Multidrug- and Extensively Drug-Resistant Uropathogenic Escherichia coli Clinical Strains: Phylogenetic Groups Widely Associated with Integrons Maintain High Genetic Diversity

In recent years, an increase of uropathogenic Escherichia coli (UPEC) strains with Multidrug-resistant (MDR) and Extensively Drug-resistant (XDR) profiles that complicate therapy for urinary tract infections (UTIs) has been observed and has directly impacted costs and extended hospital stays. The aim of this study was to determine MDR- and XDR-UPEC clinical strains, their virulence genes, their phylogenetic groups and to ascertain their relationship with integrons and genetic diversity. From a collection of 500 UPEC strains, 103 were selected with MDR and XDR characteristics. MDR-UPEC strains were mainly associated with phylogenetic groups D (54.87%) and B2 (39.02%) with a high percentage (≥70%) of several fimbrial genes (ecpA, fimH, csgA, and papGII), an iron uptake gene (chuA), and a toxin gene (hlyA). In addition, a moderate frequency (40–70%) of other genes (iutD, tosA, and bcsA) was observed. XDR-UPEC strains were predominantly associated with phylogenetic groups B2 (47.61%) and D (42.85%), which grouped with ≥80 virulence genes, including ecpA, fimH, csgA, papGII, iutD, and chuA. A moderate frequency (40–70%) of the tosA and hlyA genes was observed. The class 1 and 2 integrons that were identified in the MDR- and XDR-UPEC strains were associated with phylogenetic groups D, B2, and A, while the XDR-UPEC strains that were associated with phylogenetic groups B2, D, and A showed an extended-spectrum beta-lactamase (ESBL) phenotype. The modifying enzymes (aadA1, aadB, aacC, ant1, dfrA1, dfrA17, and aadA4) that were identified in the variable region of class 1 and 2 integrons from the MDR strains showed resistance to gentamycin (56.25 and 66.66%, respectively) and trimethoprim-sulfamethoxazole (84.61 and 66.66%, respectively). The MDR- and XDR-UPEC strains were distributed into seven clusters and were closely related to phylogenic groups B2 and D. The diversity analysis by PFGE showed 42.68% of clones of MDR-UPEC and no clonal association in the XDR-UPEC strains. In conclusion, phylogenetic groups including virulence genes are widely associated with two integron classes (1 and 2) in MDR- and XDR-UPEC strains.

The mechanisms that contribute to resistance in UPEC strains are as follows: (1) inactivation of hydrolytic enzymes by β-lactamases; (2) no hydrolytic enzyme inactivation by aminoglycoside acetyl transferase enzymes; (3) permeability alteration through active efflux pumps; (4) inactivation of the target site; and (5) resistance acquired by the horizontal transfer of genetic elements, such as insertion sequences, gene cassettes, integrons, and transposons (Peleg and Hooper, 2010). Integrons generally constitute an integrase gene (intI), an attachment site (attI), and a promoter (Pc) that induces the expression of any suitable integrated gene(s). Additionally, two classes of integrons (classes 1 and 2) have been identified and characterized in MDR-UPEC clinical strains (Stalder et al., 2012;Deng et al., 2015). These integrons carry several genes that encode proteins that participate in antibiotic resistance. These integrons are frequently embedded in mobile DNA elements, such as transposons and conjugative plasmids, to spread horizontally throughout bacterial populations (Stalder et al., 2012;Deng et al., 2015). However, the integrons in MDR-UPEC clinical strains and their relationship with virulence factors and clonality have been poorly studied. In recent years, an increase in UPEC strains with an extensively drug-resistant (XDR) profile complicating UTI therapy has been observed (Dehbanipour et al., 2016;Hadifar et al., 2016). The aim of this study was to identify MDR-and XDR-UPEC clinical strains and their virulence genes and phylogenetic groups and to ascertain their relationship with integrons and their genetic diversity.

Bacterial Strains
A total of 500 UPEC clinical strains were collected from 2010 to 2012 at the Hospital Infantil de México Federico Gómez (HIMFG) from pediatric patients with community-and nosocomial-acquired UTIs. The UPEC strains were selected considering an average bacterial count of ≥100,000 CFU/mL as determined by Kass-Sandford (Kass, 1957). UPEC strains associated with persistent UTIs were isolated from cultures from the same patient taken in different months. UPEC strains were cultured in trypticase soy broth (TSA), MacConkey medium (Mac), Luria-Bertani medium (LB), and Mueller-Hinton Broth (MHB) (Difco-Becton Dickinson, NJ, USA) at 37 • C for 24 h according to assay type. Additionally, UPEC strains were stored in cryovials at −70 • C with Brain Heart Infusion broth (BHI; Difco-Becton, Dickinson, NJ, USA) containing 25% glycerol (Sigma-Aldrich, St. Louis, MO, USA). Only one isolate from each infectious episode was included in the study, and isolates were considered to come from the same episode if they were collected <30 days apart. Thus, all MDR-and XDR-UPEC strains were isolated from 103 infectious episodes in 71 patients, and 8 patients experienced more than one episode.

Antimicrobial Susceptibility and Phenotypic Detection of ESBLs
The minimum inhibitory concentration (MIC) as determined by the MHB microdilution method was used to evaluate the antimicrobial susceptibility of 500 UPEC clinical strains according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2016). MDR strains were defined as having acquired no susceptibility to at least one antibiotic in three or more classes. XDR strains were defined as having non-susceptibility to at least one agent in all but two or fewer antibiotic classes (Magiorakos et al., 2012). The MIC for each antibiotic was compared to the standard values of the CLSI. The extended-spectrum beta-lactamases (ESBLs) were phenotypically detected as previously recommended by CLSI using the double-disc synergy test based on the synergistic effect between clavulanic acid (inhibitor of ESBLs) and β-lactam antibiotics (cefotaxime, CRO, CAZ, cefepime, cefpirome, and ATM). Additionally, ESBLs were detected using an individual disk that was tested with/without clavulanic acid (10 µg/mL) and by the Hodge test using Klebsiella pneumoniae ATCC 700603 (ESBL+) and E. coli ATCC 25922 (ESBL-) as control strains (CLSI, 2016).

Identification of Class 1, 2, and 3 Integrase Genes
Integrons in the MDR-and XDR-UPEC strains were detected by multiplex PCR, which amplified the conserved region of the integrase-encoded genes intI1, intI2, and intI3 using specific primers (Table S1). MDR-UPEC strains 502U1-0412 and 674U-0612 were used as positive controls for class 1 and 2 integrons, respectively.

Sequencing of the Amplified Integron Gene Cassettes
The variable regions of class 1 and 2 integrons from the MDR-UPEC clinical strains were amplified by PCR using a highfidelity Pfu polymerase of Thermo-Fisher Scientific (CA, USA) ( Table S1). The amplicons were cleaned and concentrated using the Zymo DNA Clean and Concentrator of Zymo Research (CA, USA) and subjected to next-generation sequencing on a NexSeq500 System (Illumina, CA, USA), which was performed at "Unidad de Secuenciación del Instituto Nacional de Medicina Genómica" (CDMX, Mexico). The sequences were analyzed and assembled using ClustalO, ORF Finder (Open Reading Frame Finder), and BLAST (Basic Local Alignment Search Tool) from the NCBI (National Center of Biotechnology Information) (Sievers et al., 2011;Soleimani et al., 2014).

PFGE Analysis in MDR-and XDR-UPEC Strains
A phylogenetic analysis of MDR-and XDR-UPEC clinical strains was performed using pulsed-field gel electrophoresis (PFGE) following the specific modifications of the protocols established by the "Laboratorio de Investigación en Bacteriología Intestinal, HIMFG" . Briefly, the samples were digested with 2 U of Xbal (Invitrogen, Life Technologies, Wyman Street, Waltham, MA, USA) at 37 • C for 4 h and separated by electrophoresis in a 1.2% ultrapure DNA agarose gel (Bio-Rad, Life Science Research, Hercules, CA, USA) that had been previously dissolved in 0.5X TBE. The samples were run on a CHEF Mapper system (Bio-Rad Life Science Research, Hercules, CA, USA) for 23 h at 200 V (6 V/cm) under two different linear ramped pulse times: 1-10 s and 10-40 s. The gels containing the macrorestriction products were stained for 40 min with 0.5 mg/mL ethidium bromide (Sigma-Aldrich St. Louis, MO, USA) and visualized under UV light using an Imaging System (ChemiDoc MP System R , Bio-Rad, Mexico City, Mexico). A lambda ladder PFGE marker (New England Biolabs, Hertfordshire, England, UK) was used as a molecular weight marker. PFGE patterns were analyzed using NTSYS-pc software (version 2.0, Applied Biostatistics, Inc., NY, USA) with the Sørensen-Dice similarity coefficient and a cluster analysis of the Unweighted Pair Group Method using Arithmetic averages (UPGMA) (Dice, 1945). The clonality of the UPEC strains was evaluated considering the Criteria of Tenover (Tenover et al., 1995).

Statistical Analysis
Fisher's exact test and Fisher's PLSD ANOVA were used to identify significant differences in the data at p < 0.05.
The tosA gene encoding the type 1 secretion A toxin was distributed at different percentages to phylogenetic groups A, B2, and D of MDR-and XDR-UPEC strains ( Table 1). The tosA gene from MDR-UPEC strains associated with phylogenetic group B2 was significantly different (p ≤ 0.0001) from that from XDR-UPEC strains ( Table 1). Fimbrial (ecpA, fimH, csgA, and papGII), iron uptake (chuA, iutD) and α-Hemolysin (hlyA) genes from MDR-and XDR-UPEC strains were highly distributed into phylogenetic groups B2 and D (Table 1). Additionally, the papGIII variant gene of the MDR-UPEC strains was only distributed into phylogenetic group B2 and D, while the papGI variant gene was not identified in any UPEC strain ( Table 1). The bcsA gene was distributed into phylogenetic groups as follows: 51.10% (23/45) to D, 43.75% (14/32) to B2 and 40% (2/5) to A of MDR-UPEC strains; however, XDR-UPEC strains carrying the bcsA gene were only distributed into phylogenetic group B2 ( Table 1).

Clonality, Phylogenetic Groups, Virulence Gene, and Integrons in the MDR-and XDR-UPEC Strains
The four clusters (II, III, IV, and V) grouping MDR-UPEC strains shared the presence of fimbrial genes (ecpA, fimH, csgA, and papGII) and an iron uptake gene (chuA). In clusters I, VI, and VII, a relationship between phylogenetic groups, integrons and virulence genes was not identified. In cluster II, two clonal groups associated with phylogenetic group D were identified: 557U (1-5 clones) characterized by the absence of the bcsA gene and 177U (1, 2, 4, and 5 clones) by the presence of the bcsA gene. In cluster III, two clonal groups associated with phylogenetic group B2 were identified: 532U (1 and 3 clones) characterized by the presence of the ESBL phenotype and 533U (2 and 4 clones) by the lack of the ESBL phenotype. In cluster IV, two clonal groups associated GM, Gentamicin; SXT, Trimethoprim-sulfamethoxazole; S, Sensitive; R, Resistant.

DISCUSSION
Uncomplicated UTIs are commonly treated with trimethoprim sulfamethoxazole, ciprofloxacin, and ampicillin. Carbapenems, nitrofutans, and cephalosporins of the 3rd and 4th generations are the most common antibiotics used to treat complicated UTIs (Foxman, 2010). The emergence of MDR-and XDR-UPEC strains has complicated the treatment of UTIs (Dehbanipour et al., 2016). In this study, MDR and XDR-UPEC strains from pediatric patients at HIMFG were analyzed for their phylogeny, integrons, and virulence profile. These strains were mainly associated with phylogenetic groups, B2 and D as described other studies; however, they also have been associated with FIGURE 1 | PFGE analysis of 82 MDR-UPEC strains associated with virulence genes and their mechanisms of resistance. A diversity analysis was performed using the Sørensen-Dice similarity coefficient in association with the UPGMA algorithm. Additionally, the dendrogram was evaluated using the cophenetic correlation coefficient obtained by the Mantel test, which indicated the dispersion of the data and showed a value of r = 0.8124. The seven clusters identified by PFGE are shown in different colors.
Frontiers in Microbiology | www.frontiersin.org FIGURE 2 | PFGE analysis of 21 XDR-UPEC strains associated with virulence genes and their mechanisms of resistance. The diversity analysis was performed using the Sørensen-Dice similarity coefficient in association with the UPGMA algorithm. Additionally, the dendrogram was evaluated using the cophenetic correlation coefficient obtained by the Mantel test, which indicated the dispersion of the data and showed a value of r = 0.8037. The four clusters identified by PFGE are shown in different colors.
Frontiers in Microbiology | www.frontiersin.org phylogenetic group A (Ejrnaes et al., 2011;Zhao et al., 2015). These UPEC strains could be considered community strains with predisposing factors that facilitate UTIs in immunocompromised patients, as previously reported (Derakhshandeh et al., 2015;Rodrigues et al., 2015). UPEC strains of Mexican origin are mainly associated with phylogenetic group B2 (López-Banda et al., 2014). Moreover, ESBLs-producing MDR-UPEC strains were mainly associated with phylogenetic groups B2, A, and D, whereas the XDR-UPEC strains were associated with A, B2, and D. The recently reported distribution of UPEC strains associated with these phylogenetic groups agrees with our data (Zhao et al., 2015). Commensal E. coli strains with the ability to cause community-acquired UTIs have been related to phylogenetic group B1, which was not identified in this study (Mosquito et al., 2015). Remarkably, variations in phylogenetic groups are related to geographical area, infection site, and antibiotic resistance (Mokracka et al., 2011). Several adhesins and siderophores from UPEC strains participate in the colonization and persistence of bladder cells (Hannan et al., 2012). The genes that encode adhesins (EcpA, FimH, PapG, and CsgA) and the protein related to iron uptake (siderophores) were widely distributed in MDR-and XDR-UPEC strains associated with two phylogenetic groups (B2 and D) and to a lesser extent with A. Recently, UPEC strains associated with phylogenetic group B2 and D showed a high presence of siderophores, autotransporters protease, and adhesion genes, while a low presence of genes encoding toxins was also observed (López-Banda et al., 2014;Yahiaoui et al., 2015;Zhao et al., 2015). In addition, the PapGII variant gene from MDR-and XDR-UPEC strains associated with phylogenetic groups D and B2 was the most prevalent compared to the papGI and papGIII variant genes. The papGII variant gene from UPEC strains associated with phylogenetic groups A and B2 is the most prevalent gene associated with cystitis in women and pyelonephritis in children and adults (Jantunen et al., 2000;Agarwal et al., 2013). However, four MDR-UPEC strains associated with phylogenetic group B2 and one strain associated with group D were associated with the papGIII variant gene, which correlates with data reported by other authors (Agarwal et al., 2013). The bcsA gene encoding cellulose is a component of the bacterial extracellular matrix in UPEC strains, and its expression is associated with the csg operon that codes for curli fimbriae (Saldaña et al., 2009). Our data showed the presence of the bcsA and csgA genes in MDR-UPEC strains associated with phylogenetic groups D, B2, and A. A study suggest that the expression of both structures is independent of the strain origin and participates in biofilm formation to protect against different antibiotics (Hung et al., 2013). A deletion in the tosA gene in CFT073 E. coli affected the colonization of the bladder and kidney in a murine model, indicating that TosA adhesin is required for the virulence of UPEC (Vigil et al., 2011a). The distribution of the tosA gene in MDR-and XDR-UPEC strains was associated with three main phylogenetic groups (A, B2, and D), which has been described in E. coli strains isolated from fecal matter, asymptomatic bacteriuria (ABU), cystitis, and pyelonephritis samples (Vigil et al., 2011b). Interestingly, a relationship between the presence of the tosA and hlyA genes was identified in MDR-UPEC strains associated with phylogenetic group B2, while XDR-UPEC strains were related to D and B2. These associations have been described in UPEC strains collected from patients with pyelonephritis (Vigil et al., 2011b). The tosA-positive MDR-and XDR-UPEC strains included in this study showed a wide profile of virulence genes compared with tosA-negative strains and were associated with phylogenetic groups B2 and D. The tosA gene is considered a genetic marker of UPEC strains carrying several virulence genes (Vigil et al., 2011b).
Nosocomial pathogens containing integron classes are related to resistance to different antibiotic groups; however, this association in UPEC strains is poorly studied (Stalder et al., 2012). Class 1 integrons in hospital E. coli strains are the most frequently reported, followed by class 2 integrons (Poey and Laviña, 2014). A total of 24.2% of class 1 integrons in Mexican E. coli strains from different clinical sources were associated with MDR (Acosta- Pérez et al., 2015); similar data were obtained in our study. The highest presence of class 1 integrons was associated with phylogenetic groups D and B2 in MDR-and XDR-UPEC strains; however, several studies described variable results regarding the association of integrons and phylogenetic groups (Gündogdu et al., 2011;Zeighami et al., 2015). Sequencing the variable region of class 1 and 2 integrons from MDR-and XDR-UPEC strains revealed the presence of genes encoding the antibiotic modifying enzymes aadA1, aadB, aacC, ant1, dfrA1, and dfrA17. MDR-and XDR-UPEC strains with resistance to SXT and GM antibiotics were mainly associated with class 1 and 2 integrons, as described by other authors (Solberg et al., 2006;Márquez et al., 2008;El-Najjar et al., 2010;Soleimani et al., 2014;Acosta-Pérez et al., 2015;Yahiaoui et al., 2015). Class 3 integrons are part of the soil/freshwater proteobacteria group, have a poor occurrence rate and were not identified in this study (Deng et al., 2015).
Genetic diversity has been widely used to characterize populations of UPEC strains associated with several diseases of the urinary tract (Luo et al., 2012;Acosta-Pérez et al., 2015;Morales-Espinosa et al., 2016). The recurrence of UTIs at HIMFG was associated with several clones in the MDR-UPEC strains of phylogenetic groups D and B2, which are responsible for a majority of persistent UTIs. In contrast, the primary persistence of recurrent UTIs was associated with phylogenetic group B2, whereas clones associated with phylogenetic group D mainly caused reinfections by UPEC isolates (Luo et al., 2012). The persistence of UPEC strains is related to invasion, bacterial community, and quiescence, including the expression of several virulence genes (Hannan et al., 2012). Four clusters (II, III, IV, and V), two phylogenetic groups (B2 and D), class 1 integrons, and genes encoding fimbrial adhesin and iron uptake are associated with MDR-and XDR-UPEC strains. The bcsA gene and phylogenetic group D, were only related to the 557U clonal group of cluster II and to the 118U group of cluster V. These UPEC strains are associated with catheterassociated bacteremia and are thus likely to function in biofilm formation on the catheter using curli and cellulose (Hung et al., 2013). Clonal group 424U of cluster IV was related to the presence of the iutD and hlyA genes; thus, these genes could be located on the same pathogenicity island, as has been reported for PAI-pheV. Interestingly, cluster V presenting four clonal groups was characterized by the presence of the tosA gene and virulence genes, including hlyA, bcsA, iutD, and papGIII. These data support the hypothesis that the TosA protein is a potential virulence marker in UPEC strains (Vigil et al., 2011a). Nonetheless, ESBL-producing clones associated with phylogenetic group B2 were only localized into the 532U clonal group of cluster III; likewise, the antibiotic resistance profiles showed that resistance to penicillin, beta-lactams, and cephalosporin was included in clonal group B2 (Mohajeri et al., 2014).
In conclusion, a high frequency of genes encoding virulence factors, a broad resistance profile associated with integrons (classes 1 and 2) and the ESBL phenotype are essential elements related to phylogenetic groups (B2 and D), which are distributed specifically in genetic clusters of MDR-and XDR-UPEC clinical strains. Additionally, these attributes confer to bacteria adaptive advantages to colonize, persist and facilitate UTIs caused by UPEC.