Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

Abstract

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and beta-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as "TEM-1B like" and "TEM-2 like" restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the beta-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of beta-lactam-beta-lactamase inhibitor combinations.

Prevalence of Clinical Isolates of Escherichia coli Producing Inhibitor-Resistant β-Lactamases at a University Hospital in Barcelona, Spain, over a 3-Year Period

“…The diversity of mutant enzymes, which appeared over a short time in nonrepetitive and nonlinked strains, cannot be explained by an epidemic phenomenon. These results suggest the independent emergence and selection of these TEM variants under antibiotic-selective pressure (3,9). We found two clonally related strains producing two different IRTs (TEM-51 and TEM-54).…”

Predictability of Phenotype in Relation to Common β-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae

“…We selected unique clinical isolates of E. coli and K. pneumoniae collected from microbiology laboratories in Sydney between 2005 and 2013, with almost all coming from Westmead Hospital (see Tables S1 to S8 in the supplemental material). These were chosen on the basis of antimicrobial susceptibility (Phoenix automated susceptibility test NMIC-101; BD Diagnostic Systems, Sparks, MD, USA) and the presence of relevant antibiotic resistance genes, as determined by PCR for bla TEM (12), plasmid AmpC genes (13), common ESBL genes (14), and for bla IMP (15). Isolates were grouped according to the bla genes identified by multiplex PCR/reverse line blot (mPCR/RLB) (16,17): bla TEM (n ϭ 33), bla CMY-2 -like genes (n ϭ 23), bla CMY-2 -like plus bla TEM (n ϭ 18), bla DHA (n ϭ 28), bla CTX-M (n ϭ 116), or bla IMP (n ϭ 100).…”

“…bla CTX-M genes were further identified as group 1 or group 9 by specific PCR amplification (19) and as specific genes by sequencing. bla TEM was amplified (12) and sequenced in all E. coli isolates categorized in the bla TEM and bla CMY-2 -like plus bla TEM groups. Additional PCR was performed to detect bla OXA-48 -like (20) and bla NDM genes (21).…”

“…The majority of resistant isolates harbored more than one resistance gene (see Tables S2 to S8 in the supplemental material). PCR and sequencing (12) revealed that all E. coli isolates containing bla TEM encode a TEM-1 enzyme (see Tables S2 and S4 in the supplemental material; all were bla TEM-1b , except JIE1805, with bla TEM-1c ). Sequencing of bla CTX-M genes revealed bla CTX-M-3 (E. coli, n ϭ 3), bla CTX-M-15 (E. coli, n ϭ 27; K. pneumoniae, n ϭ 43), bla CTX-M-14 (E. coli, n ϭ 12; K. pneumoniae, n ϭ 24), bla CTX-M-9 (E. coli, n ϭ 2; K. pneumoniae, n ϭ 3), and bla CTX-M-24 (E. coli, n ϭ 2).…”

Molecular characterisation and epidemiological investigation of an outbreak of blaOXA-181 carbapenemase-producing isolates of Klebsiella pneumoniae in South Africa

“…The resulting PCR amplicons were purified (QIAquick PCR purification kit, QIAGEN, Germany) and then sequenced. To test for the presence of extended-spectrum β-lactamase (ESBL) ( Table 1) [17][18][19][20][21][22][23][24][25][26] genes bla CTX-M , bla SHV and bla TEM , conventional PCR assays using primers designed to target internal fragments of these genes were performed and any resulting PCR amplicons of the expected size were sequenced. To describe the genomic arrangement of the identified bla OXA-181 genes in positive isolates in relation to the ISEcp1B gene, which had previously been shown to be associated with the OXA-181 gene, [7] conventional PCR was carried out using primers designed to amplify an internal fragment of the insertion element, ISEcp1B, and an internal portion of the OXA-181 gene ( Table 1).…”