High Prevalence of ST131 Subclades C2-H30Rx and C1-M27 Among Extended-Spectrum β-Lactamase-Producing Escherichia coli Causing Human Extraintestinal Infections in Patients From Two Hospitals of Spain and France During 2015

The present study was carried out to evaluate the prevalence of sequence type 131 (ST131) among 188 extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) collected in 2015 in Lucus Augusti University hospital (Lugo, Spain) and AP-HP Beaujon hospital (Clichy, France) with regard to other STs and to characterize, the types of ESBL produced, serotypes, virulence factor (VF)-encoding genes and the ST131 clades and subclades. ST131 was detected in 33 (39.1%) and 46 (47.9%) of the isolates in Lucus Augusti and Beaujon, respectively. The 109 remaining isolates displayed 57 other STs, the following STs being displayed by at least three isolates: ST10 (8 isolates), ST23 (3), ST38 (4), ST58 (3), ST88 (5), ST95 (4), ST167 (3), ST354 (5), ST361 (3), ST410 (6), ST648 (4), ST744 (3), and ST1615 (6). ST354, ST410, and ST1615 were significantly (P < 0.05) more frequent in Lucus Augusti (5.4%, 6.5%, and 6.5%) than in Beaujon (0% for the three STs). The new globally emerging clone ST1193 among extraintestinal clinical ESBL-EC was identified in one isolate from France and one from Spain. CTX-M-15 was the commonest ESBL detected in the two hospitals (44.6% in Lucus Augusti and 50.0% in Beaujon). CTX-M-14 was significantly (P = 0.0003) more frequent in Lucus Augusti (31.5%) than in Beaujon (10.4%), whereas CTX-M-1 (20.8 vs. 7.6%; P = 0.008) and CTX-M-27 (15.6 vs. 6.5%; P = 0.0389) were more frequent in Beaujon than in Lucus Augusti. The ST131 isolates showed a higher virulence score (mean 13.367) compared with the non-ST131 isolates (mean 7.661) (P < 0.001). Among the 79 ST131 isolates, most of them (52; 65.8%) belonged to subclade C2 (also known as subclone H30Rx) followed by those belonging to subclade C1 (cluster C1-M27: 16 isolates, 20.3%; cluster non-C1-M27: 6 isolates, 7.6%) and clade A (4 isolates; 5.1%). The C2 subclade isolates showed a higher VF-encoding gene score (mean 14.250) compared with the C1-M27 cluster isolates (mean 10.875) (P < 0.001). In conclusion, this study highlights the epidemiological differences between the ESBL-EC isolated from two hospitals of France and Spain obtain in 2015 and reports, for the first time, the presence of clone ST1193 in Spain.

The present study was carried out to evaluate the prevalence of clone ST131 among ESBL-EC collected in 2015 in Lucus Augusti University hospital (Lugo, Spain) and AP-HP Beaujon hospital (Clichy, France) with regard to the other STs and to characterize the types of ESBL produced, the serotypes, and the VF-encoding genes as well as the clades and the subclades of the ST131 isolates. This study allowed us to highlight the ESBL-EC epidemiological differences in the two hospitals and to report, for the first time, the presence of the new emerging global clone ST1193 among the ESBL-EC clinical isolates from Spain.

MATERIALS AND METHODS
In this study, 188 non-duplicate (one isolate per patient) ESBL-EC isolated in 2015 in Spain (92 from Lucus Augusti hospital in Lugo) and in France (96 from Beaujon hospital in Clichy) were characterized. They comprised 139 isolates from urine, 25 from blood, seven from bile, and 17 from various other sources.
All the P-values were calculated using the Fisher's exact test, except for the comparison of the means that was performed using the one-way ANOVA test. P < 0.05 were considered statistically significant.

Significant differences are indicated in bold.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org  The most frequent STs are indicated in bold.    (Table 3 and Table S1). < 0.001) status were higher within ST131 isolates than within non-ST131 isolates. In contrast, the prevalence of APEC (0 vs. 22%) status was higher among non-ST131 isolates (P < 0.001) ( Table 4).
The most prevalent virotypes identified in ST131 isolates were A (8 isolates), C2 (25), E (18) and F (14) and a new virotype similar to A (virotype A-like) displayed by seven isolates. Further, a second new virotype similar to E (virotype E-like) was found in one isolate ( Table S1). The virotype A was found more frequently among Lucus Augusti isolates (P = 0.0280), while virotype F was more frequent among Beaujon isolates (P = 0.0293).

DISCUSSION
The management of urinary tract and bloodstream infections due to E. coli has been complicated by the emergence of multidrugresistance, especially of that related to the expansion of high-risk clones such as ST131 (Nicolas-Chanoine et al., 2008;de Toro et al., 2017). Since 2006, the prevalence of ESBL-EC among E. coli causing bacteremia has raised in Lucus Augusti hospital. This increase has been due to the spread of the multidrug-resistant ST131 subclade C2 associated with the production of CTX-M-15. Thus, the number of ESBL-EC isolates increased from 1.0% during 2000-2005 to 5.5% during 2006-2011. While during the first period 0% of the ESBL-EC isolates belonged to subclade C2, during the second period this subclone represented 39.8% (Mamani et al., 2019). A similar situation has been reported in France in different hospitals (Brisse et al., 2012;Sauget et al., 2016) and worldwide (Peirano et al., 2012).
The main change with respect to previous studies conducted at the Lucus Augusti hospital, is the emergence of isolates producing CTX-M-27. Indeed, this enzyme was not produced by any of the 105 ESBL-EC isolates recovered from extraintestinal infections between 2006 and 2007 (Blanco et al., 2009) and by any of the 92 ESBL-EC bloodstream isolates collected from 2001 to 2011 (Mamani et al., 2019) and only by one of 47 ST131 ESBL-EC isolated from urinary tract infections in 2012 . Furthermore, the CTX-M-27 was only detected in one of the 44 Spanish hospitals analyzed in a study carried out in 2006Díaz et al. (2010, in one of 94 clinical ESBL-EC collected during 2008 in Vall d'Hebron hospital of Barcelona (Coelho et al., 2011), and in none of the 92 ESBL-EC obtained in eight Spanish hospitals during 2010 and 2011 (Merino et al., 2016). In the present study, CTX-M-27 was significantly more frequent in Beaujon (15.6%) than in Lucus Augusti (6.5%). However, the Beaujon's CTX-M-27 percentage appeared as remarkably higher than those found before 2014 (4.5-5.4%) in other 19 hospitals located as Beaujon in the Paris area (Robin et al., 2017;Surgers et al., 2019). Inversely, this percentage was closer to that found between 2014 and 2017 in 24 pediatric centers located in six French regions, i.e., 12.4% among 251 ESBL-EC isolated from febrile urinary tract infections (FUTIs) (Birgy et al., 2020). In addition, it has to be noted that CTX-M-27-producing isolates had already been found in 2012 in the feces of children in day care centers (DDCs) in France (Blanc et al., 2014) and also in feces of patients hospitalized in Madrid during a European survey conducted between 2014 and 2015 (Merino et al., 2018).
The increased prevalence of the CTX-M-27 in the two hospitals enrolled in the present study was mainly due to the expansion of cluster C1-M27 since 17 of the 21 positive CTX-M-27 isolates belonged to this cluster. The four remaining isolates belonged to three different STs, including ST38 (2 isolates), ST90 and ST1193. In the FUTI study, Birgy et al. (2020) also showed that their 31 CTX-M-27-producing isolates mostly belonged to cluster C1-M27 (10 isolates) and ST38 (8 isolates), and also that two belonged to ST1193.
The two ST1193 isolates identified in the present study belonged to clonotype CH14-64 and serotype O75:HNM and were positive for CTX-M-14 (the isolate from Spain) and CTX-M-27 (the isolate from France). As far as we know, this is the first report of the new emerging global clone ST1193 among clinical ESBL-EC isolates from Spain. Inversely, the ST1193 has already been described in France and shown producing either CTX-M-15 or CTX-M-27 in the pediatric FUTIs study (Birgy et al., 2020) and CTX-M-14 among the fecal isolates obtained from children in DCCs in France (Blanc et al., 2014). ST1193 has also been detected in two of 243 third-generation-cephalosporinresistant E. coli isolates obtained from patients with bloodstream infection in Denmark during 2015 (Roer et al., 2018), in three of 51 clinical ESBL-EC isolated in Germany during 2015 and 2016 (Valenza et al., 2019) and in 11 of 225 cefotaxime-resistant E. coli isolated from UTIs in South-West England during 2017 and 2018 (Findlay et al., 2019). ST 1193 is currently much more expanded in China (Xia et al., 2014;Wu et al., 2017) and the USA (Johnson et al., 2019;Tchesnokova et al., 2019).
Despite the emergence of ST1193 and ST131 cluster C1-M27, it is clear that ST131 subclade C2 associated to CTX-M-15 remains the most prevalent sublineage among ESBL-EC in the two hospitals studied here, as it is the case in most of the European hospitals (Merino et al., 2016;Sauget et al., 2016;Roer et al., 2018;Findlay et al., 2019;Valenza et al., 2019;Birgy et al., 2020). The C2 subclade isolates showed a higher virulence score (mean 14.250) compared with the subclade C1-M27 ST131 isolates (mean 10.875) (P < 0.001) and non-ST131 isolates (mean 7.661) (P < 0.001). Interestingly, the papAH, papC, papEF, cnf1, and hlyA genes were associated with the C2 subclade isolates, which mostly displayed the vitotypes A, A-like, C2, E and F. The virotype A-like is new and differs from the virotype A in the type of capsular kpsM II gene that is K5 instead of K2 (Dahbi et al., 2014). In future studies, it would be very interesting to determine the whole genome sequence of the C2 subclade isolates belonging to the new virotype A-like.
Of note, four isolates belonging to clones B2-CH38-15-ST95 and B2-CH38-294-ST95 were those with the highest number of VF-encoding genes (mean 21.000). These four UTI isolates were classified as APEC and could be of avian origin and foodborne pathogens (Vincent et al., 2010;Singer, 2015;Liu et al., 2018). ST95 is one of the most frequently ST identified among E. coli causing human extraintestinal infections, but it is rarely producer of ESBL enzymes (Kallonen et al., 2017). Nevertheless, recently Birgy et al. (2020) detected nine ST95 isolates among 251 ESBL-EC from pediatric FUTIs. The combination of so many virulence genes and resistance-encoding genes in this successful ST is very worrying.

CONCLUSIONS
Despite the enormous genetic diversity observed in our ESBL-EC collection (71 clones amongst 188 ESBL-EC), it can be concluded that the majority of the isolates belong to only three clonal complexes (CC10, CC23, and CC131) and that ST131 subclade C2 associated with the production of CTX-M-15 remains the most prevalent E. coli lineage among the ESBL-EC isolates identified in the studied Spanish and French hospitals.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
S-CF-S, VG, MD, NM, MA, IG-M, JEB, and MB undertook the laboratory work. M-HN-C and JB conceived the concept for the paper and designed the experiments. All authors provided critical input, contributed to the writing of the manuscript, and have approved the final version.