strains carrying eae and EHEC-hlyA is thought tooccur by two hypotheses. First of all, the vtx-negative strains could arise from strains that had lost their vtx genes during subculturing as several studies reported the spontaneous loss of vtx genes after multiple subcultivation steps [4,5,6,7,8]. Secondly, these vtx-negative strains could occur as inherently vtx-negative strain in the animal reservoir as such [9], designated as aEPEC.

Since several studies observed the loss after multiple subcultivation steps and long preservation times, this study was set up to determine (i) if the loss of vtx genes can already occur after the first subcultivation step after isolation, (ii) the frequency of this spontaneous loss, and (iii) to evaluate the toxin-type and serogroup dependence.

Rectal fecal samples were taken from cattle known to be infected with VTEC O157 and/or non-O157, on 3 cattle farms, to study the spontaneous loss of vtxgenes after the first subcultivation step of suspected colonies.

Enterohemorrhagic E. coli (EHEC) are a distinct class of VTEC, characterized by the presence of verocytotoxins, intimin and EHEC enterohemolysin. The key virulence determinants are the verocytotoxins 1 and 2 encoded on temperate bacteriophages. EHEC strains may convert to atypical enteropathogenic E. coli (aEPEC) strains by the loss of their vtx genes after multiple subcultivation steps or long preservation. These potential genetic changes of the pathogens have to be taken into account when interpreting screening results for the public health concern of O157 and non-O157 E. coli. To our knowledge, this is the first study conducted to examine the frequency of the loss of vtx genes after the first subcultivation step among O157 and non-O157 EHEC strains during the isolation from naturally contaminated bovine fecal samples, as other studies focused on multiple subcultivation steps and long-term preservation. In this survey, fecal samples were collected on three farms housing EHEC carrier animals. The stability of vtx genes were tested in 40positive fecal samples (20 O157 and 20 non-O157 samples) after the first subculturing step. Noteworthy, this subcultivation step is advisable and even obligatory by the ISO method 16654:2001 [15] for the detection of E. coli O157 from feed and food to ensure the purity of the strains to be characterized.

All tested strains were eae and EHEC-HlyA positive. The loss of vtx genes among O157 strains was rather rare compared to non-O157 strains, namely in 3 out of 20 O157 strains compared to 9 out of 20 non-O157 strains.Regardless of the differences in the isolation procedures, the frequency of this curing within the 3 O157 strains was rather rare (on average 1 out of 10), while for the 9 non-O157 strains, the rate of loss was higher (on average 4 out of 10 isolates). These findings are in agreement with Schmidt et al.[16] who observed that vtx genes appear to be more stably maintained in O157 strains than in non-O157. Because at least one colony still harbored vtx1 and/or vtx2 after subcultivation, it can be hypothesized that the loss already occurred during growth on the differential media. Our data may be of importance in the screening of VTEC because these toxigenic organisms of public health concern can become non-toxigenic after subcultivation. The question remains how frequently the loss of vtx genes already occurs in the intestine of cattle as Bielaszewska et al. [17] reported that an appreciable subset of patients suffering from HUS excreted EHEC that lost their vtx genes. The latter showed a common phylogeny with the EHEC of the corresponding serotype and they belong to the same MLST clonal complexes. The importance of free vtx-encoding bacteriophages should be addressed with regard to their role as vectors for horizontal virulence gene transfer [18] in the animal reservoir as Cobbaut [19] reported the presence of inherently vtx-negative strains on vtx-positive cattle herds. Therefore, the role of VT-producing E. coli may be of a greater concern than was previously assumed if there can be an alternate conversion between aEPEC and EHEC strains.

Our results provethat a loss of vtx genes in vtx-positive isolates can already occur after the first subculturing step of VTEC isolated from naturally contaminated samples. Consequently, this may lead to an underestimation of VTEC in animals, food and humans. Therefore, we advise to test different colonies instead of a single colony from a subculture for the presence of vtx genes in order to avoid false negative results.