Genetic Characterization of Plasmid-mediated qepA Gene Among ESBL-producing Escherichia Coli Isolates in Mexico

A molecular characterization of a plasmid-born qepA gene in (ESBL)-producing E. coli clinical isolates were performed. An 2.63% (11/418) were qepA positive isolates, of which a 90.0% carried CTX-M-15 (9/11) and SHV-12 (1/11). All isolates showed chromosomal mutations in the gyrA and parC genes. The clonal groups A, B and C were identied and belonged to, respectively, phylogroups A, B1 and D, as well as the sequence types 205, 405 and 617. Several plasmid proles were determined with incompatibility groups FIA, FIB and FII. The genetic environment of the qepA in plasmid pEC8020 was different from those reported previously. The plasmid sequence included genes conferring resistance to β-lactams (blaCTX-M-15), macrolides (mphA), uoroquinolones (qepA1), trimethoprim (dfrB4) and sulphonamides (sul1). Likewise, the IncF-pEC8020 plasmid carried several insertion sequences including ISCR3, IS6100 and multiple copies of IS26. This work contributes to the epidemiology and genetics of plasmid-born qepA genes of ESBL-producing E. coli. encoding aminoglycoside ribosomal methylase rmtB and TEM-1 β-lactamase genes anked by IS26 and ISCR3C insertion sequences as part of compound transposons [12, 13]. In this work, we determined the prevalence of ESBL-producing Escherichia coli isolates having the plasmid-mediated qepA gene and used molecular and whole-genome sequencing tools was determined the genetic environment of the qepA gene.


Introduction
Antimicrobial resistance is one of the main global public health problems. In low-income countries, including Latin America, the indiscriminate use of antimicrobials has increased the rates of antimicrobial resistance development. The introduction and subsequent use of broad-spectrum antibiotics, such as quinolone and cephalosporine, are widely and frequently used in healthcare systems [1].
Urinary tract infections (UTI) are the most frequent bacterial infections, that require prescription antibiotics. Escherichia coli is the most common agent of UTI and it is found around 70-90% of the cases [2,3]. The rst treatment in patients with UTI is frequently empirical and one of the most widely used antimicrobials are quinolones such as nalidixic acid, cipro oxacin and uoroquinolones [4,5]. They have an e cient and rapid absorption after oral administration and their main route of excretion is at renal level. The increase of multidrug resistant bacteria in community and hospital settings are an important issue for the health care systems [6].
The emergence and increase of quinolone resistant strains and their subsequent spread has been reported in several countries [1]. The main mechanism to quinolones resistance in E. coli is the chromosomal mutations in the type II topoisomerase genes encoding GyrA and GyrB proteins. Resistance can also be due to mutations in the genes for the topoisomerase IV ParC and ParE subunit proteins, though less frequently. However, the description of plasmid-mediated quinolone resistance (PMQR) genes that confer reduced susceptibility to quinolones was described [7]. The three main mechanisms of resistance are the qnr protein product that protects the binding site in type II DNA topoisomerases, the enzymatic modi cation of the drug by the aac(6`)-Ib-cr product, and the e ux pumps encoded by qepA and oqxAB genes [8].
These PMQR genes have been found in bacterial isolates worldwide and they reduce bacterial susceptibility to uoroquinolone, although usually not to the level of clinical non-susceptibility. They do, however, facilitate the bacterial survival and the subsequent generation mutants with a higher level or uoroquinolone resistance and probability of treatment failure [9]. Several reports describe the in the community and hospital spread of extended-spectrum βlactamase (ESBL)-producing E. coli isolates resistant to both cephalosporins and uoroquinolones [10]. Clinical isolates with multi-drug and uoroquinolone resistance mechanisms generate therapeutic failure to both cephalosporin and quinolones and /or uoroquinolones antibiotics. QepA is an e ux pump that decreases susceptibility to hydrophilic uoroquinolones, especially cipro oxacin and nor oxacin [11]. The qepA gene has been described on large conjugative IncF group plasmids with the encoding aminoglycoside ribosomal methylase rmtB and TEM-1 β-lactamase genes anked by IS26 and ISCR3C insertion sequences as part of compound transposons [12,13]. In this work, we determined the prevalence of ESBL-producing Escherichia coli isolates having the plasmid-mediated qepA gene and used molecular and whole-genome sequencing tools was determined the genetic environment of the qepA gene.

Materials And Methods
Clinical isolates included in the study The bacterial species identi cation 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 Ampli cation and DNA sequencing
The qepA, aac(6´)-Ib-cr and chrA genes were screened by single PCR with speci c primers for each gene. In the qepA positive isolates the mutations in the gyrA and parC chromosomal genes were determined by PCR using speci c primers, and con rmed by nucleotide sequencing [14]. The class 1 integrons in the 5ŕ egion were determined with the oligonucleotide Intl1 (CGTTCCATACAGAAGCTGG) vs qepA-R (CTGCAGGTACTGCGTCATG). The relationship of the qepA with the insertion sequence ISCR3C was identi ed 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 speci c oligonucleotides described previously [14,18]. The PCR products speci ed by nucleotide sequencing were puri ed 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/).

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 classi cation of four different groups, as previously described by Clermont et al [19].

Genetic characterization
Random ampli ed 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 pro les 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 -, Rif r ) 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 veri ed by their auxotrophic requirements (Pro and Met) and plasmids were analyzed according to the method described by Kieser [23].
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].
In order to identify the genetic diversity, phylogenetic group and relationship among E. coli ESBL isolates, random ampli ed polymorphic DNA (RAPD), phylogroup analysis and the MultiLocus Sequencing Typing (MLST) were used, respectively (18, 19,21). The isolates 7530, 7505, 7514 and 03212 were grouped in the clone A; followed by the 03210, 8020 and 8019 in the groups B and 7537, 7544, 09220 and 10246 in the group C. The phylogroups B1, D and A were, respectively, identi ed in the groups A, B and C. Likewise, the sequence types 205, 405 and 617, corresponding to A, B and C groups determined by RAPD ( Table 1).
The plasmid DNA was extracted from all the isolates to determine the plasmid pro les. We observed that the isolates contained plasmids of 60,100 and 130kb, 100 and 130-kb and 130-kb (Table 1) Table 1).
The genetic context of qepA gene was determined and corresponded to a complete transfer region with an IS26, followed by a truncated class 1 integrase and truncated dfr2 were located upstream qepA gene (Fig. 1). Downstream from the qepA gene we identi ed an ISCR3, a truncated intI1-groEL, the drfB4 gene, the qacED1-sul1 genes, the chromate ion transporter chrA, the transposase IS6100, three macrolide resistant genes (mphR, the erythromycin resistance repressor; mrx, a transmembrane transport protein of MFS family; and mphA, the macrolide-2'-phosphotransferase) and the IS26. Following this genetic structure, we identi ed a Tn3 family transposase, the blaCTXM-15 and two IS1 family transposases (Fig. 1b). Particularity, the class 1 integron in the 5´ region and the qepA with the insertion sequence ISCR3C was identi ed in all qepA gene positive E. coli isolates.

Discussion
The prevalence of qepA reported in recent literature uctuates between 8.3% and 10%, and is still low in any case [29,30,31]. In Egypt, qepA gene was identi ed in 10% of a collection of 39 MDR isolates. In the children´s hospital in Doha, qepA gene was identi ed in the 10% of 19 E. coli isolates from neonatal intensive care unit. Similar percentage of 8.3% qepA gene-positive was reported in a collection of 144 ESBL-producing E. coli from Tabriz University in Iran. In addition, the susceptibility results to uoroquinolone antibiotics were consistent with the identi cation of substitutions in the GyrA and ParC proteins found in all isolates [27]. As suggested by Jacoby et al [32], the horizontal transfer of PMQR determinants accelerates the selection of higher levels of quinolone resistance, which facilitates bacterial survival and subsequent generation of mutants in GyrA and ParC with higher-level to quinolone resistance that produce therapeutic failure [9].
The eleven isolates formed three speci c groups; the rst corresponds to clone A by RAPD, and belongs to phylogroup B1 and ST205. The members of this clone were isolated in three different years from at least two regions geographically separated from each other (northwest and center of Mexico). A similar characteristic was observed in the second clone, B, which belongs to phylogroup D and ST405. The third clone C belongs to phylogroup A and ST617. The genetic relationship between the members of each clone suggesting a low frequency of identi cation; however, the spread of resistance of ESBL-producing E. coli isolates to uoroquinolones in Mexican hospitals has remained constant [14,16].
Plasmid sequence analyses of pEC8020 showed an environment for the qepA gene anked by IS26 sequences not yet described. The 5´ region corresponding to the arrangement observed in plasmid pHPA (Fig. 1a) [28], with an IS26, Int1, truncated dfr2 located upstream of qepA and anked downstream by the ISCR3, and truncated intI1-groEL. Instead of truncated rmtB, blaTEM-1, and tnpR genes anking by IS26 downstream of the ISCR3 and intI1-groEL described in plasmid pHPA, we identi ed the drfB4, qacED1-sul1, chrA, IS6100, mphR, mrx, mphA and insertion sequences IS26. The arrangement of these genes was previously described as part of the transposon structure Tn6242 anked by two IS26 sequences [28].
Remarkable, the Tn6242 was identi ed after whole genome sequencing as being inserted in the chromosome of one E. coli ST405 belong to phylogroup D obtained from a urine sample [32]. Curiously, the plasmid pEC8020 was obtained from an ESBL-producing E. coli ST405 belongs to phylogroup D isolated from blood culture. We hypothesize a possible recombination between an E. coli ST405 with a chromosomal or plasmidic Tn6242, and IncFII type plasmid harbors the conserved qepA gene anked by the IS26. The mechanism of how this novel structure recruit fragments of plasmid like pHPA and the recombination with Tn6242 will needs further study in the future.
The novel genetic context previously described anked by the IS26 were identi ed in the eleven isolates by PCR and sequencing analysis. However, the Tn3, CTXM-15 and IS1 were absent in the isolates with the SHV gene.
In the case of chrA, the heterologous expression in a plasmid of chrA alone from Shewanella sp, on E. coli and Pseudomonas aeruginosa conferred increased chromate resistance [34]. Caballero et al (2012), suggested that the use of metal derivatives as antiseptics in hospital as an important factor for the selection of bacteria that acquire genes to confer and spread metal resistance among bacteria in hospitals [35].
The previous identi cation of qepA and rmtB genes, anked by the transposable element IS26 in a transferable plasmid belong to incompatibility group IncFII, such as pHPA, suggested the e cient dissemination of these genetic structures [11,33]. The description of a novel genetic structure in this work of qepA gene in plasmid pEC8020, which belongs to IncFII type, will be an e cient medium for the dissemination of resistance genes and the constant spread among Enterobacteriaceae. The emergence of multidrug-resistant gram-negative bacteria that harbor plasmids bearing qepA, crhA and CTX-M-15 genes could become a serious clinical concern for all public health care systems. Figure 1 Comparison of the genetic context of the qepA gene in plasmid pHPA and plasmid pT8020 from ESBL-producing E. coli. Open reading frames of resistance genes and genetic mobile elements are indicated in green and red, respectively. Shaded areas show the genetic context shared between the two plasmids structures.