Molecular characterization of plasmids encoding CTX-M-15 extended-spectrum -lactamase associated with the ST131 Escherichia coli clone in Belgium

Sir, Previous studies performed in a large number of Belgian hospitals showed the dissemination of a major clone of the virulent B2 group O25b:H4-ST131 CTX-M-15-producing Escherichia coli.1 This E. coli ST131 clone has been reported worldwide and represents a major public health problem.2 The present study sought to characterize blaCTX-M-15-containing plasmids associated with ST131 E. coli CTX-M-15 isolates recovered in Belgian hospitals. This specific clone was detected from clinical specimens of patients hospitalized at the Erasme hospital in Brussels since 2001, as well as in 18 other Belgian hospitals during a nationwide survey conducted in 2006.1 Nine representative CTX-M-15 E. coli isolates, recovered in 2006 from specimens (seven urine specimens, one pus specimen and one lower respiratory tract specimen) of inpatients and outpatients at eight hospitals located in seven different cities, all belonging to the ST131 clone, were selected on the basis of their antibiotic phenotypic resistance profile (variable level of resistance to cefotaxime, ceftazidime and cefepime from 2 to >256 mg/L), as well as on the presence or absence of the insertion sequence ISEcp1 upstream of the blaCTX-M-15 gene (Table 1). Plasmids were extracted and transformed into E. coli TOPO10 electrocompetent cells (Invitrogen, Merelbeke, Belgium). Conjugation experiments were not performed. MICs for clinical isolates and transformants were determined by Etest, and the presence of an extended-spectrum β-lactamase was confirmed by a double combination disc test, by PCR with primers targeting the blaCTX-M group 1 gene and by DNA sequencing. The genetic environment of blaCTX-M-15, other antibiotic resistance genes and virulence genes was analysed by PCR mapping and sequencing targeting Orf477, ISEcp1, IS26, repA, tnpR, tnpA, tnpM, int1I, blaTEM-1, blaOXA-1, aadA5, dhfrVII, mphA, sul1, aac6′-Ib-cr, catB4, aac3-IIa, tet(A), parM-stbB and vagC-vagD. Molecular characteristics of plasmids analysed AMP, ampicillin; AMC, amoxicillin/clavulanate; TZP, piperacillin/tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; SXT, trimethoprim/sulfamethoxazole; TOB, tobramycin; GEN, gentamicin; ND, non-detectable. aAccording to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The presence of ISEcp1 upstream of blaCTX-M-15, blaTEM-1 and blaOXA-1 confers resistance to β-lactam agents; aac6′-Ib-cr confers resistance to aminoglycosides and ciprofloxacin; catB4 confers resistance to chloramphenicol; aac3-IIa confers resistance to gentamicin; tet(A) confers resistance to tetracycline; parM-stbB encode plasmid segregation-stable plasmid inheritance proteins; vagC-vagD are virulence-associated genes; and ccdA-ccdB encode a toxin–antitoxin system. Molecular characteristics of plasmids analysed AMP, ampicillin; AMC, amoxicillin/clavulanate; TZP, piperacillin/tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; SXT, trimethoprim/sulfamethoxazole; TOB, tobramycin; GEN, gentamicin; ND, non-detectable. aAccording to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The presence of ISEcp1 upstream of blaCTX-M-15, blaTEM-1 and blaOXA-1 confers resistance to β-lactam agents; aac6′-Ib-cr confers resistance to aminoglycosides and ciprofloxacin; catB4 confers resistance to chloramphenicol; aac3-IIa confers resistance to gentamicin; tet(A) confers resistance to tetracycline; parM-stbB encode plasmid segregation-stable plasmid inheritance proteins; vagC-vagD are virulence-associated genes; and ccdA-ccdB encode a toxin–antitoxin system. Overall, we observed two groups of plasmids harbouring blaCTX-M-15 in the same genetic lineage. The first group included two strains with plasmids showing a 7.4 kb region upstream of blaCTX-M-15 with 100% sequence identity to pEK499. pEK499 is a plasmid of 117.5 kb that belongs to incompatibility groups IncFII and IncFIA. It harbours resistance-associated genes that confer resistance to eight antibiotic classes, including blaTEM and blaOXA-1 (β-lactam drugs), tet(A) (tetracycline), aac6′-Ib-cr (aminoglycosides and ciprofloxacin), catB4 (chloramphenicol), mphA (macrolides), dhfrVII (trimethoprim), aadA5 (streptomycin), and sulI (sulphonamide). Moreover, it encodes the virulence-associated genes vagC-vagD and the toxin–antitoxin system ccdA-ccdB located in the region of the FIA replicon.3 All of these pEK499-associated genes were present on the two plasmids included in the first group. As reported in other studies,4 the insertion sequence IS26 was also detected upstream of blaCTX-M-15, instead of the usual ISEcp1, resulting in a reduced level of resistance to broad-spectrum cephalosporins in both clinical isolates and in the transformed cells (cefotaxime MIC of 2 mg/L, cefepime MIC of 1–2 mg/L and ceftazidime MIC of 1.5 mg/L). The second group included seven E. coli isolates showing heterogeneous plasmid structures. All presented a common 5 kb region upstream of blaCTX-M-15 with 100% sequence identity to plasmids pEK516 and pC15-1a, but showed differences in other genes or regions compared with these two well-defined reference plasmids (Table 1). The pEK516 plasmid (64 kb) is a highly related variant of the pC15-1a plasmid (92 kb) and both belong to incompatibility group FII.3,5 Other differences were observed compared with pEK516 and pC15-1a reference plasmids: (i) in all but one of the group 2 plasmids the aac3-IIa gene encoding gentamicin resistance was absent; (ii) all lacked the region containing parM and stbB, which play a role in plasmid stability; and (iii) group 2 plasmids did not share the same incompatibility group as for pEK516 and pC15-1a. These features combined with the detection in five plasmids of genes present in the FIA region of pEK499 could suggest either new plasmid structures or variants of previously described plasmids. Extensive recombination events among plasmids belonging to incompatibility group F are frequent and may contribute to plasmid diversity, evolution and persistence, and are already suggested to be involved in the mosaicisms observed in CTX-M-15-bearing plasmids.2,6 In summary, we report for the first time the different structures and diversity of plasmids carrying blaCTX-M-15 in E. coli isolates belonging to the international ST131 lineage that is now widely disseminated in Belgian hospitals. The intrinsic features of this lineage and those of the acquired plasmids harbouring multiple antimicrobial drug resistance determinants may contribute to the epidemiological success of this clone in Belgium, as described in other countries.2,3,5 Further studies have to be conducted in order to gain a deeper insight into plasmid and bacterial behaviour, and thereby to be able to develop effective strategies to control their spread. This work was supported by the Fonds de la Recherche Scientifique Belge (no. 3.4.522.09F). I. M. benefited from research funding from the Fundación Canaria Dr. Manuel Morales. None to declare.