Resistance to piperacillin/tazobactam in Escherichia coli resulting from extensive IS26-associated gene amplification of blaTEM-1

Abstract

Background bla TEM-1 encodes a narrow-spectrum β-lactamase that is inhibited by β-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/β-lactamase inhibitor (P/BLI) combinations. Objectives To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome. Methods EC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of blaTEM-1 amplification was probed using PCR. Expression of blaTEM-1 mRNA was determined using quantitative PCR and β-lactamase activity was determined spectrophotometrically in a nitrocefin conversion assay. Growth rate was assessed to determine fitness and stability of the gene amplification was assessed by passage in the absence of antibiotics. Results Illumina sequencing of EC78 identified blaTEM-1B as the only acquired β-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with blaTEM-1B bracketed by IS26 elements. The chromosomal location of the IS26-blaTEM-1B amplification was confirmed by ONT sequencing. Hyperproduction of blaTEM-1 was confirmed by increased transcription of blaTEM-1 and β-lactamase activity and associated with a significant fitness cost; however, the array was maintained at a relatively high copy number for 150 generations. PCR screening for blaTEM amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a blaTEM gene. Conclusions IS26-associated amplification of blaTEM can cause resistance to P/BLI combinations. This adaptive mechanism of resistance may be overlooked if simple methods of genotypic prediction (e.g. gene presence/absence) are used to predict antimicrobial susceptibility from sequencing data.

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofblaTEM-1B

“…Tazobactam is able to inhibit the activity of class A β-lactamases [39], and therefore the presence of bla TEM-1 within the genome of an E. coli isolate should not result in resistance to TZP. However, two studies have linked amplification of bla TEM , and therefore hyperproduction of the BL, with this phenotype [15, 18], with one linking amplification and the presence of IS 26 [18]. However, the exact mechanism of amplification/hyperproduction has remained elusive.…”

“…Hansen et al 2019 associated amplification of bla TEM-1 in E. coli clinical isolate with a significant fitness cost [18];Fitness of this isolate was compared to other unrelated clinical isolates of E. coli which hyperproduced Bla TEM-1 due to promoter mutations, rather than the same isolate with and without amplification. This approach can lead to over- or under-estimation of fitness cost, as genetic background of the isolate can have an impact on the overall fitness affect, as can the environment fitness is assessed in [45, 46].…”

“…Mechanisms leading to hyperproduction include mutations in the promoter region of bla TEM , changing it from a weak promoter ( P3 ) to a stronger promoter ( P4 or P5 ) [16] or a single point mutation further upstream resulting in the overlapping, stronger promoter Pa/Pb superseding the weaker P3 promoter [17], and increased production of bla TEM . Another such mechanism proposed to cause TZP-resistance but 3 rd generation cephalosporin and carbapenem susceptibility is increase in copy number of bla TEM present in the chromosome [15, 18]. Gene amplification has been linked to the cause of temporary antibiotic resistance seen in a sub-population of bacteria and is known as heteroresistance.…”

“…While the mechanism of amplification of the bla TEM is not well known, recent studies have found that the amplified bla TEM has been co-located on a segment of DNA containing other antibiotic resistance genes, such as aadA and sulI , termed a genomic resistance module [15]. Amplification of bla TEM leading to TZP resistance via β-lactamase hyperproduction has also been suggested to be mediated by the presence of the insertion sequence, IS 26 [18]. IS 26 is often linked with the movement of antibiotic resistance genes; for example a translocatable unit (TU) containing IS 26 has been shown to be able to excise from the transposon Tn 4352 B, which itself was located on a plasmid, between two IS 26 , leaving one in the plasmid [21, 22].…”

Concurrence of Porin Loss and Modular Amplification of β-Lactamase Encoding Genes Drives Carbapenem Resistance in a Cohort of Recurrent Enterobacterales Bacteremia

“…Additionally, van Boxtel et al demonstrated that serial passaging of an ESBL or AmpC-producing isolate in the presence of a carbapenem can result in amplification of plasmid-borne β-lactamase genes (7). Increased expression of the narrow-spectrum TEM β-lactamases has similarly been reported to result in cefepime (8) and piperacillin-tazobactam resistance (9–11), indicating that β-lactamase gene dosage is a factor in increasing resistance to multiple β-lactam chemotherapies. These findings demonstrate expression level and copy number of β-lactamases without known carbapenemase activity have important effects on carbapenem susceptibility in porin deficient backgrounds (6, 7).…”