**2.5 Screening and confirmation of ESBL and integrons producers**

A double synergy test was used for ESBL-producing strains testing. This consisted of placing disks (2–3 cm diameter) of ceftriaxone and cefotaxime around an amoxicillin-clavulanic acid disk on the bacterial plate.

For molecular characterization, DNA extraction was performed using heating method [23]. A loopful of bacterial growth from Mueller-Hinton agar (Liofilchem, Italy) plate was suspended in 1 ml of sterilized water. The mixture was boiled for 10 min at +100°C and centrifuged for 10 min at 12000 rpm at +4°C. Supernatant was then collected and used for the PCR reactions as DNA matrices. Multiplex PCR assays were performed for detecting EBLS-encoding genes (*bla*TEM, *bla*SHV, *bla*OXA, and *bla*CTX-M) and the presence of the class 1, class 2, and class 3 integrons from the β-lactams-resistant DEC strains. Primers (GeneCust, France) used for these amplifications are described in **Table 1**.

Thermocycling conditions were as follows: 5 min at +94°C, followed by 35 amplification cycles of +94°C for 30 s, 59 ± 4°C for 60 s, and +72°C for 60 s with a final extension of +72°C for 10 min on a thermal cycler (Gene Amp 9700, Applied Biosystems). PCR products were revealed on 1.5% stained Redsaf agarose gel (Prolabo, France), after electrophoresis under UV light (Gel Logic 200).

The PCR assays were carried out in a 25-ml reaction mixture, which consisted of 2.5 μl of the supernatant added to 22.5 μl of reaction mixture. This mixture contained 5 U of Taq DNA polymerase (Accu Power, South Korea), deoxyribonucleic triphosphate (10 mM), buffer GC (10X), MgCl2 (25 mM), and PCR primers (10 μM). Thermocycling conditions were as follows: 5 min at +94°C, followed by 35 amplification cycles at +94°C for 30 s, + 59 ± 4°C for 60 s, and +72°C for 60 s with a final extension of +72°C for 10 min on a thermal cycler (AB Applied Biosystems). Following PCR, the reaction products were separated using electrophoresis in 1.5% agarose gel (weight/volume), stained with Redsaf solution (Prolabo, France), and visualized under UV light (Gel Logic 200) [23].

*Beta-Lactamase-Producing Genes and Integrons in* Escherichia coli *from Diarrheal Children… DOI: http://dx.doi.org/10.5772/intechopen.103169*


**Table 1.**

*List of all primers used for antibiotic ESBL genes and integrons detection.*
