Clinical isolates included in the study
A total of 418 clinical isolates of ESBL-producing E. coli were collected from patients during the period 2005–2018 from five hospitals of Mexico: ISTE-S, ISSSTESON, Hermosillo, Sonora; International Reference Laboratory (CAPERMOR), Ciudad de Mexico; Instituto Nacional de Cancerologia (INCan), Ciudad de México; Sanatorio Durango (SD-DF), Ciudad de Mexico and Hospital de Pediatria, Centro Medico Nacional Siglo XXI, Ciudad de México.
The bacterial species identification and susceptibility pattern were detected by the Dade MicroScan and VITEK 2 compact system (BioMérieux, Durham, USA) [14, 15, 16]. The of ESBL production phenotype was determined using the double-disc synergism method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) (M100-S21) [17]. The following experiments were carried out only to ESBL-producing E. coli isolates positive for the qepA gene.
PCR Amplification and DNA sequencing
The qepA, aac(6´)-Ib-cr and chrA genes were screened by single PCR with specific primers for each gene. In the qepA positive isolates the mutations in the gyrA and parC chromosomal genes were determined by PCR using specific primers, and confirmed by nucleotide sequencing [14]. The class 1 integrons in the 5´ region were determined with the oligonucleotide Intl1 (CGTTCCATACAGAAGCTGG) vs qepA-R (CTGCAGGTACTGCGTCATG). The relationship of the qepA with the insertion sequence ISCR3C was identified with the oligonucleotide qepA-F (CGTGTTGCTGGAGTTCTTC) and ISCR3C-F (CCACTGCGGTGGCACCGT). In addition, the SHV-, CTX-M-type and TLA-1 β-lactamases genes were screened by PCR, as were the PMQR qnrA, qnrB, qnrS genes using specific oligonucleotides described previously [14, 18]. The PCR products specified by nucleotide sequencing were purified with the commercial kit from Roche (Roche, USA) and sequenced by the BigDye Terminator v3.1 Cycle Sequencing Kit in the automated system (ABIPrisma 3100, Applied Biosystem, USA). The Translate Tool (http://ca.expasy.org/tools/dna.html) was used for each nucleotides sequence to obtain the amino acid sequences and were compared by BLASTp in the GenBank database (http://www.ncbi.nlm.nih.gov/).
Susceptibility determination
The minimal inhibitory concentration (MIC) of cefotaxime, ceftazidime, nalidixic acid, ciprofloxacin, levofloxacin and gentamicin were determined by micro-dilution in broth according to CLSI [17].
Phylogenetic grouping determination
Phylogenetic grouping of the E. coli isolates was performed using the triplex PCR for chuA and yjaA genes and DNA fragment TSPE4.C2, which allow classification of four different groups, as previously described by Clermont et al [19].
Genetic characterization
Random amplified polymorphic DNA (RAPD) analysis was performed to identify the genetic diversity of qepA positive E. coli isolates. The RAPD was performed using decameric primers P1254 and PCR conditions described by Betancor et al [20]. The patterns were considered to be different according to the criteria established by Tenover et al. [21] The MultiLocus Sequencing Typing (MLST) was performed in all qepA positive isolates using the MLST tools (https://enterobase.warwick.ac.uk) [22].
Plasmid analysis and mating experiments
Plasmid profiles were obtained according to the method described by Kieser [23]. Mating assays for the horizontal transfer of quinolone resistance were performed using the E. coli J53-2 (met-, pro-, Rifr) as the recipient strain, in solid-phase mating as described by Miller [24]. Transconjugants were selected on Luria-Bertani (LB) agar supplemented with rifampin (100 µg/ml) and nalidixic acid (8 mg/L), ampicillin (100 mg/L) or cefotaxime (1 mg/L). All transconjugants were verified by their auxotrophic requirements (Pro and Met) and plasmids were analyzed according to the method described by Kieser [23].
Plasmid Typing
Incompatibility groups of transconjugants were detected by PCR replicon typing. Specific primers were used including: HI1, HI2, I1, X, L/M, N, FIA, FIB, W, P, FIC, Y, FIIA, A/C, T, K, B/O, and F previously described by Carattoli et al. [25].
Plasmid sequencing and accession number.
Plasmid DNA was obtained from transconjugants pEC8020 and sequenced by pyrosequencing on Platform 454 (Roche). The assembly was obtained using the PHRED-PHRAP-CONSED and Newbler program. The prediction of open reading frames (ORFs) was done with the Glimmer3 and RAST programs and compared with the GenBank nr database. In silico plasmid analysis was performed using the Center for Genomic Epidemiology tools (https://cge.cbs.dtu.dk/) to identify antimicrobial resistance genes (ResFinder), plasmid replicons (PlasmidFinder) [26].