Characterization of two new CTX-M-25-group extended-spectrum β-lactamase variants identified in Escherichia coli isolates from Israel.
OBJECTIVE
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.
Department of Medical Microbiology, Universiteit Antwerpen, Antwerp, Belgium.
Jascha
J
Vervoort
Anna
A
Baraniak
Muriel
M
Gazin
Julia
J
Sabirova
Christine
C
Lammens
Meital
M
Kazma
Anna
A
Grabowska
Radosław
R
Izdebski
Yehuda
Y
Carmeli
Samir
S
Kumar-Singh
Marek
M
Gniadkowski
Herman
H
Goossens
Surbhi
S
Malhotra-Kumar
D002442:Ceftazidime / Q000494:pharmacology; D002443:Ceftriaxone / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D032023:Integrons / Q000235:genetics; D007557:Israel; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D002443:Ceftriaxone; D002442:Ceftazidime; D001618:beta-Lactamases; C495222:beta-lactamase CTX-M-25, E coli