*3.2. Analysis of Insertion Sequences*

The prevalent type was IS*26* (99.4%), followed by IS*Ecp1* (68%) and IS*903* (65.3%), while IS*CR1* was detected only in 19 isolates (12.6%). The wide presence of IS26 in almost all multidrug-resistant isolates is a hint that IS26 is not associated with multidrug resistance, but only with ESBL-producing isolates.

The four insertion sequences were present in all environments (Figure 2), except IS*CR1*, that was not detected in farm and feeds. This latest result contrasts with the reported by Ali et al. [44], that showed the connection between IS*CR1* and *intI1* in strains isolated from diverse dairy farms in China. However, the wide dissemination of IS among different niches has been reported by other authors. For instance, Cullik et al. [25] showed the association between *bla*CTX-M with the common elements IS*Ecp1*, IS*26*, and IS*903*, in ESBL-producing *E. coli* isolated in a German Hospital. The IS*Ecp1* type has been detected in clinical isolates from Korea, and in isolates from healthy or diseased food-producing animals, including swine and avian [45,46].

**Figure 2.** Prevalence (percentages) and distribution of insertion sequences in ESBL-producing *E. coli* according to their origin. (**A**) IS*26*; (**B**) IS*Ecp1*; (**C**) IS*903*; (**D**) IS*CR1*.

In addition, the frequent co-existence of several insertion sequences in the same strain has been detected, in agreement with other studies [25–27,47]. Genetic patterns are presented in Figure 3, showing that the majority of isolates carried two or three IS (42% and 40.7%, respectively), whereas 10% of them carried only one. The three prevalent genetic patterns were IS*26*–IS*Ecp1*–IS*903* (*n* = 55), IS*26*–IS*Ecp1* (*n* = 33), and IS*26*–IS*903* (*n* = 28) (37%, 22%, and 19%, respectively). The combination IS*26*–IS*Ecp1* has been related with the pathogen clone ST131 [25,48], and it was present in 4 isolates coming from healthy people (*n* = 2) and hospital inpatients (*n* = 2), that supposes a possible risk situation for the healthy population. Finally, it is remarkable that 7.3% of isolates contain the four IS (IS*Ecp1*–IS*26*–IS*903*–IS*CR1*), a situation that, to our best knowledge, is being described in the literature for the first time. These isolates come mainly from hospital inpatients (*n* = 9), but we also found the genetic patterns in isolates from a river (*n* = 1) and from a chicken hamburger (*n* = 1). In summary, these results show the complexity of mobile genetic elements, and suggest the facility to acquire different mechanisms to disseminate resistance genes through all environments.

**Figure 3.** Genetic patterns and prevalences among the studied ESBL-producing *E. coli*.
