A further pivotal event in the emergence of this Central Asia clade prior to its introduction into Bhutan was the fixation of the previously described E-type colicin plasmid pSSE3 (with 100 % identity in DNA sequence). This E-type colicin has been shown to be antagonistic against a variety of diarrhoeogenic E. coli, but not against commensal E. coli (Calcuttawala et al., 2015). Plasmid pSSE3, which was found in all Bhutanese isolates (Fig. 1B), also carries an immunity function that protects the host cell against the activity of the corresponding colicin. Although it was previously characterized as a ColE3 plasmid by PCR, sequence analysis of pSSE3 indicated that the colicin biosynthesis cluster was a novel recombinant hybrid with an undetermined spectrum of activity (Fig. 3). On closer examination, the first and second domains (translocation and receptor-binding) of the colicin structural gene (cea) and the lysis gene (cel) exhibited substantial amino acid sequence identity (98 %) to that of colicin E7 (ColE7). The third domain (cytotoxic) encoded by cea was most closely related to that of ColE6 (94 % protein sequence identity), predicting that the colicin's target is actually 16S rRNA rather than DNA, which is the target of the ColE7 counterpart (Kleanthous, 2010). However, the product of the colicin immunity gene (cei) exhibited only 84 % protein sequence identity to that of colicin E6.