CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel

Abstract

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.

FIG. 1.

Pulsed-field gel electrophoresis of the 20 E. coli strains after SpeI digestion. Eleven distinct clones were identified. λ lane, molecular size markers of lambda ladder. Lanes 1 to 6, clone A. Lanes 7 to 8, clone C. Lanes 9 and 10, clone I. Lanes 11 and 12, clone D. Lanes 13 and 14, clone K. Lanes 15 to 20, clones F, G, H, J, B, and E, respectively.

FIG. 2.

Electrophoresis of plasmid DNA of strains belonging to clones A, C, I, D and K (panel A), and Southern blot hybridization of the plasmid DNAs using CTX-M-2 probe (panel B). Panel A, plasmid pAFF2 (85 to 90 kb), used as a molecular size marker. Lanes 1 to 6, plasmid DNA of six strains belonging to PFGE type-A (strains 1020, 1438, 1241, 1254, 1131, and 1482, respectively). Lanes 7 and 8, 9 and 10, 11 and 12, and 13 and 14, plasmid DNA from pairs of strains belonging to the same PFGE type, strains 1031 and 1227 (clone C); strains 1299 and 1313 (clone I); strains 1266 and 1292 (clone D); and strains 1356 and 1326 (clone K), respectively. The arrows indicate plasmids that are common within the same clone. Panel B, Southern blot analysis of the plasmid DNAs digested with ApaI restriction enzyme, using bla_CTX-M-2 as a probe. DNA size standards (kb) are shown on the right.

FIG. 3.

Electrophoresis of plasmid DNA of six unique PFGE types (panel A), and Southern blot analysis of the plasmid DNAs digested with ApaI, using bla_CTX-M-2 as a probe (panel B). Plasmid pAFF2 (85 to 90 kb) was used as a molecular size marker. Panel A, lanes 1 to 6, uncut plasmid DNA isolated from strains 1027 (clone B), 1204 (clone E), 1393 (clone F), 1430 (clone G), 1455 (clone H), and 1466 (clone J), respectively. Panel B, Southern blot analysis of the plasmid DNAs digested with ApaI enzyme, using bla_CTX-M-2 as a probe. DNA size standards (kb) are shown on the right.