A phenotype of Escherichia coli and Klebsiella pneumoniae, resistant to piperacillin/tazobactam (TZP) but susceptible to carbapenems and 3rd generation cephalosporins, has emerged. The resistance mechanism associated with this phenotype has been identified as hyperproduction of the β-lactamase TEM. However, the mechanism of hyperproduction due to gene amplification is not well understood. Here, we report a mechanism of gene amplification due to a translocatable unit (TU) excising from an IS26-flanked pseudo-compound transposon, PTn6762, which harbours blaTEM-1B. The TU re-inserts into the chromosome adjacent to IS26 and forms a tandem array of TUs, which increases the copy number of blaTEM-1B, leading to TEM-1B hyperproduction and TZP resistance. Despite a significant increase in blaTEM-1B copy number, the TZP-resistant isolate does not incur a fitness cost compared to the TZP-susceptible ancestor. This mechanism of amplification of blaTEM-1B is an important consideration when using genomic data to predict susceptibility to TZP.

Resistance to piperacillin/tazobactam (TZP) in Escherichia coli has predominantly been associated with mechanisms that confer resistance to third-generation cephalosporins. Recent reports have identified E. coli strains with phenotypic resistance to piperacillin/tazobactam but susceptibility to third-generation cephalosporins (TZP-R/3GC-S). In this study we sought to determine the genetic diversity of this phenotype in E. coli (n=58) isolated between 2014–2017 at a single tertiary hospital in Liverpool, UK, as well as the associated resistance mechanisms. We compare our findings to a UK-wide collection of invasive E. coli isolates (n=1509) with publicly available phenotypic and genotypic data. These data sets included the TZP-R/3GC-S phenotype (n=68), and piperacillin/tazobactam and third-generation cephalosporin-susceptible (TZP-S/3GC-S, n=1271) phenotypes. The TZP-R/3GC-S phenotype was displayed in a broad range of sequence types, which was mirrored in the same phenotype from the UK-wide collection, and the overall diversity of invasive E. coli isolates. The TZP-R/3GC-S isolates contained a diverse range of plasmids, indicating multiple acquisition events of TZP resistance mechanisms rather than clonal expansion of a particular plasmid or sequence type. The putative resistance mechanisms were equally diverse, including hyperproduction of TEM-1, either via strong promoters or gene amplification, carriage of inhibitor-resistant β-lactamases, and an S133G bla CTX-M-15 mutation detected for the first time in clinical isolates. Several of these mechanisms were present at a lower abundance in the TZP-S/3GC-S isolates from the UK-wide collection, but without the associated phenotypic resistance to TZP. Eleven (19%) of the isolates had no putative mechanism identified from the genomic data. Our findings highlight the complexity of this cryptic phenotype and the need for continued phenotypic monitoring, as well as further investigation to improve detection and prediction of the TZP-R/3GC-S phenotype from genomic data.

Resistance to piperacillin/tazobactam in Escherichia coli is usually mediated by mechanisms providing resistance to 3rd generation cephalosporins, such as extended-spectrum β-lactamases and carbapenemases. Recent reports have identified E. coli strains with resistance to piperacillin/tazobactam but susceptibility to 3rd generation cephalosporins, achieved through hyperproduction of penicillinases, but the genetic diversity of this phenotype and the diversity of resistance mechanisms are unknown. We analysed the genomes of 63 clinical isolates of E. coli with this phenotype, isolated between 2014-2017 at a single tertiary hospital in Liverpool, UK. The phenotype was displayed in a broad range of sequence types which, after comparison with a UK-wide collection, reflected the overall diversity of E. coli clinical isolates. Resistance mechanisms were also diverse, and included predicted hyperproduction of penicillinases, either via strong promoters or gene amplification, carriage of inhibitor resistant β-lactamases, and an S133G blaCTX-M-15 mutation detected for the first time in clinical isolates.

We identified a small colony variant (SCV) of an amoxicillin/clavulanic acid-resistant derivative of a clinical isolate of Escherichia coli from Malawi, which was selected for in vitro in a subinhibitory concentration of gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase that catalyses the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed the SNP in hemA resulted in a significant fitness cost to the isolate, which persisted even in the presence of hemin. However, the phenotype quickly reverted during sequential sub-culturing in liquid growth media. As hemA is not found in mammalian cells, and disruption of the gene results in a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by E. coli .

By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae .