Population Analysis and Epidemiological Features of Inhibitor-Resistant-TEM-β-Lactamase-Producing Escherichia coli Isolates from both Community and Hospital Settings in Madrid, Spain

Punctual mutations in the TEM-1 or TEM-2 gene may lead to inhibitor-resistant-TEM (IRT) beta-lactamases with resistance to beta-lactam-beta-lactamase inhibitor combinations and susceptibility to cephalosporins. The aim of this work was to analyze the current epidemiology of IRT beta-lactamases in contemporary clinical Escherichia coli. Isolates were prospectively collected in our hospital (2007 and 2008) from both outpatients (59.8%) and hospitalized patients (40.2%). The genetic relationships of the isolates were determined by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and phylogenetic group analysis. IRT genes were sequenced and located by hybridization, and the incompatibility group of the plasmids was determined. From a total of 3,556 E. coli isolates recovered during the study period, 152 (4.3%) showed reduced susceptibility to amoxicillin-clavulanate, with 18 of them producing IRT enzymes (0.5%). These were mostly recovered from urine (77.8%). A high degree of IRT diversity was detected (TEM-30,-32,-33,-34,-36,-37,-40, and -54), and the isolates were clonally unrelated but were mostly associated with phylogenetic group B2 (55.5%). In 12 out of 16 (75%) isolates, the bla(IRT) gene was plasmid located and transferred by conjugation in 9 of them, whereas chromosomal localization was demonstrated in 4 isolates (25%). The sizes of the plasmids ranged from 40 kb (IncN) to 100 kb (IncFII, IncFI/FIIA), and they showed different restriction patterns by restriction fragment length polymorphism analysis. Unlike extended-spectrum beta-lactamase producers, the frequency of IRT producers remains low in both community and hospital settings, with most of them causing urinary tract infections. Although bla(IRT) genes are mainly associated with plasmids, they can be also located in the chromosome. Despite this situation, clonal expansion and/or gene dispersion was not observed, denoting the independent emergence of these enzymes.