Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains. Methods ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla CTX-M-94 and bla CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants. Results The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla CTX-M-94 and bla CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons. Conclusions This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.


Introduction
CTX-Ms are the most prevalent extended-spectrum b-lactamases (ESBLs) in Enterobacteriaceae causing hospital-and community-acquired infections [1][2][3]. The bla CTX-M genes are usually located on plasmids, and are derivatives of chromosomal b-lactamase genes of the Kluyvera genus due to multiple mobilization events [1,4]. Usually, these plasmids easily spread in microbial populations, also carrying other resistance genes such as those coding for aminoglycoside acetyltransferases, dihydropteroate synthases or other b-lactamases [3]. New CTX-M variants arise rapidly and so far 131 different enzymes of this family have been identified (http://www.lahey.org/studies). They can all be assigned to five different subfamilies, based on amino acid identity: the CTX-M-1, -2, -8, -9, and -25 groups [1]. New CTX-M variants within these groups emerge by the gradual accumulation of mutations, some of which affect enzyme activity and resistance phenotype, and are being selected by antibiotic pressure [5,6]. Unlike the CTX-M-1, -2 and -9 groups, the CTX-M-25-like blactamases have been rarely observed worldwide [7][8][9]. However, the situation in Israel is in stark contrast as a remarkable number of CTX-M-25-group enzymes, namely CTX-M-25, -26, 39 and -41, have been previously reported in there [10,11]. We recently identified two new CTX-M-25-group variants, CTX-M-94 and -100, in Escherichia coli isolates from patients admitted at the Tel Aviv Sourasky Medical Center, Israel. The aim of this study was to characterize CTX-M-94 and -100 and to understand the phylogenetic relationships between the novel and the known CTX-M-25-group variants.

Bacterial Strains
Two E. coli clinical strains were recovered in 2008-2009 during an epidemiological study from screening stool samples of two elderly patients hospitalized at the Tel Aviv Sourasky Medical Center, Israel. These isolates were identified using mass spectrometry (MALDI-TOF, Bruker Daltonics, Bremen, Germany). E. coli TOP10 electrocompetent cells (Invitrogen, Carlsbad, CA, USA) were utilized as hosts for cloning and site-directed mutagenesis experiments, and rifampicin-resistant E. coli A15 was used as a recipient in mating tests [12].

Genotypic ESBL Detection and bla CTX-M Characterization
Total DNA was extracted from the isolates by alkaline lysis as described previously [15]. Plasmid DNA was obtained from the isolates with the PureLink HiPure Plasmid Miniprep Kit according to the manufacturer's protocol (Invitrogen). b-lactamase genes bla CTX-M , bla SHV , and bla TEM were detected by PCR using universal primers with modified cycling conditions: 10 min at 95uC, 30 cycles of 30s at 95uC, 30s at 59uC, and 1 min at 72uC, and 10 min at 72uC [16][17][18]. The entire bla CTX-M-94 and bla CTX-M-100 genes were amplified using previously described primers CTX-M-25-F (specific for the ISEcp1 element) and CTX-M-25-R primers with modified cycling conditions: 10 min at 95uC, 30 cycles of 30 s at 95uC, 30 s at 55uC, and 1.5 min at 72uC, and 10 min at 72uC [10]. The amplicons were sequenced directly on both strands, compared to known bla CTX-M sequences (GenBank and Lahey Clinic), and analyzed using BLASTn (NCBI, http:// www.ncbi.nlm.nih.gov) and the Lasergene software (DNASTAR, Madison, WI, USA). The extrachromosomal location of bla CTX-M-94 and bla CTX-M-100 was confirmed by amplification from plasmid DNA in triplicate, as well as by plasmid electroporation into E. coli TOP10.

AmpC Detection
Production of AmpC-like b-lactamases was assessed by the combination disk test with cefotaxime and ceftazidime disks, with and without phenylboronic acid (20 ml of 20 mg/mL solution per disk). A$5 mm increase in zone diameter after addition of boronate indicated the higher-level expression of AmpC. Genes coding for acquired AmpC types were identified by PCRsequencing analysis on plasmid DNA with target-specific primer pairs [19].

Detection of Enzymatic Activity and Isoelectric Focusing of b-lactamases
The presence of functional b-lactamases was evidenced by a qualitative method utilizing the chromogenic cephalosporin nitrocefin (Calbiochem, Merck Chemicals, Nottingham, UK). Hydrolysis of nitrocefin by b-lactamases results in a distinct colour shift from yellow (l max = 390 nm at pH 7.0) to red (l max = 486 nm at pH 7.0). A nitrocefin solution (500 mg/L) was added to crude bacterial sonicates to show presence of functional b-lactamases. Sonicates positive for b-lactamase activity were subjected to isoelectric focusing (IEF) as described previously [12], in a Model 111 Mini IEF Cell (Bio-Rad, Hercules, CA, USA) The IEF gel was homogeneously covered with the nitrocefin solution to mark all bands corresponding to the different b-lactamases present in the bacterial cell extracts.

Cloning of bla CTX-M genes
Blunt-ended amplicons consisting of bla CTX-M genes and ,30 bp upstream regions were produced using Platinum Pfx DNA Polymerase (Invitrogen) and the primer pair CTX-M-25-F/ R with modified cycling conditions: 10 min at 94uC, 30 cycles of 15 s at 94uC, 30 s at 55uC, and 1.5 min at 68uC, and 10 min at 68uC. PCR products were cloned into the pCR-BluntII-TOPO vector (Invitrogen), electrotransformed into E. coli TOP10, and selected on LB agar supplemented with kanamycin (50 mg/L) and cefotaxime (2 mg/L). Transformants (pCR-CTX-M-94 and -100) were screened for presence of the bla CTX-M genes by PCR and sequencing using M13 primers according to the manufacturer's protocol.

Site-directed Mutagenesis
Single point mutations were introduced into pCR-CTX-M-94 and -100 with the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. Mutagenic primer pairs (available on request) were designed to replace alanine at position 77 with valine (A77V) and glycine at position 240 with aspartic acid (G240D). The mutagenized pCR-CTX-M-94 and -100 plasmids were electrotransformed into E. coli TOP10 and their susceptibility profiles to b-lactams were screened by Etest.

Plasmid Typing
Total plasmid DNA obtained from the clinical isolates was electroporated into E. coli TOP10 to separate coexisting multiple plasmids. Unique plasmids were identified by screening the transformants for presence of bla genes, including bla CTX-M , bla TEM and bla CMY-2 , by PCR. Plasmid profiles of the transformants carrying unique plasmids were determined by restriction digestion of their plasmid DNA with HpaI (New England Biolabs, Ipswich, MA, USA). Restriction products were separated along with a 1 Kb DNA Extension Ladder (Invitrogen) on a 0.7% agarose gel for 10 h at 2.5 V/cm with 0.5x Tris-borate-EDTA buffer. Plasmid sizes were estimated by relative mobility calculations. Presence of bla genes was confirmed by hybridization with bla CTX-M-25-group-, bla TEM -and bla CMY-2 -specific DIG-labeled probes and a DIG Luminescent Detection Kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's protocols. Replicon types of all unique plasmids were identified by PCR-based replicon typing as described previously [22]. Conjugation was carried out as described previously [12], utilizing the clinical strains producing CTX-M-94 and -100 as donors and rifampicin-resistant E. coli A15 strain as a recipient. Transconjugants were selected on LB agar with cefotaxime (2 mg/L) and rifampicin (128 mg/L), and confirmed by the phenotypic disk tests, PCR of bla genes, and MLST against the donors and recipient. Transfer efficiencies were calculated based on the ratio of CFUs/mL of transconjugants per donor cell.

Nucleotide Sequence Accession Numbers
bla CTX-M-94 and bla CTX-M-100 nucleotide sequences were assigned GenBank accession numbers HM167760 and FR682582, respectively.

E. coli Isolates Harbouring Novel CTX-M Variants
We recovered the CTX-M-94 and -100-producing E. coli strains from screening stool samples of two elderly hospitalized patients. The patients received various antibiotics, including cephalosporins (ceftriaxone), b-lactam/inhibitor combinations (piperacillin/tazobactam), cephamycins and carbapenems (imipenem). The CTX-M-94-producing E. coli belonged to ST131 and phylogroup B2. ST131 is a uropathogenic clone that has spread worldwide, usually associated with ESBLs, in particular with CTX-M-15 of the CTX-M-1 group [3,23,24]. Along with CTX-M-94, the E. coli isolate also harboured two other acquired b-lactamases, TEM-1 and the AmpC-like cephalosporinase CMY-2. The CTX-M-100-producing E. coli belonged to ST88 (clonal complex ST23) and phylogroup A. Strains belonging to phylogroup A are generally more common in the intestinal flora, and both the complex ST23 and the clone ST88 belong to the globally spread members of the phylogroup [25]. The CTX-M-100-harbouring isolate did not express any other acquired b-lactamase.
MICs of b-lactams for the CTX-M-94-and -100-producing isolates are shown in Table 1. The CTX-M-94 harbouring isolate exhibited four-to approximately 200-fold higher MICs of amoxicillin/clavulanate, cefoxitin, cefotaxime, ceftriaxone, ceftazidime (alone and with clavulanate), cefepime and aztreonam, when compared to the CTX-M-100 producer. According to the current CLSI guidelines [14], both isolates were highly resistant to amoxicillin, ampicillin, piperacillin, cefotaxime and ceftriaxone, and susceptible to piperacillin/tazobactam, cefepime and imipenem. The resistance pattern of the CTX-M-100-producing isolate was similar to that of the previously described CTX-M-39producing E. coli from the same hospital [11], and matched well the typical CTX-M-associated phenotype, with asymmetry in MICs of cefotaxime and ceftazidime and good activity of inhibitor combinations [1,5,26]. In contrast, resistance of the CTX-M-94 producer was largely influenced by CMY-2, both in level and type. AmpC b-lactamases like CMY-2 are known to hydrolyze cephamycins and are much less inhibited by class A enzyme inhibitors compared to ESBLs [27], as exemplified by high MICs of cefoxitin and clavulanate combinations, respectively (Table 1). Furthermore, AmpC b-lactamases are also capable of hydrolyzing oxyiminocephalosporins and monobactams, as exemplified by elevated MICs of cefotaxime, ceftazidime, ceftriaxone and aztreonam (Table 1) Other differences in resistance could be attributed to molecular differences between CTX-M-94 and -100 enzymes, which are discussed in the following sections.

Functional Characterization of Wild-type and Mutagenized CTX-Ms
To determine the resistance patterns conferred by the new CTX-M variants and the specific role of the F119L substitution, bla CTX-M-94 and bla CTX-M-100 with upstream regions were cloned in pCR-BluntII-TOPO and expressed in the isogenic background of E. coli TOP10 ( Table 1). The analysis revealed high-level resistance towards amoxicillin, ampicillin, piperacillin, cefotaxime and ceftriaxone for both CTX-M-94 and -100 producers without significant differences in MIC values, except for a four-fold lower ceftriaxone MIC in the CTX-M-100 producer ( mobility of the B3 b-strand is increased, resulting in enhanced protein flexibility and facilitation of hydrolysis of b-lactams with bulkier side chains. However, this increased mobility is not only associated with increased activity, but also with decreased stability of the CTX-M enzyme [5,29]. A recent in vitro-evolution study demonstrated that cefotaxime and ceftazidime probably acted as diversifying agents in the evolution of CTX-M-1 group enzymes [6].   results, Novais et al. observed a minor decrease in cefoxitin MICs and a two-fold increase in MICs of cefotaxime and ceftazidime after introducing A77V into CTX-M-15 [6]. These conflicting outcomes might be explained by differences between CTX-M-1and -25-group enzymes in amino acid residues that line the highly conserved active site. CTX-M-15, a prototypal CTX-M-1 group enzyme, differs from CTX-M-94 and -100 at positions 103 (valine and isoleucine, respectively) and 133 (valine and threonine, respectively) that lie in the a structural domain of the CTX-M enzyme and in close proximity to the V loop (based on the active site structure described in Delmas et al. [30]. Finally, the A77V substitution caused a two-fold decrease (MICs, 48 vs. 12 mg/L) and a three-fold increase (MICs, 12 vs. 32 mg/L) in ceftriaxone MICs in CTX-M-94 and -100, respectively.

Plasmid Typing
Replicon typing and HpaI plasmid profiling (Figure 3) revealed that bla CTX-M-94 was located on a ,93 kb IncFI plasmid and bla CTX-M-100 on a ,130 kb IncA/C plasmid. The bla CMY-2 gene, which was also present in the CTX-M-94-producing isolate, was located on a second ,98 kb IncI1 plasmid. The bla CTX-M-100harbouring plasmid was transferred to recipient cells with a high efficiency (5.4610 22 per donor cell). On the other hand, the bla CTX-M-94 -carrying plasmid did not transfer. Instead, the bla CMY-2 -harbouring plasmid was transferred with an efficiency of 3.8610 22 per donor cell.

Genetic Environment of bla CTX-M Genes
Both bla CTX-M-94 and bla CTX-M-100 were located 36 bp downstream of ISEcp1, the most probable factor of mobilization of these genes from the K. georgiana chromosome [31], and sequences separating the genes from ISEcp1 were identical to each other. Similar distances were previously reported for bla CTX-M-25 and bla CTX-M-26 identified in Canada and the UK, respectively [8]. Interestingly, other bla CTX-M-25 -like genes identified in Israel were found to be located 126-128 bp downstream of ISEcp1 [11]. These findings suggest that CTX-M-94 and -100 might not have emerged from other CTX-M-25-group members identified in Israel so far, but from an independent ISEcp1-mediated mobilization of a precursor K. georgiana gene.
This assumption seems to be further supported by the lack of the direct association of bla CTX-M-94 and bla CTX-M-100 with a class 1 integron, demonstrated by a PCR-sequencing based class 1 integron analysis as described previously [11]. Unlike the above mentioned bla CTX-M-25 -like genes identified in Israel, which were inserted into a class 1 integron [11], the ISEcp1-bla CTX-M-94 and ISEcp1-bla CTX-M-100 transposition modules were not present in the variable regions of the integrons identified in the bla CTX-M-94 -and bla CTX-M-100 -containing plasmids. These integrons significantly differed from each other. The one present on the plasmid with bla CTX-M-94 harboured aacC1 (aminoglycoside acetyltransferase; resistance to gentamicin), orfX (hypothetical protein), orfQ (hypothetical protein) and aadA1 (aminoglycoside adenylyltransferase; resistance to spectinomycin and streptomycin) gene cassettes, while the one on the plasmid with bla CTX-M-100 harboured aadB (aminoglycoside adenylyltransferase; resistance to gentamicin/ kanamycin/tobramycin), ereA (erythromycin esterase type 1; resistance to erythromycin) and aadA1. For now, it is unclear whether CTX-M-94 and -100 have emerged from each other or from other CTX-M-25-group members by independent mutational events. Because of limited publicly available sequence data, it is not possible to make more global assumptions on the evolutionary paths of the CTX-M-25-group enzymes.

Conclusions
This is the first report describing two novel ESBL variants of the CTX-M-25-group, CTX-M-94 and -100, identified in two E. coli isolates belonging to different clones, ST131 and ST88, respectively. Both bla CTX-M-94 and bla CTX-M-100 were found within ISEcp1 transposition units, located on ,93 kb nonconjugative IncFI and ,130 kb conjugative IncA/C plasmids, respectively. CTX-M-94 is the first CTX-M-25-group member carrying leucine-119, which was shown here to be associated with increased resistance to ceftriaxone. We have also confirmed the role of the D240G substitution in the increase of enzyme activity against ceftazidime in the context of the CTX-M-25type enzymes.