Piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1B

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.

β -lactam/β-lactamase inhibitor combinations were developed to overcome the activity of β-lactamases 1,2 , which inactivate β-lactam antibiotics by hydrolysing the β-lactam ring. βlactamase inhibitors, particularly metallo-β-lactamase inhibitors, are still urgently required and their discovery and development is the topic of intense investigation 3 . β-lactam/β-lactamase inhibitor combinations currently in clinical use include amoxicillin/clavulanic acid, ampicillin/sulbactam and piperacillin/tazobactam (TZP), with ceftolozane/tazobactam and ceftazidime/avibactam recently introduced into clinical use. TZP has broad-spectrum antibacterial activity and is routinely used for intra-abdominal infections and febrile neutropenia 4,5 . TZP usage has increased year on year in the UK, from just under 2.1% of all antibiotics prescribed in 2008-2009 to 3.6% in 2012-2013 6 . During the 2-year period between April 2012 and March 2014 10.2% of bacteraemia causing Escherichia coli isolates in England tested for TZP susceptibility were resistant 7 . Resistance to TZP has been previously linked to AmpC hyperproduction and the co-production of multiple β-lactamases, which also confer resistance to 3rd generation cephalosporins 8 . In addition, tazobactam is a poor inhibitor of metallo-β-lactamase enzymes, which cause resistance to TZP, alongside 3rd generation cephalosporins and carbapenems [9][10][11] .
A phenotype in Klebsiella pneumoniae and E. coli clinical isolates has emerged which has been classified as TZP-non-susceptible but susceptible to 3rd generation cephalosporins and carbapenems [12][13][14] , indicating a different resistance mechanism. While still relatively rare, one study in the United States found that the frequency of this phenotype was between 1.9 and 5.6% of E. coli and K. pneumoniae isolated from the bloodstream between 2011 and 2015, and specifically 4.1% of all E. coli over the study period 14 . The same study reported that risk factors associated with the TZP-non-susceptible but 3rd generation cephalosporin and carbapenem susceptible phenotype included exposure to β-lactam/β-lactamase inhibitors and cephalosporins within the previous 30 days 14 . Resistance to TZP, but 3rd generation cephalosporin and carbapenem susceptible, has been linked to the presence of β-lactamases which hydrolyse piperacillin but not 3rd generation cephalosporins. The β-lactamases such as SHV-1 and TEM-1 are usually inhibited by tazobactam, which also has an intermediate inhibitory activity towards OXA-1 15,16 . TEM has been hypothesised to overcome the inhibitory activity of tazobactam via hyperproduction of the enzyme, allowing the hydrolysis of piperacillin 17 . 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) 18 or a single point mutation further upstream resulting in the overlapping, stronger promoter Pa/Pb superseding the weaker P3 promoter 19 , increasing the production of TEM. Another such mechanism proposed to cause TZP-resistance but 3rd generation cephalosporin and carbapenem susceptibility is the increase in copy number of bla TEM present in either a plasmid or chromosome 17,20 . Gene amplification has been linked to the cause of temporary antibiotic resistance seen in a sub-population of bacteria and is known as heteroresistance. Heteroresistance is often lost after multiple generations in the absence of antibiotic selective pressure, due to the fitness cost imposed by the production of extra proteins as a result of amplification 21,22 . While the mechanism of amplification of 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 17 . 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, IS26 20 . IS26 is often linked with the movement of antibiotic resistance genes; for example a translocatable unit (TU) containing IS26 has been shown to be able to excise from the transposon Tn4352B, which itself was located on a plasmid, between two IS26, leaving one in the plasmid 23,24 . Recently, directly repeated IS26 elements, plus the interveining DNA, have been labelled as pseudo-compound transposons (PTns) 25 rather than composite transposons as they do not transpose as an entire unit. Following excision, the single IS26 and antibiotic resistance gene(s) found between the two insertion sequences forms a circular TU, which then can insert into a plasmid via a conservative Tnp26dependent but RecA-independent mechanism, Tnp26 replicative transposition or RecA-dependent homologous recombination, preferentially adjacent to another IS26 insertion sequence 23,24,26 .
Here, we identify a pair of clonal E. coli isolates, isolated from a single patient across two separate infection episodes, which display within-patient evolution to TZP resistance. In this isolate, amplification of bla TEM-1B occurs when a TU excises from a pseudocompound transposon flanked by directly repeated IS26 present in the chromosome. The TU re-inserts into the chromosome creating a tandem array of the TU and increasing the copy number of bla TEM-1B , which does not carry a fitness cost. Replicating the evolutionary event in vitro in the TZP-susceptible isolate leads to the capture of the TU in a plasmid which contains a copy of IS26.

Results
Identification of clonal isolates. Initially, we identified five isolates in the collection of TZP-resistant, 3rd generation cephalosporin and carbapenem susceptible E. coli from blood cultures at the Royal Liverpool University Hospital (RLUH) which had a corresponding TZP-susceptible isolate from the same or previous infection episode, and therefore could have evolved to become TZP-resistant within a patient. Restriction fragment length polymorphisms (RFLP) of the 16S rRNA amplicons from the five pairs of isolates indicated that three pairs of TZP-susceptible/TZP-resistant clinical isolates had identical digestion patterns ( Supplementary Fig. 1A). Two of these three pairs of isolates had an identical resistance profile generated during routine disk-based susceptibility testing, aside from TZP (Supplementary Table 1). RFLPs of genomic DNA identified one pair of isolates with identical banding patterns indicating clonality; 190693 (TZP-susceptible) and 169757 (TZPresistant) which were isolated from different infection episodes from the same patient~3 months apart ( Supplementary Fig. 1B). During the first infection episode, the TZP-susceptible E. coli was isolated and the patient was initially treated with a five-day course of TZP, followed by a seven-day course of TZP with teicoplanin and then a third seven-day course of TZP although a second blood culture was found to be negative. A second infection episode occurred~6-7 weeks after the final course of TZP was completed, and again the patient was treated initially with TZP until the TZPresistant E. coli was isolated, when the treatment was changed to meropenem. Putative clonality of these two isolates was confirmed with whole-genome sequencing; both isolates were identified as serotype H30 O86, sequence type 315 and had an average nucleotide identify (ANI) of 100%, with 36 single nucleotide polymorphisms (SNP) difference between the two isolates.
Confirmation of TZP susceptibility and resistance mechanism. We determined the minimum inhibitory concentrations (MIC) of the pair of isolates and verified that TZP-susceptible isolate was susceptible to TZP (2-4/4 µg/ml) and TZP-resistant isolate was resistant to TZP (64/4 µg/ml) according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints 27 (Table 1). Using the efflux pump inhibitor phenylalaninearginine β-naphthylamide (PAβN) as a supplement in the MIC assay, we were able to rule out overexpression of efflux pumps as a possible mechanism of resistance as there was less than a fourfold reduction 17 in MIC of both the TZP-susceptible (2/4 µg/ml) and TZP-resistant isolates (32/4 µg/ml, Table 1). Whole-genome sequencing revealed no differences in the predicted resistance genes present in the genome between the TZP-susceptible and TZPresistant isolate ( Table 2) and no mutations in the promoter region of any of the β-lactamases present within the genome (bla TEM-1B , bla OXA-1 or ampC) of the TZP-resistant isolate. We confirmed that the TZP-resistant isolate hyperproduced a β-lactamase due to the significant increase in nitrocefin hydrolysis compared to the TZPsusceptible isolate (P value = <0.0001, Fig. 1a, Supplementary Table 3). The isolates contained two β-lactamases, TEM-1B and OXA-1, which were both increased in copy number in the TZPresistant isolate compared to the housekeeping gene uidA (Fig. 1b). We, therefore determined whether TEM-1B, OXA-1 or both contributed to TZP resistance using increased amount of tazobactam to inhibit TEM-1B and 100 mM sodium chloride to inhibit OXA-1 [28][29][30] . Increasing tazobactam from 4 to 8 µg/ml and 4 to 16 µg/ml resulted in a twofold and eightfold decrease in TZP MIC respectively, while supplementation of 100 mM sodium chloride decreased the TZP MIC by twofold (Supplementary Table 2). As there was only a modest decrease in resistance in the presence of sodium chloride, it is likely hyperproduction of TEM-1B is the most important determinant of resistance to TZP.
Genomic comparison of the clonal isolates. Using a hybrid assembly of Oxford Nanopore Technologies (ONT) long and Illumina short sequencing reads, we were able to complete the genome of the TZP-susceptible isolate, which was found to be 5151952 bp in length, with a GC content of 50.64% and did not contain any plasmids (Supplementary Fig. 2A). In contrast, we were unable to complete the genome of the TZP-resistant isolate as a 530 bp segment remained unresolved, and a complete, low copy number (2.59x) 106637 bp plasmid containing an IncFII replicon (Supplementary Fig. 2B) was detected. A complete, smaller (10899 bp) circular DNA molecule was also found to be present in the TZP-resistant isolate, at a copy number of 8.51x, however, this small circular DNA molecule did not contain a plasmid replicon (Supplementary Fig. 2B). The large plasmid did not to contain any predicted antimicrobial or metal resistance genes, but did contain three bacteriocins, both colicin B and M (with cognate immunity proteins) and linocin. Comparison of the predicted resistance genes present on the chromosome of the TZP-susceptible and TZPresistant isolates highlighted that bla TEM-1B , bla OXA-1 , aac(3)-lla, aac (6')-lb-cr, tet(D) and catB3 were missing from the assembled chromosome of the TZP-resistant isolate ( Table 2). Characterisation of the small circular DNA molecule found that it contained these missing resistance genes, as well as several putative transposable elements including three copies of IS26 (Table 2), and aligned exactly to the chromosome of the TZP-susceptible isolate and was no longer present in the chromosome of the TZP-resistant isolate. The predicted catB3 resistant gene was truncated to 69.8% length and therefore unlikely to be functional, which was confirmed as both the TZP-susceptible and TZP-resistant isolates were susceptible to chloramphenicol (CHL) according to EUCAST clinical breakpoints (Table 1). Further analysis of the TZP-susceptible genome uncovered that the circular DNA molecule from the TZPresistant isolate aligned with 100% identity to an integrated pseudocompound transposon flanked by two copies of IS26 in the same orientation, which we subsequently registered as PTn6762 via The Transposon Registry 31 , however, the circular DNA molecule only contained one of the flanking IS26. The antibiotic resistance genes bla TEM-1B , bla OXA-1 , aac(3)-lla, aac(6')-lb-cr and tet(D) were identified to be present on PTn6762 (Fig. 2a). This suggested that a TU 23,24,26 , containing one flanking IS26 and the antibiotic resistance genes, was excised from the chromosomally located PTn6762 while the other flanking IS26 stayed in the chromosome (Fig. 2a). Interestingly, we also found that the tetracycline resistance regulator, tetR, on the TU had been disrupted 52 bp from the end of the tetR due to the excision which overlaps the start of the copy of IS26 remaining in the chromosome. Following excision, a copy of IS26 is present at the start of the TU which then connects to the end of the TU, containing the disrupted tetR, forming a circular DNA molecule. As the two copies of IS26 are identical and in the same orientation, and therefore containing the same 52 bp bases missing from tetR, tetR was reformed when the TU circularised completing the 657 bp gene.
Confirmation of the amplification of the TU. Firstly, we confirmed the amplification of the resistance genes found on the TU in the TZP-resistant isolate and PTn6762 in the chromosome of the TZP-susceptible isolate. Comparing the fold change in copy number of each resistant gene to the housekeeping gene uidA, we found that each resistance gene on the TU increased in copy number in the TZP-resistant isolate compared to the TZPsusceptible isolate (P value = <0.0001, Fig. 1b, Supplementary Table 3). The increase in copy number of the resistance genes found on the TU in the TZP-resistant isolate also corresponded to an increase in MIC of all antimicrobials that the genes confer resistance to (Table 1), further confirming the amplification of the Minimum inhibitory concentrations (MIC) of gentamicin (GEN), tetracycline (TET), chloramphenicol (CHL), ciprofloxacin (CIP), amoxicillin/clavulanic acid (AMC) and piperacillin/tazobactam (TZP) (with and without Phenylalanine-arginine β-naphthylamide (PAβN)) towards the TZP-susceptible and TZP-resistant isolates. Determination of MICs were performed in triplicate (n = 3).
Chromosome Translocatable unit Predicted antimicrobial resistance genes by ResFinder found on the genome of the piperacillin/ tazobactam (TZP)-susceptible and TZP-resistant isolates and their position in genome. The resistance gene catB3 was predicted by ResFinder to be present with 69.8% length, but both isolates were phenotypically chloramphenicol susceptible.
entire TU. By PCR of the left and right junctions of the chromosomally located PTn6762, with one primer specific for PTn6762 and one for the chromosome either before or after IS26 (Fig. 2b, Supplementary Table 4), we were able to confirm that PTn6762 was present in the chromosome of both the TZPsusceptible isolate and TZP-resistant isolate by yielding the expected 1640 bp and 2402 bp products, respectively (Supplementary Fig. 3). Using primers that would only yield a 1942 bp product if a tandem TU array in the chromosome was present (Fig. 2c), we were able to detect the presence of the tandem TU in the TZP-resistant isolate and absence in the TZP-susceptible isolate ( Supplementary Fig. 3). To further confirm that the TU is present in the chromosome in tandem arrays, we mapped the long-read sequences to a predicted structure of three tandem TUs and built a consensus sequence out of these reads. We were unable to build a consensus sequence for the TZP-susceptible isolate, suggesting the TU was not present in the chromosome in a tandem array. In contrast, we were able to build a consensus sequence for the TZP-resistant isolate, which showed 99.89% identity to the predicted sequence ( Supplementary Fig. 4). This suggests that the TU from PTn6762 has excised and reinserted to form a tandem array within the chromosome of the TZP-resistant isolate.
Capture of the TU. We sought to capture the excised TU, and therefore observe the excision and insertion events, using a CHL resistant, high copy number pUC plasmid (pHSG396) containing IS26 in the same orientation relative to the origin of replication as found on PTn6762 (Supplementary Methods 1). pHSG396:IS26 was transformed into the TZP-susceptible isolate and TZP-resistant derivatives were selected for by growing the isolate in the presence of TZP. We detected the insertion of a >10 kb fragment into pHSG396:IS26 after TZP selection following digestion with XhoI and EcoRI ( Supplementary Fig. 5A). Insertion of the TU from the TZP-susceptible chromosome was confirmed through PCR amplification across the two newly formed junctions on the pHSG396: IS26, with one primer specific for pHSG396 and one for either aac (6')-lb-cr (left) or tet(D) (right) on the TU for each junction (Supplementary Table 4). We yielded the expected 1458 bp (left) and 1385 bp (right) products (Fig. 2d), consistent with the insertion of the TU adjacent to IS26 and reforming PTn6762 in pHSG396 ( Supplementary Fig. 5B). Subsequent long-read sequencing of the entire pHSG396:IS26 plasmid following the capture of the TU established the presence of at least three different plasmid structures; pHSG396:IS26 without the captured TU ( Supplementary  Fig. 6A Fig. 7B). This was due to a single nucleotide insertion resulting in a frame shift and a terminal stop to the protein.
Replication of excision of the TU. We sought to determine whether the presence of another mobile IS26 and/or the use of TZP induced the excision of the TU by replicating the evolutionary event that led to the TZP-susceptible isolate becoming resistant to TZP in vitro. We found that there was a significant increase in copy number of all the resistance genes present on PTn6762, relative to the housekeeping gene uidA, following exposure to 2 × MIC of TZP (8/4 µg/ml) while there was no evidence of amplification in the same isolate grown in the absence of TZP (P value = <0.0001, Fig. 3, Supplementary Table 3). There was also an increase in copy number when the isolate containing the pHSG396:IS26 plasmid was exposed to TZP but, again, no increase in copy number when TZP was absent (P value = <0.0001, Fig. 3 Table 3). We also assessed the relative fitness of the TZP-susceptible isolate following exposure to TZP (resulting in the increase in copy number of the resistance genes present on the TU (Fig. 3)) compared to the TZP-susceptible grown in the absence of TZP (which did not result in an increase in copy number (Fig. 3)). Again, we found no significant change in fitness in LB (P value = 0.1264), ISO (P value = 0.1126) and M9 (P value = 0.3007, Fig. 4, Supplementary Table 3).

Discussion
Tazobactam is able to inhibit the activity of class A β-lactamases 32 , 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 β-lactamase, with this phenotype 17,20 , with one linking amplification and the presence of IS26 20 . Although, the exact mechanism of amplification/hyperproduction has remained elusive. Due to the emergence of a TZP-resistant but 3rd generation cephalosporins and carbapenems susceptible E. coli and K. pneumoniae phenotype [12][13][14] , as well as the increasing reliance on TZP as an empirical treatment 6  mechanism of amplification. In this study we had the opportunity to compare a pair of clonal isolates which have evolved within a patient to become TZP-resistant but remain cephalosporin/carbapenem susceptible, allowing us to build on recent studies and identify the mechanism of IS26-mediated amplification of bla TEM-1B which leads to TZP-resistance. We found multiple antibiotic resistance genes, including bla TEM-1B , co-located on a IS26-flanked pseudo-compound transposon PTn6762 on both the TZP-resistant and TZP-susceptible isolate. The resistance genes tet(D) and bla TEM-1B were both present on PTn6762 as a result of the insertion of several transposons into the same location, demonstrated by the presence of multiple insertion sequences and transposable elements on PTn6762. While tazobactam has intermediate inhibitory activity towards OXA-1 16 , we found that the presence of this β-lactamase in the TZPsusceptible isolate did not confer resistance to TZP, amplification of bla OXA-1 was involved not in resistance to TZP and TEM-1B is likely the most important determinant of TZP resistance. Although we were able to determine that efflux pumps did not have a role in TZP-resistance in this isolate, we did not investigate whether other mechanisms, such as change in permeability due to porins, contributed to TZP-resistance. Amplification of bla TEM-1B is achieved when PTn6762 is excised from the chromosome forming a TU (Fig. 5), evidenced by the hybrid assembly of the TZP-resistant isolate, increase in copy number of the antibiotic resistance genes present on PTn6762 and the capture of the TU in pHSG396:IS26. While it is a possibility that the presence of the circularised TU in the hybrid assembly is due to an artefact of the assembly, we confirmed the presence of the excised TU following capture in pHSG396:IS26. The TU then re-inserts into the chromosome adjacent to a copy of IS26 to create a tandem array of TUs, increasing the copy number of the antibiotic resistance genes present on the TU (Fig. 5). While precise excision and formation of a TU containing the antibiotic resistance gene aphA1a from a Tn4352B transposon present in a plasmid has been demonstrated before as a mechanism of movement of antibiotic resistance gene 23,24,35 , we have shown that this mechanism can directly result in gene amplification leading to antibiotic resistance. We found no evidence from the whole-genome sequencing of the TZP-resistant isolate of insertion of the TU anywhere else in the chromosome, except for a gap in sequencing where PTn6762 was originally situated in the TZP-susceptible isolate adjacent to an IS26. Through PCR of the left and right junctions of the gap in sequencing, we confirmed that PTn6762 was still present at this location in the chromosome of the TZP-resistant isolate and therefore existed as PTn6762 with tandem repeats of the TU in the chromosome, which the hybrid assembly was unable to resolve. Rodriguez-Villodres et al. 36 replicated the amplification event leading to TZP-resistance in E. coli isolates containing bla TEM through exposure to increasing concentrations of TZP 36 . They found an increased copy number of bla TEM in TZP-resistant isolates and regions of amplification, in which one of the amplified regions contained IS26 36 . Using the CHL-resistant pUC vector, pHSG396, containing a copy of IS26 from the TZP-susceptible chromosome, we were also able to replicate the evolutionary event and capture the excised TU adjacent to the IS26 copy in the plasmid, providing evidence of the insertion event, but we found no evidence of tandem repeats of the TU in the pHSG396:IS26 plasmid. Therefore, the TU preferentially re-inserts into the chromosome adjacent to the chromosomally located IS26 via a conservative Tnp26-dependent but RecAindependent mechanism, Tnp26 replicative transposition or RecA-dependent homologous recombination 24 producing tandem repeats of the TU. Again, while this mechanism has previously been demonstrated in terms of movement of antibiotic resistance genes 23,24 , we have shown that this mechanism will cause gene amplification resulting in antibiotic resistance in a clinical isolate. This mechanism is of concern as IS26 has been associated with the transfer of bla NDM-1, in a recent nosocomial outbreak in Germany 35 , and other carbapenemases 37 and therefore represents a risk of clinical resistance to any carbapenemase inhibitor currently in development 38 which needs to be investigated further. The method of capture of the TU used in this study can be used to investigate whether the same mechanism will result in gene amplification and subsequent reduction in efficacy to other β-lactam/β-lactamase inhibitors, as well as to further confirm the role of TZP in the induction of excision of the TU, and therefore gene amplification. Abdelraouf et al. 15 identified five TZP-resistant but 3rd generation cephalosporin susceptible K. pneumoniae which did not contain bla TEM , but instead, the most common type of β-lactamase was SHV-1 15 . These isolates showed a different phenotypic resistance profile to the E. coli isolates containing bla TEM assessed in the same study; exhibiting higher MICs against the 3rd generation cephalosporins tested and susceptibility to TZP was not restored with increasing concentrations of tazobactam, potentially indicating a different mechanism of resistance 15 . Further work should be undertaken with both E. coli and K. pneumoniae to fully understand both the resistance profile and the different mechanisms of resistance involved in this phenotype to aid diagnostics.
Hansen et al. 20 associated amplification of bla TEM-1 in an E. coli clinical isolate with a significant fitness cost 20 ; Fitness of this isolate was compared to other unrelated clinical isolates of E. coli which hyperproduced 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 39,40 . Adler et al. 22 , however, identified a fitness cost associated with IS26-mediated amplification of an antibiotic resistance cassette from a plasmid in all lineages tested 22 . In this study, we were able to compare the fitness of the paired clinical isolates and in vitro evolved isolates with and without amplification of the TU and found that there was no significant difference in fitness cost, despite amplification of a >10 kb region with multiple functionally transcribed genes and, in terms of the TZP-resistant clinical isolate, has acquired a large plasmid. If this lack of fitness cost is translated into a physiological environment, it may result in the TZP-resistant phenotype persisting. While there was no observed fitness effect of amplification on the single isolate in this study, the effect of amplification on bacterial fitness needs to be extensively investigated as it may not be a global phenomenon Resistance to the β-lactam/β-lactamase inhibitor TZP can be the result of gene amplification, and subsequent hyperproduction, of bla TEM-1B . The mechanisms involved in this gene amplification are the IS26-associated excision and re-insertion into the chromosome, of a TU containing bla TEM-1B derived from a chromosomally located pseudo-compound transposon PTn6762, which is either selected by or in response to exposure to TZP. The TU is capable of re-inserting into the chromosome, creating a tandem array of TUs and increasing the copy number of bla TEM-1B . In this clinical isolate and an in vitro evolved isolate, we found that there was no effect on fitness due to the amplification and subsequent carriage of high numbers of the TU. This mechanism of amplification, and the subsequent hyperproduction, of bla TEM-1B is an important consideration if treatment failure involving TZP occurs, as well as other β-lactam/β-lactamase inhibitor combinations, and when using genomic data to predict resistance/susceptibility to β-lactam/β-lactamase inhibitor combinations.

Methods
Ethics statement. All E. coli isolates used in this study were collected at the RLUH (Liverpool, UK) as part of routine clinical diagnostics procedures. The bacterial isolates were identified in the hospital biobank database as having interesting resistance profiles by the Consultant Microbiologist. Isolates were retrieved from the Microbiology Laboratory by Thomas Edwards (Liverpool School of Tropical Medicine), who has an NHS research passport enabling work in the hospital laboratories. Antimicrobial susceptibility data and the treatment data were anonymised, unlinked to patient identifiers and data produced in this study was not used for the treatment or management of patients, therefore requirement for ethical approval and informed patient consent was not required. This was confirmed using the online NHS Research Ethics Committee review tool http://www.hradecisiontools.org.uk/ethics/.
Bacterial isolates, media and antibiotics. Clinical isolates of E. coli isolated from blood cultures between 2010 and 2017 at the RLUH (Liverpool, UK) which were found to be carbapenem and cephalosporin susceptible but TZP resistant using the disk diffusion method of antimicrobial susceptibility testing (AST) were initially identified from isolate records. Isolate records were then searched for a corresponding carbapenem, cephalosporin and TZP susceptible isolates, isolated in the same or a previous infection episode from the same patient. Using these criteria, we identified five paired clinical isolates of E. coli. All isolates had been stored at the time of blood culture isolation in glycerol broth at −80°C.
Piperacillin, tazobactam (both Cayman Chemical, USA), gentamicin (GEN), and amoxicillin trihydrate:potassium clavulanate (4:1, AMC) was solubilised in molecular grade water (all Sigma, UK), while CHL and tetracycline (TET) (both Sigma, UK) were solubilised in ethanol (VWR, USA) and ciprofloxacin (CIP) was solubilised in 0.1 N hydrochloric acid solution (both Sigma, UK). All stock solutions of antibiotics were filter sterilised through a 0.22 µm polyethersulfone filter unit (Millipore, USA). In all assays, unless stated, tazobactam was used at a consistent concentration of 4 µg/ml and the piperacillin concentration was altered.
Restriction enzyme digestion. RFLP analysis of 1 µg of 16S rRNA PCR amplicon from the 10 putative clonal isolates were digested with AlwNI, PpuHI and MslI (all New England Biolabs (NEB), USA) and 1 µg of long fragment genomic DNA extracts of 153964, 152025, 190693 and 169757 were digested with SpeI and MslI (both NEB, USA) for 1 h at 37°C. Both RFLP digest reactions were incubated for 5 min at 80°C and immediately run on a 1% agarose gel. Enzyme digest of 500 ng of plasmid DNA extracted from 190693 and 169757 was performed with PpuMI and XhoI (both NEB, USA) and immediately run on a 1% agarose gel following incubation for 1 h at 37°C. RFLP was performed on a single DNA extract from each bacterial isolate.
MIC for TZP, GEN, CIP, CHL, AMC and TET were performed using the broth microdilution method, in cation adjusted Mueller Hinton Broth (CA-MHB, Sigma, UK), following EUCAST Guidelines 44 . Efflux pump inhibition was performed using PAβN as a supplement in CA-MHB at a final concentration of 50 µM, inhibition of TEM-1B was determined with piperacillin with increasing concentrations of tazobactam (4, 8 and 16 µg/ml) and inhibition of OXA-1 was determined with TZP plus 100 mM sodium chloride (Sigma, UK). Each MIC was performed in triplicate using independent bacterial cultures, each with three technical replicates.
Nitrocefin assay. β-lactam hydrolysis was evaluated using a colorimetric nitrocefin assay. Cell lysates were obtained from triplicate cultures of 190693 and 169757 in LB, adjusted to an optical density at 600 nm (OD 600 ) of 0.1. Cultures (10 ml) were centrifuged at 14,000 × g for 5 min, the supernatant discarded, and the pellet resuspended in 5 ml phosphate-buffered saline (PBS). The cultures were sonicated for three intervals of ten seconds, on ice, using a Soniprep 150 plus (MSE centrifuges, UK). The lysed cultures were centrifuged at 14,000 × g for 5 min, and the supernatant taken as the culture lysate.
A total of 90 µl of this lysate was then added to 10 µl of 0.5 mg/ml nitrocefin solution (Sigma, UK) in a 96-well microplate, in triplicate. The absorbance of the plate was read at an optical density of 450 nm (OD 450 ) every 20 s for 25 min, using a SPECTROstar OMEGA spectrophotometer (BMG lab systems, Germany). This assay was performed in triplicate using independent bacterial cultures.
Whole-genome sequencing and bioinformatics. Whole-genome sequencing was performed from a single DNA extract for each isolate. Illumina MiSeq 2 × 250 bp short-read sequencing of long fragment DNA extractions from isolates 190693 and 169757, as well as adapter trimming of the sequencing reads, were provided by MicrobesNG (MicrobesNG, UK).
The same long fragment DNA extracts were processed using the SQK-LSK109 ligation and SQK-RBK103 barcoding kit and sequenced on an R9.4.1 flow cell with an ONT MinION. Sequencing reads were basecalled during the sequencing run using MinKNOW (v19.05.0), de-multiplexing and adapter trimming of the basecalled reads were performed using Porechop (v0.2.4) and finally sequencing reads were filtered for a quality score of 10 via Filtlong (v0.2.0).
Each qPCR reaction contained 6.25 µl QuantiTect® SYBR Green PCR buffer (Qiagen, UK), 0.4 µM forward and reverse primers (Supplementary Table 4), 1 ng of extracted DNA, and molecular grade water to a final volume of 12.5 µl. Reactions were processed using a Rotor-Gene Q (Qiagen, Germany), using the following protocol; an initial denaturation step of 95°C for 5 min, followed by 40 cycles of; DNA denaturation at 95°C for 10 s, primer annealing at 58°C for 30 s, and primer extension at 72°C for 10 s with fluorescence monitored in the FAM channel. High resolution melt analysis was carried out over a temperature range of 75°C to 90°C, increased in 0.1°C increments, in order to confirm specific amplification. Fluorescence thresholds were set manually for calling Ct values, at 5% of the difference between baseline and maximum fluorescence.
The mean qPCR Ct value for the uidA gene from each strain was taken using at least three replicates qPCR reactions, and the ΔΔCT method was utilised to determine to fold change using at least triplicate qPCR reactions for each AMR gene from a single DNA extract of each isolate.
In vitro evolution of susceptible isolate. The clinical isolate 190693 (TZP-susceptible isolate) and 190693 transformed with pHSG396:IS26 were subcultured into 10 ml LB and 10 ml LB plus 35 µg/ml CHL, respectively, and incubated at 37°C for 18 h at 200 rpm. Following incubation 10 µl of 190693 was subcultured into 10 ml LB and 10 ml LB plus 8/4 µg/ml TZP and 10 µl of 190693 with pHSG396:IS26 was subcultured into 10 ml LB plus 35 µg/ml CHL and 10 ml LB plus 35 µg/ml CHL and 8/4 µg/ml TZP and incubated at 37°C for 24 h at 200 rpm. Each evolution experiment was performed in triplicate using independent cultures. Genomic DNA from each of the four cultures from each replicate were extracted for qPCR following the protocol described in Supplementary Methods 1, except a single replicate of each of the three biological replicates were performed.
TU capture. The TZP-susceptible isolate 190693 transformed with pHSG396:IS26 was grown in the presence of TZP and CHL as previously stated. Following selection, the culture was serially diluted 1/10 in PBS down to 10 −7 dilution and 50 µl of each dilution was plated out on to LB agar supplemented with 35 µg/ml CHL and 16/4 µg/ml TZP. Five single colonies were selected and subcultured into 10 ml LB plus 35 µg/ml CHL and 16/4 µg/ml TZP for 18 h at 37°C and 200 rpm and the plasmid extracted following the protocol in Supplementary Methods 1. The purified plasmids were transformed into NEB® 5-alpha competent E. coli (NEB, US) following the protocol in Supplementary Methods 1 and plated out on to LB agar supplemented with 35 µg/ml CHL and 16/4 µg/ml TZP and incubated at 37°C for 18 h. A single colony from each transformation was subcultured into 10 ml LB supplemented with 35 µg/ml CHL and 16/4 µg/ml TZP and incubated at 37°C, 200 rpm for 18 h and the plasmid extracted following the protocol in Supplementary Methods 1. The initial pHSG396:IS26 plasmid extract and pHSG396: IS26 plasmid selected in TZP and extracted from NEB® 5-alpha E. coli were digested with XhoI (NEB, US) and EcoRI for 1 h at 37°C, followed by a 20 min incubation at 65°C and run on a 1% agarose gel. A single DNA extract of pHSG396:IS26 selected in TZP was sequenced on a MinION, adapters removed, and sequencing reads filtered as stated above, except using a quality score of 50. Long-read sequencing reads of the captured TU in pHSG396:IS26 were mapped to three predicted plasmid structures, pHSG396:IS26, pHSG396:IS26 plus TU and tandem pHSG396:IS26 plus TU and used to build a consensus sequence using the method described above.
Comparative fitness. The relative fitness of 169757 (TZP-resistant) and 190693 (TZP-susceptible) grown in the presence of 8/4 µg/ml TZP, compared to 190693 and 190693 grown in the absence of TZP, respectively, were assessed comparatively in LB, ISO and M9. Each culture was diluted to an OD 600 of 0.1 in the respective media, then further diluted 1/1000 in the same media and 150 µl of each diluted culture added to a flat bottom, 96-well microtitre plate in duplicate as well as 150 µl of the media as a negative control. The 96-well plate was incubated at 37°C and the OD 600 of each well was measured with 100 flashes every 10 min over 24 h, with orbital shaking at 200 rpm between readings, using a Clariostar Plus microplate reader (BMG Labtech, Germany). The relative fitness compared to either 190693 or 190693 grown in the absence of TZP between absorbance values 0.02 and 0.08 and a minimum R value of 0.9905 was estimated using BAT version 2.1 59 . Comparative fitness was performed in triplicate with independent bacterial cultures, with two technical replicates for each replicate.
Statistical analysis. Statistical analysis of comparison for the qPCR assay of the antibiotic resistance genes was performed using the two-way ANOVA with Uncorrected Fisher LSD test. Statistical analysis of the nitrocefin assay was performed using the two-way ANOVA test. Statistical analysis of relative fitness of 169757 and 190693 grown in the presence of TZP was performed using ordinary one-way ANOVA with Uncorrected Fisher LSD test. All statistical tests were performed using GraphPad Prism version 8.2.1. Means and standard error of the mean of all data presented in this study are available in Supplementary Table 3.