Detection of Clones B2-ST131-C2 and A-ST617 in Escherichia coli Producing Both CTX-M-15 and CTX-M-27 from Tunisian Community Patients

During a two-month period (2017–2018), 336 urine samples positive for Escherichia coli were collected from Tunisian patients. Of the 336 samples, 266 were collected from community patients and 70 from hospital settings. In all, 15 ESBL producers were identified (8 and 7, respectively) and assigned to 13 pulsotypes, including four ESBL-producing E. coli (ESBL-E) with E1 and E2 profiles (2 isolates each) from community patients. The two strains E1 were identified as B2-ST131 subclade C2 and the two isolates E2, A-ST617. The four strains carrying both CTX-M-15 and CTX-M-27, exhibited the multireplicon IncFII/F1A/F1B with the same formula F31:A4:B1. Two isolates with patterns E3 and E4 (Dice coefficient, 78.7%) isolated from community and hospital settings of two geographic areas were assigned to the emerging ST131 C1-M27 subclade and contained the replicon F1:A-:B20. The remaining ESBL-E divided into different sequence types/associated CTX-M: 2 ST131-C2/CTX-M-15 and ST744/CTX-M-55, ST617/CTM-15, ST2973/CTX-M-55, ST6448/CTX-M-15, ST224/CTX-M-15, ST1431/CTX-M-15, and ST38/CTX-M-27, one isolate each. Our study reports for the first time the presence in the Tunisian community of two clones of E. coli, including the virulent clone ST131-C2 harboring both CTX-M-15 and CTX-M-27, and confirms the spread of the emergent clone ST131-C1-M-27, notably in community urinary tract infections.


Introduction
Resistance to extended-spectrum cephalosporins is widespread among the Enterobacterales species and is mostly due to the dissemination of extended-spectrum ß-lactamases (ESBLs), which are becoming a major global public health concern [1,2]. Initially reported in the late 1980s, CTX-M-type ESBLs (CTX-M for CefoTaXimase from Munich) have emerged since the 2000s, and are now considered as pandemic enzymes [1,3]. Many bla CTX-M variants exist and most of them can be clustered into five groups based on sequence homologies; each cluster of CTX-M genotypes has a corresponding progenitor gene sharing homology with different environmental Kluyvera spp., from which the bla CTX-M genes originated [1,3]. In clinical isolates and by far, the most prevalent in the CTX-M-1 group is CTX-M-15. In the CTX-M-9 group, CTX-M-9 and CTX-M-14 are the most common enzymes, but recent surveillance studies showed that CTX-M-27 is emerging in different parts of the world [1,3]. The role of mobile genetic elements and horizontal gene transfer in the dissemination of the bla CTX-M genes has been well documented [4]. The bla CTX-M genes are often present on 2 of 11 conjugative plasmids belonging to the IncF incompatibility group, which may be multireplicons harboring IncFII, IncFIA and IncIB. These conjugative plasmids can also carry other resistance genes, thus playing a major role in the spread of multidrug resistance [4,5].
Escherichia coli is the predominant bacteria found in human infections, mainly in urinary tract infections (UTI). It is the most prevalent causative agent found associated with global ESBLs [3]. CTX-M-producing E. coli associated with UTI are often encountered within specific lineage belonging to the highly virulent phylogenetic group B2 of the sequence type (ST) 131 [6]. This high-risk ST131 lineage has been disseminated worldwide with sublineages classified into three clades (A, B, and C) and different C subclades [7]. Phylogenetic studies have revealed a frequent association of CTX-M-15 with the subclade C2 [8]. More recently, a C1 subclade associated with the bla CTX-M-27 gene reported as C1-M27 was detected in Japan, and rapidly emerged in different parts of the world [1,9], including North Africa [10,11].
In Tunisia, the presence of ESBL-producing E. coli (ESBL-E) has been increasingly reported in community-acquired UTI since 2013 [12,13]. In the present work, during a two-month period, urine samples were collected from patients of two private biological laboratories situated at Sfax. With the aim of comparing their possible epidemiological relationships during the same period, urine samples of patients from healthcare facilities of two distant Tunisian locations (Sfax and Tunis) were also included in the study.
In this study, 15 ESBL-E (eight in community and seven in hospital) were characterized with regard to their ESBL and plasmid contents. Their clonal epidemiological relationships were analyzed by phylogroup, pulsotype and sequence-type determination.

Prevalence of ESBL-Producing E. coli
Between December 2017 and January 2018, 266 non-redundant E. coli strains were collected from the urine samples of community patients (one isolate per patient) divided into 60 and 206 from two private laboratories, CA and CB respectively. Among them, eight isolates produced ESBL as confirmed by phenotypic and molecular characterizations (see Section 4. Materials and Methods), giving a prevalence rate of 1.7% (in CA) and 3.4% (in CB).
During the same period, 70 non-repetitive isolates of E. coli were randomly recovered among a collection of urine positive cultures from three healthcare facilities. One of them (polyclinic, H1) situated at Sfax provided samples mainly collected from patients of a urology unit. The two others (H2, regional hospital and H3, psychiatric clinic) were located in Tunis; both locations (Sfax and Tunis) are ca. 270-km away. Among them, seven ESBL producers were isolated, giving an overall prevalence rate of 10% divided into 7.5% in H1 (3 among 40 isolates), and 20% (3/15) and 6.7% (1/15) in H2 and H3, respectively. Thus, a total of 15 ESBL-producing E. coli (ESBL-E) were recovered in our study.

Analysis of β-Lactamase Content
All ESBL-E gave positive PCR amplifications with specific primers for the bla CTX-M genes. Surprisingly, four isolates contained two groups of CTX-M: CTX-M-15 (group 1) and CTX-M-27 (group 9). Five E. coli carried CTX-M-15, three CTX-M-55, and the three remaining carried CTX-M-27. Six and four isolates also exhibited OXA-1-like and TEM-1-like ß-lactamases, respectively, including one that carried both enzymes. No other ßlactamases genes screened (plasmid-mediated cephalosporinase and carbapenemase genes) were identified.

Antibiotic Resistance Pattern
The double-disk synergy test was positive for the 15 isolates. All CTX-M-producing E. coli remained susceptible to carbapenems and only one strain (ID# 67HR) exhibited a resistance to cefoxitin (cephamycin), which could be attributed to an outer membrane impermeability or to overexpression of the chromosomal cephalosporinase. Cefotaxime MICs values were high (≥1024 µg/mL) except for two isolates (ID# 17MA and 151) with MICs of 256 µg/mL. Ceftazidime MICs ranged from 4 to 64 µg/mL ( Table 1). The ESBL-E also exhibited resistances to trimethoprim and cotrimoxazole (100%), fluoroquinolones (87%) and to aminoglycosides as follows: gentamicin, tobramycin and amikacin, 33%, 47% and 27%, respectively. All remained susceptible to fosfomycin and colistin (≤2 µg/mL), and two isolates (ID# 917 and 46SR) were resistant to nitrofurantoin (Table 1, Figure 2 and Table S1 in Supplementary Material).

Discussion
In Tunisia, investigations of ESBL-producing bacteria in community-infected patients are rare. In our study, the rate of ESBL-producing E. coli among UTI in the community reached 3.4%, and it is consistent with recent data [13], confirming that the proportion of such strains has increased in this country since 2013 [12]. Higher prevalence rates were found in healthcare facilities. The clinical data associated with the acquisition of these bacteria were not collected in this study, but previous studies reported risk factors including length of hospitalization, presence of an indwelling urinary catheter, prior exposure to antibiotics and underlying illness [16]. According to the Laboratory of Antibio-Resistance

Discussion
In Tunisia, investigations of ESBL-producing bacteria in community-infected patients are rare. In our study, the rate of ESBL-producing E. coli among UTI in the community reached 3.4%, and it is consistent with recent data [13], confirming that the proportion of such strains has increased in this country since 2013 [12]. Higher prevalence rates were found in healthcare facilities. The clinical data associated with the acquisition of these bacteria were not collected in this study, but previous studies reported risk factors including length of hospitalization, presence of an indwelling urinary catheter, prior exposure to antibiotics and underlying illness [16]. According to the Laboratory of Antibio-Resistance in Tunisia (LART), which is a national hospital surveillance system, the global rate of E. coli resistance to broad-spectrum cephalosporins with a positive synergy test was 17.4 % in 2017 (10.7 to 30.7%, depending on the hospital) [17].
The main finding of our work is the detection of two clones of E. coli producing concomitantly two groups of CTX-M. In the literature, very few studies mentioned the presence of such strains, and only with a low proportion in the ST131 lineage [18][19][20] and, to our knowledge, there is to date no report from Tunisia. The virulent ST131-C2 E. coli is a predominant lineage among extraintestinal pathogenic E. coli (ExPEC), which has played a major role in the worldwide dissemination of CTX-M-15 [6]. This pandemic clone has been reported in human beings but also from non-human sources including animals, the food chain and the environment in Tunisia [11,[21][22][23], as well as in Algeria, a neighboring country [24]. E. coli A-ST617 related to the clonal complex 10 (Cplx10) is less often encountered among the CTX-M-15-producing non-ST131 isolates. However, it has emerged in Africa (Nigeria) since the beginning of the 2010s [25]. It has also been found in 2015 from a colonized patient newly admitted in a hospital of Tunis [23]. A systematic review conducted among ExPEC lineages reported proportions for ST617 higher from Africa than studies from Asia and Europe [26]. To the best of our knowledge, we report for the first time in Tunisia, the presence and the spread among four community-infected patients of two multidrug-resistant E. coli clones of pulsotypes E1 (ST131-C2) and E2 (A-ST617), carrying not only CTX-M-15 but also the additional CTX-M-27. In our study, three other isolates with only CTX-M-15 belonging to lineages ST131-C2 and A-ST617 were detected in hospital, but their pulsotypes showed no recent clonal relationship with the clones E1 and E2.
We also describe the presence of subclade ST131-C1-M-27 among Tunisian infected patients (from community and hospital). This clone was described in 2013-2015 in wastewater in Tunisia [11]. Recently, it has been reported with a rate of intestinal carriage of 9% in Tunisian food handlers, and probably with a similar rate among the general population [10]. This presence in both hospital and non-hospital settings, and in different regions, clearly shows the increasing dissemination of this clone in this country, but it remains little reported elsewhere in Africa.
With regard to the remaining lineages of ESBL-E detected, this study showed a diversity of ESBL and ST. Firstly, D-ST38 (Cplx38) is a widely distributed lineage in association with bla CTX-M-15 and bla CTX-M-14 [27]. However, the presence of bla CTX-M-27 in ST38, as reported in our survey, is less frequent. This association was described in the United States [28], and also in Tunisian wastewater [29] and other African countries [30]. CTX-M-15 is the predominant ESBL described in Tunisia, as also reported in two other phylogroup B1 clones, ST224 and ST1431. E. coli strains belonging to B1-ST224/CTX-M-15 are mainly identified in animals [31], but also from clinical isolates of a tertiary hospital in Tanzania [32]. ST1431/CTX-M-15 was described from wild boar in Algeria [33]. In our study, three ST: i.e., A-ST744 (ST10 Cplx), A-ST2973 and B1-ST6448, were associated with the CTX-M-55 variant. This gene first detected in Thailand in 2005 [34] has successfully disseminated in China and worldwide [1,35]. E. coli ST744 harboring the bla CTX-M-1 group has been sporadically described in human beings, but is more commonly found in animal sources in various parts of the world [36], and has also been reported in association with a colistin resistance gene in a French veterinary study [36]. A-ST2973/CTX-M-55 and B1-ST6448/CTX-M-55 has been sporadically isolated in Tunisia from river water and wastewater [29,37].
Most of ESBL-E found in our study were resistant to different antibiotic families. In the literature, ESBL enzymes are often identified in multidrug-resistant strains [1]. A linkage between the clone ST131-C and quinolone resistance has also been well documented [6,7]. The CTX-M-15 and CTX-M-27 are ESBL variants that contain a residue change in position 240 (Asp to Gly) conferring higher level resistance to ceftazidime [38]. Although ceftazidime MICs did not show higher values in the two clones with both enzymes compared to other strains with a single ESBL, their concomitant presence may have given them a selective advantage and favored their selection and dissemination under antibiotic pressure.
The bla CTX-M genes are largely mediated by conjugative plasmids in Enterobacterales [5,39]. Plasmids, especially those belonging to the incompatibility group IncFII, additionally harboring FIA/FIB replicons, have been described as contributing to the successful spread of some pandemic clones, including the major CTX-M-15-producing E. coli ST131 [8,40]. In our study, the lineages B2-ST131-C2 and A-ST167 harboring bla CTX-M-15 and bla CTX-M-27 genes contained a multireplicon IncFII/FIA/FIB with the same combination (FII:FIA:FIB), i.e., F31:A4:B1, suggesting a possible horizontal transfer. In the literature, this F31:A4:B1 replicon carrying only the bla CTX-M-15 gene has been identified in ST131-C2, and also ST617 isolates in different countries worldwide, including Tunisia [15,41]. Since no other common replicons were found in these two clones, we hypothesize the possible additional acquisition of the bla CTX-M-27 gene in this replicon. Investigations are needed to characterize it further and to verify whether the acquisition of the bla CTX-M-27 gene is due to independent events. The two E. coli ST131 C1-M27 contained the replicon F1:A-B20, and in the literature the F1:A2:B20 plasmid is often associated with the C1-M-27 clade isolates [42], but the replicon F1:A-B20 without FIA is poorly documented. However, its presence in strains found in different geographical areas (Tunis and Sfax) could suggest that it is well established in Tunisia.

Strains Collection and Identification of E. coli
Over the study period, a total of 646 urine samples were recovered from community patients from two private biological Laboratories in Sfax (Laboratories CA and CB). During the same period, of the 846 urine positive cultures collected from patients of three healthcare facilities, 200 were randomly selected. The samples originated from a polyclinic located in Sfax (named H1) and from two hospitals in Tunis (H2 and H3). The identification of E. coli was firstly carried out by using the API ® 10S strip method (BioMerieux) and confirmed by using the Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight/Mass Spectrometry (MALDI-TOF/MS, Brüker Daltonics), as previously described [43].

Molecular Typing of ESBL-Producing E. coli
The clonal relationship among ESBL-E was investigated after XbaI enzyme digestion and pulsed-field gel electrophoresis (PFGE) analysis using the CHEF DRII ® apparatus (Bio-Rad), as detailed elsewhere [47]. The PFGE patterns were first compared after visual inspection, then a dendrogram was constructed using the DICE similarity coefficient by the Antibiotics 2022, 11, 1329 8 of 11 UPGMA method (1% tolerance limit and 1% optimization) performed with the software package Bionumerics ® 6.3 (Applied Maths).

Plasmid Analysis
The identification of group incompatibility plasmids were subjected to PCR-based replicon typing (PBRT) method [51]. The presence of IncFII, IncFIA, IncFIB were characterized using the PCR amplification procedure of Villa et al. [15], and after sequencing, the IncF replicon typing scheme was assigned to the FAB formula by using the web tool described by Carattoli et al. [39].

Conclusions
In this study, we identified, for the first time, in Tunisia and in community-acquired infections, the presence of both CTX-M-15 and M-27 in at least two clones of E. coli including the virulent clone, ST131-C2. The two clones share a common plasmid F31:A4:B1, which may have contributed to the spread of these two ESBLs which are known to confer high level resistances to extended-spectrum cephalosporins. This study also reported the presence of subclade ST131-C1-M-27 among Tunisian community-infected patients and confirmed its implantation in the country. Overall, our results highlight the need for a sustained monitoring and reinforcement of hygiene measures to prevent human transmission and the spread of multi-drug resistant bacteria not only in hospital but also in the community setting in Tunisia.