A Descriptive Analysis of Urinary ESBL-Producing-Escherichia coli in Cerdanya Hospital

Urinary tract infections caused by extended-spectrum β-lactamase Escherichia coli (ESBL-EC) are increasing worldwide and are a current concern because treatment options are often limited. This study investigated antimicrobial susceptibility, antimicrobial resistance genes (ARGs), and the biological diversity of urinary ESBL-EC isolates at Cerdanya Hospital, a European cross-border hospital that combines French and Spanish healthcare models. Bacterial identification and susceptibility were determined using the Microscan WalkAway® system and ESBL production was examined by the double-disk synergy method. Isolates were sequenced using the Ion S5™ next-generation sequencing system, with the whole-genome sequences then assembled using SPADEs software and analyzed using PubMLST, ResFinder, FimTyper, PlasmidFinder, and VirulenceFinder. A phylogenetic analysis was performed by constructing an assembly-based core-SNV alignment, followed by a phylogenetic tree constructed using Parsnp from the Harvest suite. All isolates studied were multidrug-resistant and could be classified into 19 different sequence types characterized by a high genetic diversity. The most prevalent ESBL-enzymes were CTX-M-14 and CTX-M-15. High-risk international clones (ST131, ST10, and ST405) were also identified. The results demonstrated the absence of a single predominant clone of ESBL-MDR-EC at Cerdanya Hospital.


Introduction
Urinary tract infections (UTIs) are among the most common human infections and can be both nosocomial and community-acquired. These infections affect individuals of all age groups and are, particularly in elderly, a cause of morbidity in out-patients as well as hospitalized patients. The so-called uropathogenic Escherichia coli (UPEC) strains are the primary etiological agents; more than 90% of these infections are community-acquired, with the rest being hospital-acquired of E. coli had reduced susceptibility to second-generation cephalosporins (i.e., cefoxitin, cefuroxime) and 3GC (i.e., cefotaxime and ceftazidime), and were screened for ESBLs. Those isolates recovered from the same patients were included in the study, given the different antimicrobial susceptibility profiles of the isolates (Table S1).

Bacterial Culture, Antimicrobial Susceptibility Testing, and ESBL Detection
Isolates were recovered by conventional microbiological methods. E. coli isolates were identified using MicroScan (MicroScan WalkAway ® , Beckman Coulter, Inc., Brea, CA, USA). Antibiotic susceptibility testing was performed by broth microdilution and interpreted according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints [16]. The isolates were considered as MDR following the criteria defined by Magiorakos et al. [17]. ESBL production was confirmed using the modified double-disk synergy test [18]. Among the 33 presumptively ESBL-producer isolates, seven did not display ESBL genes after WGSA and subsequently were removed from the study.

Whole-Genome Sequencing and Bioinformatic Analysis
Genomic DNA of the confirmed ESBL-EC isolates was extracted using the DNeasy tissue kit following the manufacturer's instructions (Qiagen, Hilden, Germany) and sent to CosmosID (Rockville, MD, USA) for WGS using the Ion S5™ next-generation sequencing system (Thermo Fischer Scientific, Waltham, MA, USA). Genomes were assembled with SPAdes [19] and genome quality was assessed using Check-M-point [20].
The phylogenetic analysis was performed by constructing an assembly-based core-SNV alignment using Parsnp from the Harvest suite [29] with default parameters, with the exception of parameter -c. Isolate 8778 was selected as the reference genome and the total number of SNPs was obtained using HarvestTools. The WGS tree was visualized using iTOL 5 (https://itol.embl.de/upload.cgi, accessed on 28 January 2022) [30].

Sequence Data Deposition
All raw reads generated were deposited in the National Centre for Biotechnology Information (NCBI) under the BioProject number PRJNA746954.

Bacterial Isolates and Antimicrobial Susceptibility
E. coli isolates in this study (n = 26) were obtained from 30 patients seen at the Hospital de Cerdanya. From two patients, two and three isolates, respectively, were included. Twenty-one isolates were obtained from women who presented community-acquired infections and cystitis as the most frequent diagnosis. Primary care and internal medicine were the most common hospital services. The remained isolates were from men treated at the emergency services, mainly with community acquired infection and diagnosed with acute bacterial prostatitis (https://uroweb.org/guideline/urological-infections, accessed on 28 January 2022) (Table S1).
Antimicrobial susceptibility testing identified all E. coli isolates as MDR. In addition to cefotaxime resistance, 14 of the isolates were resistant to ceftazidime, 21 to ciprofloxacin (cipR), and 16 to trimethoprim/sulfamethoxazole. Six isolates (30.3%) were resistant to amoxicillin/clavulanic acid, 12 to tobramycin, and nine to gentamycin. All isolates were susceptible to imipenem and to the combination of the β-lactam antibiotics piperacillin and tazobactam.
An analysis of the ARG content of the isolates showed that the β-lactam resistance gene bla CTX-M was the most prevalent, found in 21 isolates, followed by bla OXA-1 and bla SHV-12 in six and five isolates, respectively. From the CTX-M family, bla CTX-M-14 (n = 9), bla CTX-M-15 (n = 6), and bla CTX-M-3 (n = 2) were the most frequent; concerning the TEM family, bla TEM-1B (n = 12) and bla TEM-1C (n = 2) were the most common.
CipR was associated with mutations in the quinolone-resistance-determining region (QRDR) in the chromosomal gyrA and parC genes. In gyrA, S83L was the predominant mutation (n = 22). A second mutation (D87N) was detected in 19 of these isolates and only one carried the mutations S83L and D87Y. In parC, S80I was the most common mutation (n = 15), with three of these isolates also carrying a second mutation: three E84V, one E84G, one E62K, and one A56T. Among the isolates with mutations in parE, S458A was identified in eight, I529L in three, and S458T in two. The triple mutation gyrA (S83L and D87N or D87Y) and parC (S80I) was found in 15 isolates. CipR was also related to the presence of aac(6')-Ib-cr, found in seven isolates. All isolates with bla CTX-M-15 simultaneously contained the ARGs bla OXA-1 and aac(6')-Ib-cr.
Trimethoprim/sulfamethoxazole resistance was mainly associated with the combinations of drfA (n = 18) and sul (n = 19) genes. Sul1 alone was present in six isolates, sul1 plus sul2 in five, and sul1 plus sul3 in three.
Among the aminoglycosides, resistance was mainly related to the presence of the aadA5 and aph(6)-Id (n = 9, each), aadA2b (n = 7), and aac(6 )-Ib-cr (n = 7) genes. Details of the antimicrobial susceptibility tests and AGR content of all the isolates are provided in Table S1.

Plasmid Typing
PlasmidFinder analysis confirmed the presence of a large diversity of plasmid replicons in the studied isolates. Among the 18 different replicons identified, the most common were IncFIB (n = 23), IncFII (n = 17), IncFIA (n = 10), IncI1 (n = 9), IncX1 (n = 6), IncFIC, and Col (n = 5, each). Most of the isolates contained more than one replicon; 13 harbored three, while seven had four, and two more than four replicons. IncN was detected once, but IncM and IncA/C were absent. Only one isolate was negative for replicons, although it was nonetheless resistant to some antimicrobial agents.
It should be noted that all isolates producing CTX-M-15 presented FIA, FIB, and FII, and some of them presented IncX1, while those producing CTX-M-14 presented a variation in the content of IncI1, IncX1 and IncF replicons, with FIA as the less prevalent (Table S1).

Molecular Epidemiology of the Strains
According to the MLST analysis, 19 different STs were found. The most prevalent were ST131 and ST44 (n = 3, each), followed by ST10, ST88, and ST405 (n = 2, each). The other STs comprise only one isolate each. ESBL-MDR-EC isolates were assigned mainly to the phylogenetic group A (n = 9), followed by B2 (n = 6), and B1 (n = 2). The most common replicons were from the IncF family and they were present in all isolates belonging to high-risk clones (ST131, ST405, and ST10) ( Table 1 and Table S1). A high clonal diversity was determined among the isolates, which were organized in three phylogenetic groups (C1, C2, and C3) and four subgroups (C1a, C1b, C3a, and C3b) (Figure 1). A core genome (in 60%) consisting of 5,333,101 bp, including 189,993 SNPs, was determined by mapping and alignment of the isolates to the reference strain E. coli 8778 (Figure 1).
The phylogenetic group C1 included four isolates recovered between April 2016 and November 2017 and was found to have caused community-acquired UTIs. In C1, with its subgroups C1a and C1b (Figure 1), between 18 and 35,974 SNPs were detected. The C1a phylogroup included two isolates (one susceptible to tobramycin and the other resistant) recovered from the same patient. Both isolates belonged to the high-risk clone ST405, serotype O102:H6, an unknown phylogroup. Besides the fimH27 allele, they carried bla CTX-M-3 and bla TEM-1B , and were trimethoprim/sulfamethoxazole-resistant, which was conferred by both sul1 and sul2. C1a isolates were also CipR and harbored mutations in gyrA (S83L and D87N) and parC (S80I). The replicon-types identified in this ST belonged to the IncF family (Table 1). Phylogroup C1b included two isolates, one belonging to ST2003 and the other to ST80. Both isolates carried bla CTX-M-14 and were CipR, associated with mutations in parC (S80I) and gyrA (S83L), with the latter being either D87N or D87Y.
ESBL-MDR-EC phylogroup C2 included six isolates, recovered between June 2016 and December 2017, that caused both community (n = 4) and healthcare-associated (n = 2) UTIs. Isolates within this clade showed high diversity (SNP range 88-70,988). The high-risk clone ST131 was identified in three isolates, and the highly pathogenic E. coli ST95 in one. This isolate belonged to phylogroup B2 and serotype O50:H4, and carried fimH27 and bla CTX-M-14 . Moreover, it was trimethoprim/sulfamethoxazole-and ciprofloxacin-susceptible. The other isolates were ST1236 and ST117.
Isolates from ST131 belonged to the lineage B2, O25:H4 serotype; among them, two had the fimH30 allele, carried bla CTX-M-15 with bla OXA-1 , and were CipR associated with the aac(6')-Ib-cr gene and with mutations in parC (S80I and E84V), gyrA (S83L and D87N), and parE (I529L). In addition, they were related with hospital-acquired infections. The other ST131 isolate carried the fimH161 allele and bla CTX-M-14 and bla TEM-1C . Only one ST131 isolate was trimethoprim/sulfamethoxazole-resistant, via sul1 and dfrA17; CipR was associated with mutations in gyrA (S83L) and parE (S458A and I529L). EC 7987, isolated from a French patient in October 2016, showed a low genetic diversity (88 SNP) and was similar to EC 13012, isolated from a Spanish patient in September 2017. Both isolates were from patients with healthcare-associated UTIs. However, despite the similarity of their ARGs and VFs, it was not feasible to determine the mode of transmission, due to the lack of detailed epidemiological data.

Discussion
UTIs represent a clinical problem in both community and hospital settings [7]. Communityacquired UTIs are the most common infection caused by ESBL-producing E. coli and have been increasing worldwide [31], which is consistent with the findings of this study, where 92% of the isolates were related to community-acquired infections. This phenomenon might represent a high risk of transmission to hospitalized patients who can become infected or colonized by these isolates [31]. The prevalence of ESBL-MDR-EC in UTIs and the difficulties in treating these patients constitute a challenge in infectious disease. In addition, the published studies differ greatly in their patient populations and antibiotic policies. There is also a need for studies that consider both community and hospital settings.
The prevalence of ESBL-MDR-EC in UTIs diagnosed at Hospital de Cerdanya between 2016 and 2017 (around 6%) was similar to that reported by other Spanish and French hospitals [10,13].
Moreover, ESBL-EC with an MDR profile is increasing at both the hospitalized and community level [7]. Accordingly, all isolates of this work were classified as MDR, which is difficult to treat in patients and constitutes a challenge in infectious disease management.
Overall, the antimicrobial resistance of the studied isolates was observed to be high to second-and third-generation cephalosporins (mainly cefotaxime and ceftazidime), ciprofloxacin and trimethoprim/sulfamethoxazole, similar to the results reported by other authors [31,32].
Among ESBL-producers, resistance to cefotaxime was found in more isolates than resistance to ceftazidime (100% and 53.8%, respectively), in accordance with the results reported by Oteo et al., who also found more resistance to cefotaxime than to ceftazidime (66.7% and 33.6%, respectively) [33].
ARGs content obtained by WGSA showed strong agreement with the phenotype-based resistance to all tested antimicrobial agents.
CTX-M-15 and CTX-M-14 β-lactamases are the most frequently occurring ESBLs worldwide [1]. In our work, CTX-M enzymes were the most prevalent among the studied isolates. While bla CTX-M-15 is considered to be predominant, followed by bla CTX-M-14 , among our isolates, bla CTX-M-14 was more frequent than bla CTX-M-15 , in agreement with previously reported data from Spain [34] and China [35], which identified bla CTX-M-14 as the main ESBL in E. coli of adults with acquired UTIs. It may be noted that no acquired AmpC βlactamase-producing E. coli were present in our collection, in agreement with a Portuguese study [36], performed with both clinical and non-clinical isolates [37][38][39].
The global spread of E. coli isolates producing bla CTX-M-27 was recently reported [40] . While these have been found in different parts of the world such as Germany, Spain, France, Japan, and China, they were not identified among the isolates analyzed in this study [10,35,[41][42][43].
Findings of our study showed that two patients with UTIs caused by ESBL-MDR-EC recurred with ESBL-profile in the subsequent episode, in agreement with Ahn et al. [44] who found that out of 60 patients, 43 (71.7%) recurred with ESBL-producing E. coli in the first UTI recurrence episode.
The other ESBL enzyme found was SHV-12, which has been described worldwide as one of the most common SHV-type ESBL found in Enterobacteriaceae [1]. In the light of these results, we conclude that ESBLs, CTX-M-14, CTX-M-15, and SHV-12 were predominant in the Hospital de Cerdanya, similarly to what was previously described in a tertiary-care hospital in Barcelona [45].
CipR was noticed in most of the ESBL-MDR-EC. This is in agreement with other reports from India, Saudi Arabia, and Spain, which found a strong association between ESBL-producing Enterobacteriaceae from UTIs and CipR [46][47][48]. Phenotypic CipR was mainly associated with known chromosomal mutations in the gyrA (S83L, D87N) and parC (S80I) genes [47,49]. In addition to these mutations, we also evidenced the quinolone resistance determinant aac(6')-Ib-cr, which is increasingly found in ESBL-producing Enterobacteriaceae [49]. Among quinolones, ciprofloxacin is one of the most prescribed antibiotics, which might explain the linkage between CipR and carriers of aac(6']-Ib-cr and CTX-M ESBL [32].
Plasmid analysis revealed that the majority of the ESBL-MDR-EC replicons were from the large size F incompatibility group (IncF), mainly in the CTX-M-14 and CTX-M-15 ESBLs-producers. Furthermore, IncF has been previously associated with the increasing emergence and global spread of bla CTX-M-15 and aac(6']-Ib-cr [49,50].
Retrieved from the WGSA, we detected that most of the isolates contained more than one replicon in the CTX-M-producers; in addition to the IncF type, a variation of the IncI1 and IncX1 was evidenced. The presence of a great diversity of replicons was also described by Yasir et al. [47], who found IncF type replicons, IncY, and Col156.
Concerning VFs, all the ESBL-MDR-EC studied contained one or more VFs, which underlines the ability of E. coli to colonize and cause infections. Different bacterial adhesins, toxins, and iron uptakes were found. With regard to adhesins, fimH, afa (ACD), pap, and iha were detected. Extraintestinal pathogenic E. coli (ExPEC) are complex from a phylogenetic point of view, displaying a high genetic diversity. Nevertheless, a wide range of virulence factors (VF) have been described, and it should be noted that genome plasticity is considerable. Strains causing uncomplicated UTIs normally express several VFs, as we found in our work [51].
Additionally, we observed a high diversity of E. coli isolates circulating at the Cerdanya Hospital according to MLST and phylogenetic analysis, as well as the presence of the highrisk clones ST131, ST405, and ST10 [7,52]. Similar to previous reports from Tanzania [32] and Spain [53], the most common clonal complexes were ST10 and ST131, both carriers of CTX-M-14 and CTX-M-15 as the main ESBLs.
As a cross-border hospital, Hospital de Cerdanya treats both Spanish and French patients. Cerdanya is located in what has traditionally been a cattle-raising region, and recently, Day et al. reported that although these high-risk clones occur mainly in humans, ST10 has been frequently identified in isolates from cattle and poultry [9].
The epidemic clone ST131 was the most prevalent among our isolates, which included those associated with CTX-M-15 β-lactamase, fluoroquinolone resistance, and the fimH30 allele. These features, as well as an extensive virulence gene content and ARGs, were previously found in other isolates from France and Spain [7,34]. Furthermore, E. coli isolates from clone ST131 belonged to the phylogenetic group B2. Other studies have shown that E. coli ST131/CTX-M-15/B2/fimH30 is the most disseminated E. coli clonal group worldwide and that it contains an extensive antimicrobial resistance profile [36,54,55].

Conclusions
ESBL-MDR-EC were isolated from UTI patients diagnosed at Hospital de Cerdanya, a cross-border hospital serving a large geographical area and several municipalities. Among these isolates, there was no single predominant clone. Instead, the high-risk international clones ST131, ST405, and ST10 ESBL-MDR-EC were detected. The isolates of patients from Hospital de Cerdanya included E. coli ST131/CTX-M-15/B2/fimH30, one of the most widely disseminated E. coli clonal groups worldwide, which is associated with an extensive antimicrobial resistance. The most prevalent enzymes were CTX-M and TEM. Overall, our study provides further evidence of the potential for the widespread dissemination of ESBL-MDR-EC and their acquisition of ARGs circulating in the Cerdanya area.
Study limitations: Our study was based on a relatively small number of isolates and it was limited to ESBL-MDR-EC isolates of UTI. Thus, much remains to be learned about the clonal diversity of UTI-causing E. coli isolates, including those circulating in the area served by the Hospital de Cerdanya.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms10030488/s1, Table S1: Description of the isolates of this study.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by infectious disease commission at the Cerdanya Hospital (Acta comisión de infecciosa, 27 February 2020).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding authors upon request.